Biosynthesis of violacein, structure and function of L-tryptophan oxidase VioA from chromobacterium violaceum

Article abstract of DOI:10.1074/jbc.M116.741561

Violacein is a natural purple pigment of Chromobacterium violaceum with potential medical applications as antimicrobial, antiviral, and anticancer drugs. The initial step of violacein biosynthesis is the oxidative conversion of L-tryptophan into the corresponding α-imine catalyzed by the flavoenzyme L-tryptophan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-methylpropanoic acid) and a product-related (2-(1H-indol-3-ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was synthesized for subsequent kinetic and x-ray crystallographic investigations. Structures of the binary VioA?FADH₂ and of the ternary VioA?FADH₂ ?2-(1H-indol-3-ylmethyl)prop-2-enoic acid complex were resolved. VioA forms a "loosely associated" homodimer as indicated by small-angle x-ray scattering experiments. VioA belongs to the glutathione reductase family 2 of FAD-dependent oxidoreductases according to the structurally conserved cofactor binding domain. The substrate-binding domain of VioA is mainly responsible for the specific recognition of L-tryptophan. Other canonical amino acids were efficiently discriminated with a minor conversion of L-phenylalanine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan, 5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient substrates of VioA. The ternary product-related VioA structure indicated involvement of protein domain movement during enzyme catalysis. Extensive structure-based mutagenesis in combination with enzyme kinetics (using L-tryptophan and substrate analogs) identified Arg⁶⁴ , Lys²⁶⁹ , and Tyr³⁰⁹ as key catalytic residues of VioA. An increased enzyme activity of protein variant H163A in the presence of L-phenylalanine indicated a functional role of His163 in substrate binding. The combined structural and mutational analyses lead to the detailed understanding of VioA substrate recognition. Related strategies for the in vivo synthesis of novel violacein derivatives are discussed.

Full text of DOI:10.1074/jbc.M116.741561

crossmark
THE JOURNAL OF BIOLOGICAL CHEMISTRY VOL. 291, NO. 38, pp. 20068–20084, September 16, 2016
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc. Published in the U.S.A.
Biosynthesis of Violacein, Structure and Function of
L-Tryptophan Oxidase VioA from Chromobacterium violaceum*
Received for publication, June 9, 2016, and in revised form, July 25, 2016 Published, JBC Papers in Press, July 27, 2016, DOI 10.1074/jbc.M116.741561
Janis J. Füller‡, René Röpke§, Joern Krausze¶, Kim E. Rennhack‡, Nils P. Daniel‡, Wulf Blankenfeldt¶ʈ, Stefan Schulz§,
Dieter Jahn‡1 , and Jürgen Moser‡
From the ‡Institute of Microbiology and ʈInstitute of Biochemistry, Biotechnology and Bioinformatics, Technische Universität
Braunschweig, Spielmannstrasse 7, D-38106 Braunschweig, the §Institute of Organic Chemistry, Technische Universität
Braunschweig, Hagenring 30, D-38106 Braunschweig, and the ¶Structure and Function of Proteins, Helmholtz Centre for Infection
Research, Inhoffenstrasse 7, D-38124 Braunschweig, Germany
Violacein is a natural purple pigment of Chromobacterium
violaceum with potential medical applications as antimicrobial,
antiviral, and anticancer drugs. The initial step of violacein bio-
synthesis is the oxidative conversion of L-tryptophan into the
corresponding ␣-imine catalyzed by the flavoenzyme L-trypto-
phan oxidase (VioA). A substrate-related (3-(1H-indol-3-yl)-2-
methylpropanoic acid) and a product-related (2-(1H-indol-3-
ylmethyl)prop-2-enoic acid) competitive VioA inhibitor was
synthesized for subsequent kinetic and x-ray crystallographic
investigations. Structures of the binary VioA⅐FADH2 and of the
ternary VioA⅐FADH2⅐2-(1H-indol-3-ylmethyl)prop-2-enoic acid
complex were resolved. VioA forms a “loosely associated”
homodimer as indicated by small-angle x-ray scattering experi-
ments. VioA belongs to the glutathione reductase family 2 of
FAD-dependent oxidoreductases according to the structurally
conserved cofactor binding domain. The substrate-binding
domain of VioA is mainly responsible for the specific recogni-
tion of L-tryptophan. Other canonical amino acids were effi-
ciently discriminated with a minor conversion of L-phenylala-
nine. Furthermore, 7-aza-tryptophan, 1-methyl-tryptophan,
5-methyl-tryptophan, and 5-fluoro-tryptophan were efficient
substrates of VioA. The ternary product-related VioA structure
indicated involvement of protein domain movement during
enzyme catalysis. Extensive structure-based mutagenesis in
combination with enzyme kinetics (using L-tryptophan and sub-
strate analogs) identified Arg64, Lys269, and Tyr309 as key cata-
lytic residues of VioA. An increased enzyme activity of protein
variant H163A in the presence of L-phenylalanine indicated a
functional role of His163 in substrate binding. The combined
structural and mutational analyses lead to the detailed under-
standing of VioA substrate recognition. Related strategies for
the in vivo synthesis of novel violacein derivatives are discussed.
for the synthesis of the natural bisindole compounds violacein,
rebeccamycin, and staurosporine. The water-insoluble purple
pigment violacein is mainly produced by the bacteria Chromo-
bacterium violaceum and Janthinobacterium lividum in tropi-
cal habitats where it might be responsible for the shielding
against UV radiation (1–3). Various biological activities of
violacein, including antibacterial, antiviral, as well as cytotoxic
effects against several tumor cell lines, have been demonstrated
(4). The related compounds rebeccamycin and staurosporine
show strong antitumorigenic activity because of DNA topoi-
somerase or protein kinase inhibition (5, 6).
Synthesis of violacein, rebeccamycin, or staurosporine is
based on a closely related initial pathway in which orthologous
enzymes catalyze the oxidation of a Trp moiety into the related
indole-3-pyruvic acid (IPA)2 imine by the enzymes VioA,
RebO, or StaO (Fig. 1) (7–9). Subsequently, oxidative coupling
of two imines by VioB, RebD, or StaD results in the formation of
a short-lived compound that was proposed to be an IPA imine
dimer (7, 10). For the synthesis of rebeccamycin and staurospo-
rine, this reactive intermediate is spontaneously converted into
chromopyrrolic acid (11–13). By contrast, violacein biosynthe-
sis requires a key intramolecular rearrangement. The postu-
lated IPA imine dimer is the substrate of VioE, which is
catalyzing a [1,2]-shift of the indole ring to produce protode-
oxyviolaceinic acid (7, 14). Fig. 1 gives a schematic overview
about the related pathways as follows: common enzymatic
reactions and the involved cofactors are highlighted (gray shad-
ing), subsequent steps for the synthesis of violacein (VioE,
VioD, VioC, and one autocatalytic step) (3, 7), rebeccamycin
(RebP, RebC, RebG, and RebM) (15, 16), and staurosporine
(StaP, StaC, StaG, StaN, StaMA, and StaMB) (16) are indicated.
Synthesis of violacein starts with the FAD-dependent oxida-
tion of L-Trp to IPA imine catalyzed by VioA. The VioA protein
from C. violaceum shares a substantial degree of sequence con-
servation with RebO or StaO proteins (ranging from 18 to 22%
identity; Clustal Omega (17)). Furthermore, sequence identity
values of 14–22% were observed for the comparison of VioA
with L-amino acid oxidases (LAAOs) (3, 18). LAAO-catalyzed
two-electron oxidations are well studied from prokaryotic and
The amino acid tryptophan (Trp) is a versatile metabolite
that is shunted into several microbial secondary pathways. The
oxidative dimerization of two Trp scaffolds is an essential step
* This work was supported by the Government of Niedersachsen, Graduate
School MINAS Microbial Natural Products. The authors declare that they
have no conflicts of interest with the contents of this article.
Theatomiccoordinatesandstructurefactors(codes5G3S,5G3T,and5G3U)have 2 The abbreviations used are: IPA, indole-3-pyruvic acid; IAA, 3-(1H-indol-3-yl)-
been deposited in the Protein Data Bank (http://wwpdb.org/).
1 To whom correspondence should be addressed. Tel.: 49-531-391-5800; Fax:
49-531-391-5854; E-mail: d.jahn@tu-bs.de.
2-methylpropanoic acid; IEA, 2-(1H-indol-3-ylmethyl)prop-2-enoic acid;
LAAO, L-amino acid oxidase; SAXS, small angle x-ray scattering; NSD, nor-
malized spatial discrepancy.
20068 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
FIGURE 1. Biosynthetic pathway of violacein (black), staurosporine (blue), and rebeccamycin (red). Orthologous enzymes VioA, StaO, or RebO and VioB,
StaD, orRebDareinvolvedintheinitialpathwayforthesynthesisofviolacein, staurosporine, andrebeccamycin, respectively(highlightedin gray). ATrpmoiety
(L-Trp) is converted into indole-3-pyruvic acid imine (IPA imine) and subsequently dimerized into a short-lived compound proposed as IPA imine dimer.
Synthesis of rebeccamycin initially requires the chlorination of L-Trp to 7-Cl-Trp by virtue of RebH and RebF. Violacein biosynthesis then includes a VioE-de-
pendent [1,2]-shift of the indole ring (curved arrow) resulting in protodeoxyviolaceinic acid. Subsequent VioD and VioC catalysis followed by a final autocata-
lytic oxidation step leads to the product violacein. A deoxyviolacein derivative is synthesized without involvement of VioD. For staurosporine and rebeccamy-
cin biosynthesis, the IPA imine dimer is spontaneously converted into chromopyrrolic acid. Subsequent enzymes StaP, StaC, StaG, StaN, StaMA, StaMB or RebP,
RebC, RebG, RebM then facilitate the synthesis of staurosporine or rebeccamycin.
eukaryotic enzyme sources (19, 20). However, the synthesized
imines are subsequently deaminated by virtue of an attacking mopyrrolic acid is observed. This non-enzymatic chromopyr-
water molecule into the respective ␣-keto acids (21, 22). rolic acid formation is an essential step during staurosporine or
In the absence of VioE, the off-pathway metabolite chro-
By contrast, violacein biosynthesis relies on the reactive IPA rebeccamycin biosynthesis (Fig. 1) (3, 11, 13). In the presence of
imine as a direct substrate of VioB. Furthermore, the postulated VioE, the reactive IPA imine dimer instead is converted to pro-
IPA imine dimer reaction product is also labile, which might todeoxyviolaceinic acid. Structural and biochemical investiga-
reflect the need for an activated substrate for the unusual [1,2]- tions for VioE revealed that the sophisticated [1,2]-indole rear-
shift of the indole ring during VioE catalysis. However, present rangement is catalyzed in the absence of any cofactor or metal
date in vitro investigations revealed that the direct interaction ion (67).
of VioA and VioB (or of VioB and VioE) is not an absolute
prerequisite for protodeoxyviolaceinic acid synthesis (7).
Violacein biosynthesis is completed by sequential action
of two flavin-dependent oxygenases, VioD and VioC. VioD
In a recent publication, 50% FAD occupancy was determined hydroxylates one indole ring at the 5-position to yield pro-
for recombinantly purified VioA protein. Kinetic characteriza- toviolaceinic acid. Subsequently, VioC hydroxylates the sec-
tion of this protein was performed in a tandem peroxidase assay ond indole ring at the 2-position and spontaneous oxidative
with an optimal pH of 9.25. Formation of the unstable IPA decarboxylation then leads to the formation of violacein (Fig.
imine goes along with a reduced flavin on VioA, which is sub- 1) (7).
sequently reoxidized by molecular oxygen leading to stoichio-
Under physiological conditions, the violacein biosynthetic
metric peroxide formation. The detection of hydrogen perox- pathway also results in the formation of deoxyviolacein (23).
ide revealed kcat and Km(Trp) values of 203 minϪ1 and 31 ␮M for Deoxyviolacein, lacking the hydroxyl group at position 5, is
VioA (7).
synthesized without involvement of VioD, which might indi-
The heme-containing oxidase VioB catalyzes the coupling of cate a certain degree of flexibility for VioC substrate recog-
two IPA imine molecules to form the postulated intermediate nition (Fig. 1).
IPA imine dimer (7). For this molecule, only indirect experi-
In this study, recombinantly produced VioA from C. viola-
mental evidence is available due to the short-lived nature of this ceum is analyzed in a combined biochemical and x-ray crystal-
compound (often referred to as compound X in the literature) lographic approach. Structure-based site-directed mutagenesis
(7, 18). Catalase activity has been demonstrated for VioB, and it along with kinetic experiments in the presence of artificial sub-
was proposed that the heme iron center is responsible for VioB strates or active site inhibitors reveal the molecular mechanism
catalysis (7).
of VioA.
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20069

Crystal Structure of VioA
FIGURE 2. Purification and characterization of VioA analyzed by SDS-PAGE, UV-visible spectroscopy, analytical gel permeation chromatography and
SAXS. A, SDS-PAGE analysis of GST-tagged VioA affinity purification using Protinoா glutathione-agarose 4B. Lane M, molecular mass marker; relative molecular
masses (ϫ1000) are indicated. Lane 1, supernatant after ultracentrifugation; lane 2, flow-through of the GST affinity chromatography; lanes 3 and 4, washing
steps; lanes 5–7, elution fractions of untagged VioA with a calculated molecular mass of 47,159 Da (black arrow) after on-column cleavage with PreScission
protease; lane 8, GST tag elution with buffer containing reduced glutathione. B, UV-visible absorption spectra of the prosthetic FAD/FADH2 group of VioA.
Purified VioA (15 ␮M) was incubated with different concentrations of L-Trp (0–10 ␮M) under anaerobic conditions. Increasing L-Trp concentrations resulted in
a sequential decrease of the characteristic absorption maxima of FAD at 387 and 457 nm (black arrows) and an increased absorption at 328 nm (gray arrow) due
to the formation of FADH2. C, bar chart comparing the calculated molecular weight of a VioA monomer or dimer with the experimentally derived values obtained
from analytical gel permeation chromatography (GPC) experiments (also in the presence of the inhibitor IEA) and small angle x-ray scattering experiments (SAXS). D,
analytical gel permeation chromatography of purified VioA (75 ␮M) using a SuperdexTM 200 Increase 5/150 GL column (GE Healthcare) on an Äkta purifier system.
Protein standards were apoferritin (Mr ϭ 443,000), ␤-amylase (Mr ϭ 200,000), alcohol dehydrogenase (Mr ϭ 150,000), albumin (Mr ϭ 66,000), carbonic anhydrase
(Mr ϭ29,000), andcytochromec(Mr ϭ12,000). Columncalibration(inset)andproteinsampleanalysiswereperformedataflowrateof0.45ml minϪ1. Elutionvolumes
were recorded at 280 nm (for protein standards) or at 450 nm (for the VioA⅐FAD complex). Samples of VioA were measured in the absence (black line) or presence (red
line) of 500 ␮M IEA. E, SAXS analysis of the ternary VioA structure. Experimental SAXS data obtained from a soluble VioA sample (black dots) were related to the
simulatedSAXScurvesofapotentialmonomer(blue), amoreglobulardimer(red), oranelongateddimer(green). TheoreticalSAXScurveswerecalculatedonthebasis
of the binary VioA crystal structure. The ␹ values reflect the fit quality with respect to the experimental data (maximum fit quality observed for the globular dimer).
Superposition of the three-dimensional low resolution electron density (gray, calculated from the SAXS scattering curve) with the globular dimer (red, calculated from
the binary VioA x-ray structure) indicates a high degree of structural complementarity.
Results
nm (Fig. 2B, solid line), as is characteristic for FAD-containing
flavoproteins (24). Spectroscopically, an overall FAD content of
55% was determined using the molar extinction coefficient of
the oxidized flavin, in agreement with the outcome of a recent
publication (7). In addition, the FAD content of purified VioA
was also estimated on the basis of single-turnover VioA activity
experiments. Strict anoxic conditions were required to prevent
the reoxidation of the resulting FADH2 cofactor due to electron
transfer onto molecular oxygen. Purified samples of VioA at a
concentration of 15 ␮M were incubated in the presence of vary-
ing L-Trp concentrations ranging from 0 to 10 ␮M. The
recorded absorption spectra revealed a clear decrease of the
characteristic FAD signal (Fig. 2B, compare solid line versus
Production and Purification of VioA—The L-Trp oxidase
VioA from C. violaceum was efficiently overproduced in Esch-
erichia coli as a soluble GST-VioA fusion protein (Fig. 2A, lane
1). The tagged protein was immobilized on a glutathione-aga-
rose column. Subsequently, the target VioA protein was liber-
ated by on-column cleavage with PreScissionTM protease (Fig.
2A, lanes 2–7). Overall, 40 mg of the homogeneous target pro-
tein was obtained from 1 liter of cell culture. SDS-PAGE anal-
yses indicated a relative molecular weight of 45,000 (Fig. 2A,
lanes 5–7, calculated molecular mass, 47,159 Da) and the integ-
rity of purified VioA was confirmed by N-terminal sequencing.
UV-visible absorption spectroscopy of a purified VioA sam-
ple revealed characteristic absorption maxima at 387 and 457 dotted lines). When the remaining FAD concentrations were
20070 JOURNAL OF BIOLOGICAL CHEMISTRY VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
FIGURE 3. Identification of VioA inhibitors. D-Trp, citrate, o-aminobenzoate, IEA, and rac-IAA were analyzed as potential inhibitors in the coupled VioA
horseradish peroxidase assay containing 0.2 ␮M VioA in the presence of 1 mM L-Trp (A). Alternatively, potential inhibitors were analyzed in an HPLC-based
substrate depletion assay using 1 ␮M VioA and 500 ␮M L-Trp (B). A, representative inactivation kinetics monitoring the formation of the quinonimine dye at a
wavelength of 505 nm. A VioA activity assay (solid line) and competition experiments in the presence of 10 mM D-Trp, o-aminobenzoate, citrate, IEA, and IAA,
respectively, are displayed (gray line and consecutive dotted lines). B, representative substrate depletion assays analyzed on a Jasco HPLC system using
␮BondapakTM C18 3.9 ϫ 150 mm column. Reactions were incubated for 3 min at 30 °C. A control experiment in the absence of VioA (a), a standard VioA activity
experiment (b), and inhibition experiments in the presence of 5 mM citrate (c), 5 mM IEA (d), or 5 mM rac-IAA (e) are displayed. C, relative activities of VioA
inhibition experiments (coupled assay and depletions assay) using different inhibitor concentrations. The activity of the standard VioA assay in the presence of
1 mM L-Trp was set as 100%. Dashes indicate experiments not performed. Results are presented as means Ϯ S.D. of three independent biological samples,
measured as triplicates.
plotted against the applied L-Trp concentration, an FAD con- revealed a moderate VioA inhibition (72% relative activity, 10
tent of ϳ50% was estimated for the recombinantly purified mM D-Trp). In the present literature, citrate and o-aminoben-
VioA.
zoate have been described as efficient inhibitors of LAAOs (21,
Tight binding of the FAD cofactor was demonstrated in an 25, 26). In agreement with these distantly related investigations,
aerobic dialysis experiment. Under the employed turnover con- a relative activity of 15% in the presence of 10 mM citrate was
ditions, the FAD cofactor is constantly regenerated due to the observed. By contrast, in the presence of 10 mM o-aminobenzo-
presence of O2. A VioA sample was incubated in the presence of ate only a moderate inactivation indicated by a residual activity
a large volume of buffer containing 40 mM L-Trp. The resulting of 71% was determined (Fig. 3C). Because citrate does not
VioA dialysate after a subsequent buffer exchange step (to closely resemble the L-Trp molecule, a substrate-related and a
remove VioA educt and product) did not reveal a decreased product-related L-Trp derivative was newly synthesized for
FAD content (data not shown).
subsequent inhibition experiments. In compound 3-(1H-indol-
Inhibition of VioA and Synthesis of a Substrate- and Product- 3-yl)-2-methylpropanoic acid (IAA), the amino group of L-Trp
related Inhibitor—The identification of efficient active site is replaced by a methyl group, whereas the non-chiral com-
inhibitors is a well established strategy for the detailed under- pound 2-(1H-indol-3-ylmethyl)prop-2-enoic acid (IEA) is car-
standing of enzyme mechanisms. Inhibitor concentrations of rying a methylene group, instead. Accordingly, the IEA mole-
up to 10 mM were analyzed in the coupled VioA activity assay cule is sharing a closely related spatial orientation (due to the
(9) or alternatively in VioA substrate depletion kinetics (com- sp2-hybridized carbon) as the IPA imine reaction product of
pare Fig. 3, A–C). Experiments in the presence of D-Trp VioA (Fig. 3C). IEA and racemic IAA were synthesized from
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20071

Crystal Structure of VioA
Identification of the Physiological VioA Dimer—Analytical
size exclusion chromatography revealed a dimeric structure of
VioA as indicated by a relative molecular mass of 94.000 Ϯ
7.000 (Fig. 2D). Identical chromatographic results were also
obtained in the presence of 500 ␮M IEA (Fig. 2D). Examination
of potential VioA contacts in the ternary crystal structure (and
in the binary structure) of VioA using the PISA server (Protein
Interfaces, Surfaces and Assemblies) (33) revealed a globular
dimer or alternatively an elongated dimer burying a surface
area of 721.7 or 667.3 Å2 (compare Fig. 2E, inset: top, globular
dimer; middle, elongated dimer; bottom, monomer). Identical
dimers were also observed for VioA⅐FADH2.
FIGURE 4. Synthesis of potential VioA inhibitors IEA and rac-IAA.
Subsequently, small angle x-ray scattering (SAXS) experi-
indole and methyl 2-(bromomethyl)acrylate (1) following a ments were performed to characterize the dimer of VioA in
procedure from Holzapfel et al. (27). Methyl 2-(bromomethyl) solution. This technique makes use of a dilute protein solution
acrylate was obtained in two steps from methyl acrylate and and allows for the reconstruction of a low resolution electron
paraformaldehyde, followed by bromination with PBr3 (28, 29). density map. Almost identical scattering curves for VioA and
Reduction of the methylene group was performed using mag- for VioA in the presence of 3.75 mM IEA were obtained. In Fig.
nesium in MeOH, and saponification of the corresponding 2E, the comparison of the experimental VioA scattering curve
esters 2 and 4 led to the desired products in good yields (Fig. 4) (black dots) with the theoretical scattering curves for the glob-
(30, 31). Kinetic experiments revealed a residual specific VioA ular dimer (red), the elongated dimer (green), and for the VioA
activity of 61 and 53% in the presence of 1 mM IEA and IAA. At monomer (blue) is shown. A significantly higher quality of fit
inhibitor concentrations of 10 mM, residual activities of 7 and was obtained for the globular dimer (␹ ϭ 1.638) when com-
1% were determined. Results for the efficient inhibitors citrate, pared with the elongated dimer (␹ ϭ 9.881) or the monomer
IEA, and IAA were independently confirmed in substrate (␹ ϭ 14.173). From SAXS measurements, an apparent molecu-
depletion activity assays (Fig. 3C). The results of these inhibi- lar mass of 80,000–105,000 was determined, in agreement with
tion experiments were further substantiated in a direct biomo- the related gel filtration experiments (Fig. 2C). The ab initio
lecular interaction study.
model derived from the SAXS experiments described the shape
Microscale Thermophoresis—Thermophoresis is a phenom- of the globular dimer well (Fig. 2E, inset). These findings
enon that describes the movement of molecules along a tem- allowed us to clearly assign the globular dimer with a rather
perature gradient (also termed Ludwig-Soret effect) (32). In this small interface as the physiological unit of VioA.
study, we made use of microscale thermophoresis to analyze
Overall Structure of VioA—Each VioA monomer is com-
the direct binding of citrate, IEA, and IAA to VioA (labeled with posed of 17 ␣-helices and 17 ␤-strands and folds into three
the fluorescence dye NT-647-NHS). Fluorescence was mea- distinct domains. The FAD-binding domain is composed of
sured at a spot in the capillary that was heated by an IR laser residues 4–38, 185–256, and 380–418 and forms a classical
beam. Inhibitor binding was observed as a change in the ther- nucleotide-binding fold (with the P-loop sequence motif
mophoretic properties of the fluorescently labeled protein due 11GXGXXG(X)21D38 (34)) of the glutathione reductase family
to complex formation with VioA at various ligand concentra- 2. The substrate-binding domain includes residues 49–86,
tions. The following dissociation constants were determined in 177–180, and 262–366, and the helical domain contains resi-
agreement with the results of the above mentioned inhibition dues 87–175 (Fig. 5, A, B, and G).
study: IAA 32.2 Ϯ 10.3 ␮M Յ IEA 42.6 Ϯ 13.6 ␮M Յ citrate
The VioA dimer is associated via a discontinuous protein
45.3 Ϯ 7.8 ␮M. According to these results, the newly synthe- interface that involves amino acid residues of the FAD-binding
sized compounds IEA and IAA were considered as promising and of the substrate-binding domain. The dimer assembly is
candidates for the co-crystallization of VioA.
based on two symmetry-related interfaces mainly mediated by
Determination of the Binary and Ternary VioA Structure— residues Arg319, Ala323, Glu324, with His4, Arg35, Tyr206, Asp226
,
,
The sitting-drop vapor diffusion technique was used to crystal- respectively. Among these, conserved residues Arg319, Tyr206
lize VioA, and the phase problem was resolved using single- and Asp226 are engaged in a polar network, including a biden-
wavelength anomalous diffraction data of a samarium nitrate- tate salt bridge interaction of Arg319 and Asp226. This “loosely
derivatized crystal. Electron density in the active center of the associated” dimer forms a central cavity (along the 2-fold sym-
native VioA structure indicated the presence of the flavin cofac- metry of the dimer) with a volume of ϳ13 ϫ 10 ϫ 8 Å3 (Fig. 5A).
tor (occupancy 100%). Crystals of VioA were also grown in the From this cavity, the adenosyl moiety of the tightly bound FAD
presence of IEA. The molecular replacement technique was cofactor is accessible.
used to solve the structure of the ternary VioA complex in the
A Dali search of the Research Collaboratory for Structural
presence of the flavin and IEA. Table 1 summarizes the data Bioinformatics (RCSB) Protein Data Bank (35) indicated the
collection and refinement statistics. The finalized models with strongest structural similarity to the LAAO from Calloselasma
a resolution of 1.8 and 2.4 Å (native structure and ternary com- rhodostoma (Protein Data Bank code 1F8S). Both proteins
plex) were deposited in the Protein Data Bank with accession share a sequence identity of only 14%. However, structural
numbers 5G3T and 5G3U.
superposition revealed a root mean square deviation of solely
20072 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
TABLE 1
Data collection and refinement statistics
The values in parentheses account for the shells of highest resolution. AU, asymmetric unit.
Samarium nitrate
Dataset
derivative
Native VioA
VioA⅐IEA
Synchrotron source, beamline
SLS, X06DA
Desy, Petra III P1 1
BESSY, BL 14.1
Wavelength (Å)
1.770
1.033
0.918
Temperature (K)
100
100
100
d
max Ϫ dmin (Å)
19.92–2.08 (2.15–2.08)
P21
19.98–1.80 (1.86–1.80)
P21
45.98–2.38 (2.42–2.38)
P212121
Space group
Unit cell parameters
a, b, c (Å)
67.88, 87.07, 78.02
90.00, 112.95, 90.00
49,742 (4,528)
0.98 (0.91)
67.09, 89.167, 144.43
90.00, 92.66, 90.00
157,109 (15,444)
0.98 (0.97)
69.27, 81.46, 167.12
90.00, 90.00, 90.00
38,865 (3,822)
1.00 (1.00)
␣, ␤, ␥ (°)
Unique reflections
Completeness
Multiplicity
24.4 (19.6)
6.9 (6.9)
6.6 (6.9)
Mean I/␴(I)
26.3 6.6)
10.9 (2.0)
11.0 (2.0)
Rmerge
0.1097 (0.5100)
0.999 (0.961)
0.1721 (0.2010)
0.1903 (0.2389)
2
0.1034 (0.9474)
0.998 (0.817)
0.1567 (0.2271)
0.1926 (0.2694)
4
0.1616 (0.7014)
0.998 (0.932)
0.1769 (0.2243)
0.2133 (0.2549)
2
CC1/2
Rwork
Rfree
No. of protein molecules per AU
No. of amino acid residues ordered/total
819/850
1,652/1,700
824/850
No. of non-hydrogen atoms per AU
Total
6,908
6,357
168
14,478
12,858
259
6,837
6,398
142
Protein
Ligands
Average B (Å2)
Root mean square deviation from ideal
Bonds (Å)
28.8
26.5
31.2
0.002
0.59
0.008
0.94
0.004
0.94
Angles (°)
Ramachandran plot
Favored (%)
97.3
2.0
98.1
0.0
97.4
0.0
Outliers (%)
PDB code
5G3S
5G3T
5G3U
2.7 Å (calculated via Dali server; 483 residues included). Most the substrate-binding domain. The dimethylbenzene ring is
relevant structural differences are localized in the helical positioned in a partially hydrophobic pocket surrounded by
domain of VioA and include the deletion of two strands and one highly conserved VioA residues Trp359, Val363, and Gly61. In
helical segment (following strand 5 of VioA) as well as the inser- both VioA complex structures, atom N5 of the isoalloxazine
tion of the short helix I (residues 167–170). Beside this, the ring is in direct contact with a structurally conserved water
substrate-binding domain of VioA is linked to the FAD-binding molecule that is bound to Lys269 (Fig. 5, C and F). For the struc-
domain via loop region 367–375 (containing unresolved resi- ture of LAAO from C. rhodostoma, the equivalent water mole-
dues 369–371) and strand 16 (residues 376–378) instead of a cule was discussed as a potential nucleophile for subsequent
helical segment found in LAAO. In Fig. 5G, the topology dia- imine deamination (21). However, synthesis of violacein is
gram (with labeled residues of each secondary structural ele- strictly based on the formation of the IPA imine as the final
ment) is highlighting the FAD-binding domain (light blue), sub- product of VioA catalysis. Accordingly, a nucleophile function
strate-binding domain (blue), and the helical domain (dark of the water molecule must be excluded for VioA. Instead, this
blue).
water might be essential for the appropriate positioning of the
FAD Binding—In the binary and ternary VioA structure, the isoalloxazine ring of FAD.
prosthetic group FAD is bound and deeply buried in an identi-
Active Site of VioA—The structure of VioA in the presence of
cal extended conformation as observed for several LAAO the flavin cofactor and IEA reveals a detailed picture of VioA
enzymes (Fig. 5, A, B, H, and I) (21). The FAD molecule forms an catalysis. In the final electron density map, the isoalloxazine
array of polar contacts, including main chain atoms of amino ring adopts a bent conformation (Fig. 5E) as observed for many
acids Ser15, Met39, Arg46, Gly63, Arg64, Leu208, and Met398, side reduced flavoproteins, including the complex structure of
chain atoms of residues Ser15, Asp38, Arg46, and Asp389, and LAAO from C. rhodostoma (36). For crystallization purposes,
also a series of hydrogen bonding interactions via spatially con- the VioA⅐cofactor complex was always purified in the presence
served water molecules (data not shown). A partially related of the reducing agent DTT, which results in a reduced
polar network is observed in the structure of LAAO from C. VioA⅐FADH2 sample. Crystal growth under oxic conditions
rhodostoma. However, these interactions rely on the conserva- then resulted in faintly yellow crystals indicative of the minor
tion of only three residues (Asp38, Arg46, and Arg64 VioA num- oxidation of the FAD complex. However, subsequent reduction
bering) but otherwise make use of a closely related array of of the prosthetic FAD group due to radiation damage during
polar contacts involving several structurally conserved water synchrotron data collection has been well described in the lit-
molecules that are positioned via equivalent main chain atoms erature (37). Accordingly, the observed crystal structure was
of non-conserved amino acids. As described for most flavopro- referred to as VioA⅐FADH2⅐IEA. For this structure, no differ-
teins, an Mg2ϩ ion is involved in ionic bonds to the diphosphate ences of the overall flavin geometry were observed when com-
group of FADH2. The isoalloxazine ring of FAD points toward pared with the binary VioA⅐FADH2 complex.
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20073

Crystal Structure of VioA
20074 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
In the ternary structure, atom N5 of the prosthetic group a bridging interaction between atom N5 of the isoalloxazine
resides out of the plane of the isoalloxazine ring and points and Lys269. This channel is mainly delineated by conserved res-
toward the IEA ethene group. This inhibitor was synthesized idues Lys269 and Tyr309 and by the partially conserved residues
as a potential transition state analog that closely resembles Glu59, Tyr87, and Phe307. The observed channel might suggest a
the IPA imine reaction product. Accordingly, the VioA⅐ direct path for O2 to the catalytic site. Indeed, the position of
FADH2⅐IEA structure might represent a snapshot in the course the conserved water molecule would be also appropriate for the
of VioA catalysis. The electron density of the IEA ligand clearly binding of O2 and the subsequent formation of H2O2. Analo-
revealed the sp2-hybridized ethene group atoms of IEA. By con- gously, a related LAAO structural analysis revealed a (spatially
trast, the natural L-Trp substrate contains an sp3-C␣ atom unrelated) substrate channel for the oxidative half-reaction
(compare Fig. 5E and the structure with a modeled L-Trp ligand (36).
depicted in Fig. 5F).
Domain Movement of VioA—Structural comparison of the
The substrate-binding site of VioA is located in a deeply bur- overall VioA⅐FADH2⅐IEA and the VioA⅐FADH2 complex
ied cavity (Fig. 5, A, B, H, and I). IEA is accessible via a substrate reveals a significant domain movement (compare gray and blue
entry channel that is delineated by residues Met155, Asp158
,
C␣ representation in Fig. 5H). The structural alterations
Ile159, Phe365, Cys395, and Trp397. The isoalloxazine ring of the observed in the presence of IEA can be roughly described as a
cofactor is located at the re face of the inhibitor, and the imid- movement of the helical domain toward the FAD-binding
azole moiety of IEA is extending away from the cofactor with domain, resulting in a partial closure of the substrate entry path
the N1 atom pointing toward the entry path. The inhibitor’s as indicated by a 3.5-Å closer positioning of the C␣ atoms of
carboxylate forms a salt bridge with the guanidinium group of Met155 and Phe365. These overall movements result in slight
Arg64 and is further engaged in hydrogen bonds with the alterations of the side chain conformation of active site residues
hydroxyl of Tyr309 and with the imidazole ring of His163 (Fig. 5, Val363 and Trp397. Most significant changes are observed for
C and D). A closely related carboxylate binding mode is also residues His163 and Arg64, which are located in a 0.4-Å closer
observed in the structure of LAAO (33). The C2 atom of IEA position in the VioA⅐FADH2⅐IEA structure. The observed con-
(representing the site of oxidative attack in the VioA substrate) formational alterations might be relevant for catalysis and/or
is positioned at a distance of ϳ3.4 Å from the N5 atom of the for the appropriate timing of the IPA imine product release.
isoalloxazine ring. Such distance often is observed for flavoen- The presented structures of VioA have been further explored in
zymes performing a direct hydride transfer mechanism. The biochemical experiments to provide a solid basis for the under-
methylene group of IEA (as part of the imine mimic) points standing of the catalytic mechanism and the substrate recogni-
toward the planar indole ring of Trp397. The indole moiety of tion of VioA.
IEA resides in a hydrophobic pocket that is mainly formed by
Substrate Specificity of VioA—A series of recently described
side chains of residues Tyr143, Ala145, Ile159, and Val363
.
activity assays (9) with the 20 canonical L-amino acids revealed
Possible Channel for the Oxidative Half-reaction of VioA— solely the conversion of L-Trp. This is in agreement with our
VioA catalysis is based on the FAD-dependent formation of the experiments in the presence of L-tyrosine, L-valine, L-proline,
IPA imine (reductive half-reaction) in a process that is coupled L-histidine, L-leucine, and L-alanine (detection limit Ͻ9.9 pmol
to the O2-dependent formation of H2O2 (oxidative half-reac- min mgϪ1, data not shown). Such efficient discrimination
Ϫ1
tion). Because of the tightly bound prosthetic flavin group, the might be essential under physiological conditions, because
second substrate O2 must gain access to the active site of VioA. VioA catalysis results in highly toxic peroxide formation.
In the ternary VioA structure, the catalytic cavity is completely
Our experiments using L-phenylalanine revealed a relative
locked due to the binding of IEA. Accordingly, the deeply bur- activity of 1.8% when compared with the natural substrate. To
ied flavin appears to be inaccessible to O2 via the substrate some extent, the benzyl group of L-phenylalanine might adopt a
entrance channel. Inspection of the VioA protein surface (for related spatial conformation in the active site of VioA under the
both crystal structures) indicated a second water-filled channel employed in vitro assay conditions. Efficient discrimination of
(constriction radius ϳ2.0 Å) (Fig. 5I, right) giving direct access D-Trp was observed in agreement with a stereospecific hydride
to the structurally conserved water molecule (3.1 Å) involved in abstraction mechanism.
FIGURE 5. Crystal structure of VioA from C. violaceum. A, schematic representation of the physiological dimer of the VioA⅐FADH2⅐IEA complex viewed along
the 2-fold symmetry dimer axis (black lens). Individual VioA domains are colored light blue (FAD-binding domain), blue (substrate-binding domain), and dark
blue (helical domain); the respective domains of the second protomer are rendered transparent. The co-crystallized FADH2 (orange) and the inhibitor IEA
(wheat) are shown as ball-and-stick model and the Mg2ϩ ion is displayed as a green sphere. The transparent van der Waals surface is depicted in white. B,
schematic representation of an isolated VioA protomer from A with indicated N and C termini. C, close-up of the active site of VioA. The IEA inhibitor (wheat),
FADH2 (orange), and key VioA residues (blue ball-and-sticks) and the respective hydrogen bonding and salt bridge interactions are shown (dashed lines); water
molecule is displayed as red sphere. D, LIGPLOT (66) representation of the active site showing non-polar and polar contacts (with indicated distances in green
(Å)). E, conformation of the co-crystallized IEA and the flavo moiety of FADH2. Portion of the 2Fo Ϫ Fc electron density map is shown as gray mesh at a contour
level of 1.5 ␴. F, the substrate L-Trp (violet) was modeled into the VioA active site. The distance between the hydrogen atom located at the sp3-hybridized C␣
atom (light gray) and atom N5 of FAD (2.3 Å) is indicated (dashed line). G, topology diagram of VioA. Helices are depicted as cylinders and ␤-strands as arrows
(colorcodeasinA). Undefinedregionsarecoloredin grayandunresolvedresidues369–371areindicatedbythe dottedline. H, superpositionofthebinary(light
gray) and ternary (dark blue) VioA structure (same orientation as in B). The ribbon representation is showing significant movement of the helical domain toward
the FAD-binding domain. I (center), a longitudinal section of the VioA monomer represented as van der Waals surface colored according to its surface
electrostatics (Ϫ10 (red) to ϩ10 (blue) KbT/ec). The left half is highlighting the substrate-binding pocket with the bound IEA molecule and the proposed
substrate entry path (indicated by an orange asterisk). The right half additionally shows the FAD/FADH2-binding pocket and the proposed channel for the
second substrate O2 (indicated by a yellow ϫ) required for the oxidative half-reaction of VioA. The orientation of this right half is roughly as in B and H).
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20075

Crystal Structure of VioA
FIGURE 6. Synthesis of tryptophan analogs 5-fluoro-Trp and 7-aza-Trp.
To further elucidate the substrate specificity of VioA, a series
The bidentate salt bridge of the substrate’s carboxylate with
of four artificial substrates (modified at C1, C5, and C7; com- the guanidinium group of Arg64 (Fig. 5, C and D) is of prime
pare Fig. 7A) were analyzed in the coupled enzyme assay. For importance because mutant proteins R64Q and R64S did not
this purpose, 5-fluoro-Trp and 7-aza-Trp were synthesized reveal any detectable VioA activity (Fig. 7C). The spatial posi-
starting from commercially available 5-fluoroindole and tioning of this carboxylate by additional hydrogen bonding
7-azaindole, respectively. First, a Mannich reaction to the cor- interactions to Tyr309 and His163 is further supported by resid-
responding gramines was performed, followed by addition of ual activities of 5, 8, and 85% for variant proteins Y309A,
methyl iodide (38). The resulting tetramethylammonium H163N, and H163A (Fig. 7C). The moderately reduced activity
iodide salts were obtained with very good yields. Introduction of mutant H163A clearly indicates that VioA catalysis does not
of the amino acid side chain was realized using N,N-diphenyl- rely on an imidazole-mediated general acid/base mechanism.
ethylglycinate (39). Deprotection of the amino group and
For mutant W397A, no enzymatic activity was determined.
saponification led to the desired product in good yields (Fig. 6). The ternary VioA structure indicates the shielding of the sub-
For compound 7-aza-Trp, a relative VioA activity of 23% was strate’s amino group via the planar indole ring system of Trp397
determined when compared with the natural substrate (Fig. 7, (Fig. 5, C and D). Obviously, this Trp plays a substantial role for
A and C). Interference of the 7-aza modification of the indolyl the accurate positioning of the substrate as a prerequisite for
ring with the side chain of Ile159 might be responsible for the subsequent enzyme catalysis. This hypothesis is in agreement
decreased activity. A significantly higher VioA activity of 76% with the observed residual activity of 63% for mutant W397Y
was observed for 1-methyl-Trp in agreement with the ternary (Fig. 7C). To some extent, the planar tyrosyl side chain might be
VioA structure (Fig. 7, A and C). This modification does not able to cope with the spatial orientation of the indole side chain
result in any steric clashes because a substituent at N1 extends of Trp397
into the active site entry channel of VioA (Fig. 5I, left). The most
.
In both VioA structures, Lys269 is in contact with N5 of the
efficient VioA substrates were compounds 5-methyl-Trp and isoalloxazine ring of the cofactor via a bridging water molecule
5-fluoro-Trp showing activities of 133 and 87% (Fig. 7, A and C). (Fig. 5, C and D). This indirect hydrogen bonding interaction of
Obviously, a methyl or a fluoro substituent (providing inverse Lys269 is of central importance for the catalytic mechanism of
inductive effects) is well tolerated in the active site pocket. A VioA because mutant proteins K269Q and K269S revealed
certain degree of flexibility for the side chain of residue Asp311 residual activities of solely 2 and 1% (Fig. 7C). This residue
might be responsible for the broadened substrate specificity of might be essential for the accurate positioning of the isoalloxa-
VioA. These results are further supported by a recent investi- zine ring. However, a potential role during the O2-dependent
gation showing a relative activity of 86% in the presence of the reoxidation of FADH2 must also be considered (40).
artificial substrate 5-hydroxy-Trp (7). Basically, substrate dis-
Val363 is part of the defined binding pocket that is relevant for
crimination only occurs in the presence of natural amino acids, the specific recognition of the hydrophobic indole ring of the
whereas all chemically synthesized L-Trp derivatives were effi- substrate (Fig. 5, C and D). This is supported by our mutational
ciently converted by VioA.
analysis, because variants V363Q and V363A yielded relative
Mutagenesis of Catalytic Residues of VioA—In a structure- activities of 17 and 57%. Replacing Val363 by a larger and more
based mutagenesis approach, the impact of conservative (or polar glutamine residue might result in a steric restriction of the
alternatively of a more drastic) mutations was analyzed with hydrophobic active site. By contrast, the related alanine muta-
respect to amino acids Arg64, His163, Lys269, Tyr309, Val363, and tion might result in a widening of this pocket, reflected by an
Trp397. The integrity of all site-directed mutant proteins was increased activity of 57% (Fig. 7C). Nevertheless, integrity of the
confirmed with thermal shift experiments, and the amount of overall hydrophobic active site pocket of VioA might be rele-
co-purified FAD cofactor was determined (Fig. 7, B and C).
vant to obtain full enzymatic activity.
20076 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
FIGURE 7. Characterization of VioA mutant proteins. A, Trp analogs as artificial substrates for VioA. B, SDS-PAGE analysis of purified recombinant VioA wild
type and mutant proteins. Lane M, molecular mass marker, relative molecular masses (ϫ1000) are indicated. Lane 1, VioA wild type, and lanes 2–12 showing
VioA mutants R64Q, R64S, H163N, H163A, K269Q, K269S, Y309A, V363Q, V363A, W397Y, and W397A. C, relative activities of VioA mutant proteins in the
presenceofTrpanalogsshowninA. Theactivityofthewildtypeproteininthepresenceof L-Trpwassetas100%. TheFADcontentandthemeltingtemperature
(obtainedfromthermalshiftexperiments)forallproteinsareindicated. FormutantsH163A, V363Q, V363A, andW397Y, theactivityinthepresenceof L-Trpwas
also set as 100%, and the activities from substrate analog kinetics were related to that (percent values in parentheses). Activities below the detection limit of the
employedassayareindicatedas n.d. (notdetectable). Dashesindicateexperimentsnotperformed. Resultsarepresentedasmeans ϮS.D. ofthreeindependent
biological samples, measured as triplicates.
Mutant Activities in the Presence of Non-natural Substrates— for variant H163A, discrimination of L-tyrosine, L-valine, L-pro-
As summarized in the table depicted in Fig. 7C, the activity of line, L-histidine, L-leucine, and L-alanine was shown, whereas a
mutants R64Q, R64S, H163N, K269Q, K269S, Y309A, and relative enzymatic activity of 5.4% was determined in the pres-
W397A significantly decreased using artificial substrates 7-aza- ence of L-phenylalanine, corresponding to a 3-fold increase
Trp, 1-methyl-Trp, 5-methyl-Trp, and 5-fluoro-Trp so that val- with respect to the wild type enzyme (Fig. 7C). This indicates
ues partially below the detection limit were determined. When that His163 plays an essential role for the appropriate position-
the activity of the “more active” mutants H163A, V363Q, ing of the (artificial) substrate in the active site of VioA. A
V363A, and W397Y in the presence of L-Trp was set as 100% slightly altered network for the recognition of the substrate’s
(values in parentheses, respectively) the following order of sub- carboxylate group might cause an improved binding of L-phe-
strate activities was obtained: 5-methyl-Trp (70–160%) Ͼ nylalanine in the active site of VioA.
5-fluoro-Trp (51–79%) Ͼ 1-methyl-L-Trp (24–77%) Ͼ 7-aza-
Discussion
Trp (7–22%). Basically, these mutant activities mirror the out-
come of the wild type enzyme assays in the presence of the
VioA and orthologous enzymes StaO and RebO catalyze the
different artificial substrates. Nevertheless, the individually FAD-dependent oxidation of a Trp scaffold within the biosyn-
determined values do not only reflect the simple addition of a thesis of important natural bisindole compounds (Fig. 1). The
mutant and of an artificial substrate effect.
catalytic cycle for the VioA-dependent IPA imine formation
Site-directed mutagenesis of key residues often goes along involves the reoxidation of the prosthetic group by O2 with
with an altered specificity of the respective enzymes (41). formation of H2O2. The resulting imine reaction product is
Accordingly, active mutant proteins V363Q, V363A, H163A, susceptible for subsequent deamination as it is observed for the
and W397Y were further analyzed with respect to substrate related catalysis of LAAO enzymes (21, 42). By contrast, viola-
specificity.
cein biosynthesis requires efficient transfer of the labile inter-
Widening the Substrate Specificity of VioA—VioA proteins mediate from the active site of VioA to the subsequent enzyme
H163N, V363Q, V363A, and W397Y were analyzed in the pres- of the pathway.
ence of L-tyrosine, L-valine, L-proline, L-histidine, L-leucine, and
This investigation reveals a detailed picture for the under-
L-alanine and L-phenylalanine, respectively. None of these standing of VioA substrate specificity. Efficient discrimination
combinations resulted in any detectable enzymatic activity, of the canonical amino acids might be essential under in vivo
indicative of a preserved VioA substrate specificity. By contrast, conditions because the H2O2 “side product” and also unwanted
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20077

Crystal Structure of VioA
imine products might be deleterious for the bacterial cell. How- system of Trp397. Then, the exact orientation of C in the
ever, in the presence of unnatural artificial VioA substrates, Michaelis complex facilitates the oxidative attack. Thi␣s transi-
substantial flexibility due to the conversion of 7-aza-Trp,
1-methyl-Trp, 5-methyl-Trp, and 5-fluoro-Trp was indicated.
Obviously, evolution of the VioA active site did not rely on
clearly defined determinants for the recognition of the indole
ring system.
tion state might be further activated by distortion of the sub-
strate. By analogy to the VioA⅐FADH2⅐IEA complex, the tetra-
hedral C␣ of the substrate might adopt a more planar geometry
with partial sp2 character. Such a distorted geometry would not
only reflect the sterical characteristics of the IPA imine reaction
product but would also explain the observed competitive inhi-
bition in the presence of the transition state analog IEA. For-
mation of this activated ternary complex is accompanied by
protein domain movements as indicated by the structural tran-
sition from the VioA⅐FADH2 to the VioA⅐FADH2⅐IEA complex.
These conformational rearrangements result in a partial clo-
sure of the active site cavity and also include the repositioning
of residues His163 and Arg64. These alterations might be a pre-
requisite for the direct transfer of the C␣ hydride onto N5 of the
isoalloxazine ring. This process is guided by the movement of
the lone pair of electrons from the ␣-amino nitrogen to the C␣,
leading to the formation of the protonated imine reaction prod-
uct. To some extent, the ternary VioA complex also resembles a
well protected enzyme⅐product complex in which the IPA
imine is shielded from the deamination via attacking water
molecules. Subsequently, channeling of the IPA imine only in
the presence of the subsequent enzyme of the pathway would
be an elegant solution to the metabolic problem. However,
characterization of such a type of transient interaction might
require the trapping of the overall VioA protein dynamics.
With respect to this hypothesis, the conformational rearrange-
ments of VioA might also be relevant for the appropriate timing
of the release of the IPA imine reaction product.
Our structural investigation showed a well defined network
for the specific interaction of the polar “head group” of the VioA
substrate (Fig. 5, C and D). Such type of a fixed L-Trp only shows
limited conformational flexibility at the C atom. The hydro-
phobic indole moiety is not specifically␤recognized in the
hydrophobic part of the active site pocket. Obviously, confor-
mational restraints at C in combination with the rigid and
planar indoyl substituen␤t play a key role for VioA substrate
recognition.
With respect to the overall biosynthetic pathway, com-
pounds 7-aza-Trp, 1-methyl-Trp, 5-methyl-Trp, and 5-fluoro-
Trp are interesting candidates for the in vivo synthesis of new
violacein derivatives. As outlined in Fig. 1, variant Trp mole-
cules with a modified indole ring at position 1, 5, or 7 are theo-
retically processed into the respective violacein (or deoxyviola-
cein) derivatives (compare numbering indicated for violacein
and deoxyviolacein). Initial in vivo pathway reconstitution
experiments in our laboratory already indicated the conversion
of synthetic artificial Trp derivatives to new enzymatic prod-
ucts and intermediates. Structural elucidation of the novel
compounds produced and investigations toward their possible
biosynthetic pathway are ongoing. On the basis of these results,
synthetic biology approaches for the engineering of variant
violacein, rebeccamycin, or staurosporine pathways might be a
promising strategy for the synthesis of novel bisindole thera-
peutics. Successful engineering of staurosporine and rebecca-
mycin variant molecules has been demonstrated recently (43,
44).
The second half-reaction of VioA catalysis is responsible for
the reoxidation of the prosthetic group by O2. For this purpose,
a smaller entry channel gives direct access to the bridging water
molecule that links Lys269 to the N5 atom of FADH2 (compare
Fig. 5, C and D, and yellow ϫ in Fig. 5I, right). The position of
this water molecule (found in both structures) might also rep-
resent the site for H2O2 generation as a result of flavin reoxida-
tion. Mutagenesis of Lys269 might indicate almost complete
VioA inactivation due to a significant influence on the reduc-
tive and also on the oxidative half-reaction of VioA. A related
binding position for the oxidative half-reaction of LAAO from
C. rhodostoma has been also described in the literature (36).
Additional experimental work is required to further elucidate
the oxidative half-reaction and the precise sequential order of
VioA catalysis.
The outcome of the combined structural and biochemical
investigation allowed us to propose a detailed molecular mech-
anism of VioA catalysis. Enzyme kinetics of mutant proteins
functionally identified Arg64, His163, Lys269, Tyr309, and Trp397
as essential residues of VioA catalysis. Inspection of the spatial
orientation of these amino acids in both structures of VioA did
not reveal an appropriate catalytic base for the specific depro-
tonation of the C␣-H-bond of the L-Trp substrate. This is incon-
sistent with an acid-base mechanism via a substrate carbanion.
However, the C␣ of L-Trp (as indicated by the bound IEA mol-
ecule) is well oriented with respect to a direct hydride transfer.
In Fig. 5F, the C␣-H-bond (of the modeled substrate complex) is
pointing toward the N5 atom of the cofactor exactly as observed
for many related flavoenzymes performing a hydride transfer
mechanism (21, 45, 46). Accordingly, the following Reaction
sequence for VioA catalysis was postulated. The zwitterionic
L-Trp substrate approaches the catalytic site via the substrate
entry channel (compare orange asterisk in Fig. 5I, left and right).
Subsequently, deprotonation of the ␣-amino group is required
for the formation of the Michaelis complex. This proton
abstraction might be supported by the optimal pH of 9.25 (7)
Experimental Procedures
Plasmid Construction and Site-directed Mutagenesis
The vioA gene from C. violaceum ATCC 12472 was amplified
from genomic DNA (German Collection of Microorganisms
and Cell Cultures (DSMZ)) using primers CAC GGA TCC
ATG AAG CAT TCT TCC GAT ATC and GTA CTC GAG
TCA CGC GGC GAT GCG CTG C (restriction sites under-
lined) and cloned into the BamHI and XhoI sites of pGEX-6P-1
(GE Healthcare), yielding the plasmid pGEX-vioA. Mutations
and also by the active site architecture where the polar ␣-amino in the vioA gene were introduced using the QuikChangeTM
group points perpendicularly to the hydrophobic indole ring site-directed mutagenesis kit (Stratagene) using the following
20078 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
oligonucleotides (exchanged nucleotides in bold): R64Q, GAG Determination of Protein Concentration
CTG GGC GCG GGG CAG TAC TCC CCG CAG C and GCT
GCG GGG AGT ACT GCC CCG CGC CCA GCT C; R64S,
GAG CTG GGC GCG GGG TCT TAC TCC CCG CAG C and
GCT GCG GGG AGT AAG ACC CCG CGC CCA GCT C;
H163N, CGA CAT CGT CGG CAA GAA CCC GGA AAT
CCA GAG CG and CGC TCT GGA TTT CCG GGT TCT TGC
CGA CGA TGT CG; H163A, CGA CAT CGT CGG CAA GGC
GCC GGA AAT CCA GAG CG and CGC TCT GGA TTT CCG
GCG CCT TGC CGA CGA TGT CG; K269Q, GGC TCG CTG
CCG CTG TTC CAG GGT TTC CTC ACC TAC GG and CCG
TAG CTG AGG AAA CCC TGG AAC AGC GGC AGC GAG
CC; K269S, GGC TCG CTG CCG CTG TTC TCT GGT TTC
CTC ACC TAC GG and CCG TAG GTG AGG AAA CCA
GAG AAC AGC GGC AGC GAG CC; Y309F, GGG CGA CAA
GTA CCT GTT CTT CTT TAC CGA CAG CGA GAT GGC C
and GGC CAT CTC GCT GTC GGT AAA GAA GAA CAG
GTA CTT GTC GCC C; Y309A, GGG CGA CAA GTA CCT
GTT CTT CGC GAC CGA CAG CGA GAT GGC C and GGC
CAT CTC GCT GTC GGT CGC GAA GAA CAG GTA CTT
GTC GCC C; V363Q, CAA GTA TTG GGC GCA TGG CCA
GGA GTT CTG CCG CGA CAG CG and CGC TGT CGC
GGC AGA ACT CCT GGC CAT GCG CCC AAT ACT TG;
V363A, CAA GTA TTG GGC GCA TGG CGC GGA GTT
CTG CCG CGA CAG CG and CGC TGT CGC GGC AGA
ACT CCG CGC CAT GCG CCC AAT ACT TG; W397Y, CAC
CGA GCA CTG CGG CTA TAT GGA GGG CGG CCT GC
and GCA GGC CGC CCT CCA TAT AGC CGC AGT GCT
CGG TG; and W397A, CAC CGA GCA CTG CGG CGC GAT
GGA GGG CGG CCT GC and GCA GGC CGC CCT CCA
TCG CGC CGC AGT GCT CGG TG.
The concentration of purified VioA was determined at 280
Ϫ1
nm using an extinction coefficient of ⑀280 ϭ 71,695 MϪ1 cm
.
N-terminal Amino Acid Sequence Determination
Automated Edman degradation was used to confirm the
identity of purified VioA (47–49).
Thermal Shift Experiments
The temperature of the unfolding transition midpoint was
determined in thermal shift experiments as described else-
where (50).
FAD Cofactor Quantification
The FAD content of purified VioA samples was quantified by
UV-visible absorption spectroscopy using an extinction coeffi-
Ϫ1
cient of ⑀450 ϭ 11,300 MϪ1 cm
.
Alternatively, single turnover experiments were performed
under anoxic conditions (oxygen partial pressure below 1 ppm,
oxygen detector, Coy Laboratories, Grass Lake, MI) to estimate
the amount of co-purified FAD. All buffers were prepared by
repeated cycles of evaporation and nitrogen flushing. Purified
samples of VioA at a concentration of 15 ␮M were supple-
mented with increasing concentrations of L-Trp (0–10 ␮M) in a
total volume of 600 ␮l and incubated for 2 min at 22 °C. The
absorption spectrum of all samples was measured using sealed
cuvettes (550–250 nm). Employed concentrations of L-Trp
where then plotted versus the photometrically determined FAD
concentration to estimate the amount of copurified FAD in the
original VioA sample.
Synthesis of VioA Inhibitors and Trp-derivatives, General
Remarks
Heterologous Production and Purification of VioA
Recombinant VioA was produced in E. coli BL21 (␭DE3) cells
containing plasmid pGEX-vioA. An overnight culture was used
Commercially available starting material and solvents were
purchased from Sigma, Fisher/Acros, Alfa Aesar, or Carl Roth
and were used without further purification. Technical solvents
were distilled before use. All reactions involving water-sensitive
chemicals were carried out in heat gun-dried glass equipment
with magnetic stirring under an argon atmosphere. TLC was
performed on Polygram௡ SIL G/UV254 plates (Macherey-Na-
gel) with detection by UV (254 nm) or by immersion in aqueous
solution containing sulfuric acid, ammonium molybdate tetra-
hydrate, and Cer(IV)sulfate tetrahydrate, followed by heating.
Flash chromatography was performed on Silica Gel M60 (0.04–
0.063 mm, 230–400 mesh ASTM) (Macherey & Nagel) under
nitrogen pressure. NMR spectra were recorded on a Bruker
DRX-400 spectrometer (1H 400 MHz, 13C 101 MHz). Chemical
shifts are reported in parts per million relative to tetramethyl-
silane in case of CDCl3 and MeOD, and relative to trimethylsi-
lylpropanoic acid in case of D2O, as an internal standard (␦ ϭ 0).
Coupling constants J are given in Hertz.
Ϫ1
to inoculate 500 ml of LB medium containing 100 ␮g ml
ampicillin. Protein production was induced at an A578 of ϳ0.5
by addition of 50 ␮M isopropyl 1-thio-␤-D-galactopyranoside.
After 21 h at 25 °C and 180 rpm, cells were harvested by cen-
trifugation (15 min, 3000 ϫ g, 4 °C) and washed with 25 ml of
buffer 1 (50 mM Tris-HCl, pH 9.0, and 100 mM NaCl), and the
sedimented cells were stored at Ϫ20 °C. The pellet was thawed
on ice, resuspended in 7.5 ml of buffer 1, and BenzonaseTM
(Merck Millipore, Darmstadt, Germany) was added according
to the manufacturer’s instructions. Cells were disrupted by a
single passage through a French press cell at 19,200 p.s.i. After
ultracentrifugation for 1 h at 110,000 ϫ g at 4 °C, the cell-free
extract was incubated for 2 h with 2 ml of Protino௡ glutathione-
agarose 4B (Macherey & Nagel). After washing two times with
10 ml of buffer 1, the VioA-loaded column was incubated with
200 units of PreScissionTM protease (GE Healthcare) in 2 ml of
buffer 1 for 16 h at 4 °C under slight agitation to liberate the
target protein from the GST tag. VioA was eluted in 6 ml of
buffer 1, and protein containing fractions were identified by
SDS-PAGE. The target protein was stored in buffer 1 contain-
Synthesis of Inhibitors IEA and IAA
IEA (2-(1H-Indol-3-ylmethyl)prop-2-enoic Acid) (3)—To a
ing 5% (v/v) glycerol at 4 °C or at Ϫ20 °C. For subsequent crys- stirred solution of ester 2 (750 mg, 3.48 mmol) in 10 ml of
tallization experiments, VioA purification was performed in the MeOH was added slowly a solution of KOH (590 mg, 10.52
presence of 2 mM DTT.
mmol) in 3 ml of water and 5 ml of MeOH at 0 °C. The mixture
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20079

Crystal Structure of VioA
was stirred for 30 min with cooling. After stirring for 24 h at dried with MgSO4, and the solvent was evaporated under
room temperature, the solvent was evaporated under reduced reduced pressure. Column chromatography on silica (CHCl3/
pressure. The aqueous phase was acidified with HCl to pH 4 MeOH/NH4OH 90:10:1 3 40:10:1) gave the product (1.33 g,
and extracted with ethyl acetate. After drying with MgSO4 and 6.92 mmol, 93%) as a beige-colored solid. 1H NMR (ppm) (400
filtration, the solvent was evaporated under reduced pressure. MHz, CDCl3): 8.47 (s, 1H), 7.36–7.31 (m, 1H), 7.25–7.20 (m,
Purification on a silica gel column (pentane/ethyl acetate 2:1, 1H), 7.12 (d, 3J ϭ 2.6, 1H), 6.95–6.88 (m, 1H), 3.58 (s, 2H), 2.28
Rf ϭ 0.23) gave the desired product as a brownish solid (541 mg, (s, 6H). 13C NMR (ppm) (100 MHz, CDCl3): 157.5 (d, 1J ϭ 234.4,
1
2.69 mmol, 77%). H NMR (ppm) (400 MHz, CDCl3): 11.00 F-Cq), 132.4 (Cq), 128.0 (d, 3J ϭ 9.9, Cq), 125.1 (CHarom), 113.2
(br.s., 1H), 7.97 (s, 1H), 7.55–7.51 (m, 1H), 7.35 (ddd, 3J ϭ 8.1, (d, 4J ϭ 4.9, Cq), 111.3 (d, 3J ϭ 9.7, CHarom), 110.0 (d, 2J ϭ 26.4,
4J ϭ 1.0, 5J ϭ 0.8, 1H), 7.19 (ddd, 3J ϭ 8.3, 3J ϭ 6.9, 4J ϭ 1.4, 1H), CHarom), 103.87 (d, 2J ϭ 23.5, CHarom), 54.1 (-CH2-), 44.9
7.11 (ddd, 3J ϭ 7.8, 3J ϭ 7.1, 4J ϭ 1.0, 1H), 7.02 (d, 3J ϭ 2.3, 1H), (2x-CH3).
6.36–6.34 (m, 1H), 5.63–5.60 (m, 1H), 3.79–3.76 (m, 2H). 13
C
Tetramethylammonium Iodide Salts (8A, 8F)—Based on the
NMR (ppm) (100 MHz, CDCl3): 172.7 (Cq), 138.9 (Cq), 136.4 method of Wartmann and Lindel (39), 1.5 eq of methyl iodide
(Cq), 128.0 (ϭCH2), 127.2 (Cq), 122.9 (CHarom), 122.1 (CHarom), was added at 0 °C to an 0.15 M solution of the corresponding
119.4 (CHarom), 119.0 (CHarom), 112.7 (Cq), 111.1 (CHarom), gramine in THF and stirred for 5 min at this temperature. The
27.2 (-CH2-).
reaction mixture was stirred for 1 h at room temperature and
Methyl 3-(1H-Indol-3-yl)-2-methylpropanoate (4)—Follow- then diluted to a tripled volume with pentane. The resulting
ing the general method described by Youn et al. (30), magne- suspension was filtered, and the residue washed with pentane.
sium turnings (180 mg, 7.41 mmol) were added to a solution of The products were used without further purification.
2 (750 mg, 3.48 mmol) in 20 ml of absolute MeOH and stirred
Addition of the Amino Acid Side Chain, rac-Ethyl 2-((diphe-
for 30 min with gas development under an argon atmosphere. nylmethylene)amino)-3-(5-fluoro-1H-indol-3-yl)propanoate
The temperature was then kept at 10 °C for 2 h. This mixture (9F)—Based on the procedure of Wartmann and Lindel (39),
was poured into a cooled HCl solution (3 M) and stirred until the ethyl N-(diphenylmethylene)glycinate (902 mg, 3.38 mmol)
turbidity disappeared. Adjusting the pH to 8.5–9 by addition of was added at 0 °C under argon atmosphere to a suspension of
3 M ammonium hydroxide solution was followed by extraction NaH (135 mg, 3.38 mmol, 60% suspension in mineral oil) in 21
with diethyl ether. The organic phase was dried with MgSO4, ml of THF, and the resulting mixture was stirred for 30 min
and the solvent evaporated under reduced pressure. The pro- under cooling. A suspension of 7F (1.13 g, 3.38 mmol) in 10 ml
cedure yielded a yellow liquid that was used in the next step of THF was added and stirred for an additional 3 h at 0 °C. The
without further purification.
reaction was quenched with saturated aqueous NH4Cl solution
IAA (rac-3-(1H-Indol-3-yl)-2-methylpropanoic Acid) (5)— and extracted with ethyl acetate. After drying with MgSO4 and
To a stirred solution of KOH (424 mg, 7.55 mmol) in 2 ml of filtration, the solvent was evaporated under reduced pressure.
MeOH and 1.2 ml water, a solution of 4 (546 mg, 2.52 mmol) in Flash column chromatography (pentane/ethyl acetate 5:1 3
4 ml of MeOH was added at 0 °C. The reaction was performed 3:1, Rf ϭ 0.4) gave the diphenyl protected ester 9F (722 mg, 1.74
as described for 3. Purification on a silica gel column (pentane/ mmol, 52%) as a clear yellow resin-like liquid. 1H NMR (ppm)
ethyl acetate 4:1 to 1:1) gave the pure product (409 mg, 2.01 (400 MHz, CDCl3): 8.19, 8.02 (s), 7.83–7.79 (m), 7.63–7.56 (m),
mmol, 80%) as a beige-colored solid. 1H NMR (ppm) (400 MHz, 7.52–7.45 (m), 7.39–7.33 (m), 7.33–7.22 (m), 7.21–7.14 (m),
MeOD): 7.55–7.52 (m, 1H), 7.33 (ddd, 3J ϭ 8.0, 4J ϭ 1.0, 5J ϭ 0.8, 7.11–7.08 (d), 6.99 (d), 6.96–6.81 (m), 6.66–6.59 (m), 4.35 (dd),
1H), 7.08 (ddd, 3J ϭ 8.1, 3J ϭ 6.9, 4J ϭ 1.2, 1H), 7.00 (ddd, 3J ϭ 4.23–4.09 (m), 3.38 (ddd), 3.24–2.99 (m), 1.25 (t), 1.23 (t). 13C-
3
4
7.9, J ϭ 7.0, J ϭ 1.2, 1H), 7.03 (s, 1H), 3.19–3.10 (m, 1H), NMR (ppm) (100 MHz, CDCl3): 172.1 (Cq), 170.6 (Cq), 157.6 (d,
2.87–2.77 (m, 2H), 1.17 (d, 3J ϭ 6.6, 3H). 13C NMR (ppm) (100 1J ϭ 234.40, F-Cq), 139.4 (Cq), 137.6 (Cq), 135.8 (Cq), 132.4
MHz, MeOD): 180.7 (Cq), 137.8 (Cq), 128.6 (Cq), 123.7 (-CH2-), (CHarom), 130.0 (CHarom), 128.8 (CHarom), 128.3 (CHarom),
122.1 (CHarom), 119.4 (CHarom), 119.2 (CHarom), 113.5 (Cq), 128.1 (d, 3J ϭ 11.70, Cq), 127.9 (CHarom), 127.5 (CHarom), 124.9
112.1 (CHarom), 41.8 (-CH-), 30.3 (-CH2-), 17.4 (-CH3).
(CHarom), 112.2 (d, 4J ϭ 4.71, Cq), 111.4 (d, 3J ϭ 9.65, CHarom),
110.1 (d, 2J ϭ 26.37, CHarom), 103.8 (d, 2J ϭ 23.46, CHarom), 65.1
(-CH-), 60.9 (-CH2-), 29.2 (-CH2-), 14.2 (-CH3).
Synthesis of Trp-derivatives 7-Aza-Trp and 5-Fluoro-Trp
7-Azagramine (7A) was synthesized according to the proce-
rac-Ethyl 2-((Diphenylmethylene)amino)-3-(1H-pyrrolo[2,3-
dure of Pierce et al. (38). Purification by flash column chroma- b]pyridin-3-yl)propanoate (9A)—As described for 9F, 7-azag-
tography (CHCl3/MeOH/NH4OH 90:10:1 3 40:10:1) yielded ramine salt (8A) (850 mg, 2.67 mmol) was used to yield the
the product as a yellow solid (2.34 g, 13.34 mmol, 79%). Analyt- desired product (350 mg, 0.88 mmol, 33%) after column chro-
ical data were in agreement with the literature (38).
matography (pentane/ethyl acetate 1:1 3 1:2, Rf ϭ 0.4) as a
5-Fluorogramine (7F)—A solution of 5-fluoroindole (1.0 g, clear brown viscous liquid. 1H NMR (ppm) (400 MHz, CDCl3):
7.40 mmol) in 1 ml of AcOH was added to a solution of dimeth- 9.57, 9.24 (s), 8.29, 8.22 (dd),7.95 (dd), 7.83–7.89 (m), 7.62–7.55
ylamine (567 mg, 1.42 ml, 12.58 mmol, 40% aqueous solution) (m), 7.52–7.45 (m), 7.40–7.34 (m), 7.33–7.23 (m), 7.22–7.12
and formaldehyde (333 mg, 0.9 ml, 11.09 mmol, 37% aqueous (m), 7.10–7.05 (m), 6.88 (dd), 6.60 (dd), 4.37–4.32 (m), 4.24–
solution) in 5 ml of acetic acid at 0 °C under an argon atmo- 4.00 (m), 3.80 (dd), 3.45–3.04 (m), 1.28–1.19 (m). 13C NMR
sphere. The mixture was stirred without cooling for 3 h, poured (ppm) (100 MHz, CDCl3): 172.0 (Cq), 170.6 (Cq), 142.8
onto 20 ml of ice-water, and adjusted to pH 11 with 2 M NaOH. (CHarom), 139.3 (Cq), 137.6 (Cq), 135.8 (Cq), 132.4 (CHarom),
The resulting suspension was extracted with diethyl ether and 130.3 (CHarom), 130.1 (CHarom), 128.8 (CHarom), 128.3
20080 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
(CHarom), 128.1 (CHarom), 127.5 (CHarom), 127.4 (CHarom), (dd, 2J ϭ 15.4, 3J ϭ 7.9, 1H). No 13C NMR spectra were recorded
123.3 (CHarom), 120.2 (Cq), 115.7 (CHarom), 110.8 (Cq), 66.1 due to low solubility.
2-Amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoic Acid
(11A)—Amino ester 10A (140 mg, 0.600 mmol) was obtained as
a yellow solid after purification on RP-18 (MeOH/H2O 70:30 3
50:50) (90 mg, 0.436 mmol, 75%). 1H NMR (ppm) (400 MHz,
D2O): 8.22 (dd, 3J ϭ 4.8, 4J ϭ 1.4, 1H), 8.12 (dd, 3J ϭ 8.0, 4J ϭ 1.5,
1H), 7.18 (dd, 3J ϭ 7.9, 3J ϭ 4.9, 1H), 4.02 (dd, 3J ϭ 7.5, 3J ϭ 5.0,
1H), 3.41 (dd, 2J ϭ 15.4, 3J ϭ 5.0, 1H), 3.31 (dd, 2J ϭ 15.3, 3J ϭ
7.5, 1H). No 13C NMR spectra were recorded due to low
solubility.
(-CH-), 61.0 (-CH2-), 29.3 (-CH2-), 14.2 (-CH3).
Deprotection of the Amino Group
As described by Wartmann et al. (39), the intermediate esters
9A and 9F were dissolved in THF (0.1 M), treated with 2 M HCl
(3.5 eq), and stirred for 1 h at room temperature. The reaction
mixture was diluted with ethyl acetate and neutralized with
saturated aqueous NaHCO3. The aqueous phase was extracted
three times with ethyl acetate, and the combined organic
phases were dried with MgSO4. The solvent was evaporated
under reduced pressure.
VioA Activity Assays
VioA enzyme kinetics were determined in a coupled peroxi-
dase assay mainly as described previously (7, 9). A reaction mix-
ture of 600 ␮l containing 50 mM Tris-HCl, pH 9.0, 0.2 ␮M VioA,
1 mM 4-aminoantipyrine, 1 mM phenol, and 15 units/ml horse-
radish peroxidase was pre-incubated at 30 °C. Then, VioA
catalysis was initiated by the addition of 1 mM L-Trp, and the
formation of the red quinoneimine dye was monitored at a
wavelength of 505 nm in the linear range of the activity assay
(ϳ3 min). The specific activity of VioA was determined by cal-
culation of the slope in the linear range. An extinction coeffi-
cient of ⑀505 ϭ 6400 MϪ1 cmϪ1 (9) was used to determine the
specific activity of VioA.
Ethyl 2-amino-3-(5-fluoro-1H-indol-3-yl)propanoate (10F)—
Following the above described procedure, protected 5-fluoro-
amino ester 9F (520 mg, 1.26 mmol) gave the desired product
after flash column chromatography (DCM/MeOH 7:1) as a yel-
low viscous oil (260 mg, 1.04 mmol, 83%). 1H NMR (ppm) (400
MHz, CDCl3): 8.19 (s, NH, 1H), 7.27–7.23 (m, 2H), 7.11 (d, 4J ϭ
2.4, 1H), 6.93 (dt, 3J ϭ 9.0, 4J ϭ 2.4), 4.16 (dq, 3J ϭ 7.2, 2J ϭ 2.5,
2H), 3.78 (dd, 3J ϭ 7.5, 3J ϭ 5.0, 1H), 3.21 (dd, 2J ϭ 14.45, 3J ϭ
4.98, 1H), 3.03 (dd, 2J ϭ 14.49, 3J ϭ 7.47, 1H), 1.25 (t, 3J ϭ 7.15).
13C NMR (ppm) (100 MHz, CDCl3): 175.2 (Cq), 157.9 (d, 1J ϭ
234.5, F-Cq), 132.8 (Cq), 127.9 (d, 3J ϭ 9.7, Cq), 124.7 (CHarom),
111.8 (d, 3J ϭ 9.6, CHarom), 111.5 (d, 4J ϭ 4.7, Cq), 110.6 (d, 2J ϭ
26.4, CHarom), 103.8 (d, 2J ϭ 23.6, CHarom), 61.0 (-CH2-), 54.9
(-CH-), 30.6 (-CH2-), 14.2 (-CH3).
Alternatively, the activity of VioA was directly measured
with an HPLC-based substrate depletion assay as described
elsewhere (7). Samples of 100 ␮l were taken from a reaction
mixture (1 ml) containing 50 mM Tris-HCl, pH 9.0, 1 ␮M VioA,
and 500 ␮M L-Trp at various time points (every 20 s for 3 min).
Samples were quenched by the addition of 20 ␮l of 50% (v/v)
trichloroacetic acid and centrifuged for 10 min at 12,100 ϫ g,
and the supernatant was analyzed on a ␮BondapakTM C18
3.9 ϫ 150-mm column (125 Å 10 ␮m, Waters/Millipore) using
a linear gradient of 0–50% acetonitrile over 15 min in 0.1% TFA
at a flow rate of 0.5 ml minϪ1. The employed Jasco HPLC system
was equipped with a diode array detector (MD-1515), which
was used to monitor the absorbance from 200 to 900 nm. The
amount of L-Trp of each sample was quantified using Borwin௡
peak integration routine.
Ethyl 2-amino-3-(1H-pyrrolo[2,3-b]pyridin-3-yl)propanoate
(10A3)—Following the above described procedure, protected
7-aza-amino ester 9A (340 mg, 0.86 mmol) gave the desired
product after flash column chromatography (CHCl3/MeOH/
NH4OH 90:10:1 3 40:10:1) as a brownish clear solid (150 mg,
0.64 mmol, 75%). 1H NMR (ppm) (400 MHz, CDCl3): 10.20 (s,
NH, 1H), 8.28 (dd, 3J ϭ 4.8, 4J ϭ 1.5, 1H), 7.95 (dd, 3J ϭ 7.9, 4J ϭ
1.6, 1H), 7.22 (s, 1H), 7.08 (dd, 3J ϭ 7.9, 3J ϭ 4.8, 1H), 4.15 (dq,
3J ϭ 7.2, 2J ϭ 2.1, 2H), 3.81 (dd, 3J ϭ 7.2, 3J ϭ 5.3, 1H), 3.24 (dd,
2J ϭ 14.6, 3J ϭ 5.2, 1H), 3.09 (dd, 2J ϭ 14.5, 3J ϭ 7.5, 1H), 1.22 (t,
3J ϭ 7.2, 3H). 13C NMR (ppm) (100 MHz, CDCl3): 175.1 (Cq),
148.7 (Cq), 142.8 (CHarom), 127.4 (CHarom), 123.6 (CHarom),
120.2 (Cq), 115.5 (CHarom), 109.7 (Cq), 61.0 (-CH2-), 55.1
(-CH-), 30.7 (-CH2-), 14.2 (-CH3).
Both types of activity assays were also performed in the pres-
ence of potential VioA inhibitors at concentrations ranging
from 0.5 to 10 mM. All VioA activity assays were completed by
control experiments in the absence of L-Trp. Tandem peroxi-
dase assays of selected experiments were also performed with
varying concentrations of horseradish peroxidase (15–150
units/ml) to ensure that catalysis of VioA is rate-determining.
Saponification to Amino Acids
To a solution of KOH (3 eq) in MeOH/H2O (5:3) was added
a solution of the respective ester in MeOH (0.6 M), and the
resulting mixture was stirred overnight. MeOH was evaporated
under reduced pressure, and the aqueous residue was washed
with diethyl ether. The aqueous phase was acidified with AcOH
to pH 4–5. Reversed phase column chromatography on RP-18
(100 g, MeOH/H2O) gave the desired amino acid.
VioA Dialysis under Turnover Conditions
FAD binding was investigated in a dialysis experiment under
aerobic conditions. A 10 ␮M VioA sample (100 ␮l) was dialyzed
against 1 liter of buffer 1 containing 40 mM L-Trp for 16 h using
a Slide-A-Lyzer MINI dialysis unit (10,000 molecular weight
cutoff, Thermo Scientific). L-Trp and the VioA reaction prod-
uct were removed by a buffer exchanged step (illustraTM
2-Amino-3-(5-fluoro-1H-indol-3-yl)propanoic Acid (11F)—
Reaction performed with amino ester 10F (156 mg, 0.623
mmol) yielded the desired product after purification on RP-18
(MeOH/H2O 60:40 3 50:50) as a slight yellow solid (84%). 1H
NMR (ppm) (400 MHz, D2O): 7.51 (dd, 3J ϭ 8.8, 4J ϭ 4.6, 1H), NAPTM-5 column SephadexTM, GE Healthcare, using buffer 1).
7.44 (dd, 2J ϭ 10.0, 4J ϭ 2.2, 1H), 7.13–7.03 (m, 1H), 4.07 (dd, The residual FAD content of the VioA sample was determined
3J ϭ 7.6, 3J ϭ 4.8, 1H), 3.46 (dd, 2J ϭ 15.2, 3J ϭ 4.84, 1H), 3.32 by UV-visible absorption spectroscopy.
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20081

Crystal Structure of VioA
Analytic Gel Permeation Chromatography
Protein Crystallization
The native molecular mass of VioA was determined on an
Purified VioA was concentrated to 150–200 ␮M using a
Äkta purifier system equipped with a SuperdexTM 200 Increase VivaspinTM 4 centrifugal concentrator (30,000 molecular
5/150 GL column (GE Healthcare). The system was equili- weight cutoff PES, Sartorius). Crystals were prepared as sitting
brated with buffer 1 or alternatively with buffer 1 containing drops by mixing 1 ␮l of protein solution with 1 ␮l of reservoir
500 ␮M Trp derivative IEA at a flow rate of 0.45 ml minϪ1. The solution. Native VioA crystals grew at 4 °C from 80 mM Tris-
HCl, pH 8.5, 24% (w/v) PEG 4000, 20% (v/v) glycerol after 5
weeks. A heavy metal derivative was produced by soaking VioA
crystals with 10 mM Sm(NO3)3 for 2 weeks. Co-crystals of VioA
and IEA grew after 5 days at 17 °C in sitting drops in the pres-
ence of 100 mM HEPES, pH 7.0, 10% PEG 6000, 100 mM lithium
chloride, and 3.75 mM IEA. Crystals were cryo-protected in the
presence of 20% glycerol (v/v) and flash-cooled in liquid
nitrogen.
column was calibrated using protein standards (gel filtration
markers kit for protein molecular mass 12,000–200,000 Da,
Sigma) according to the manufacturer’s instructions. Samples
of VioA (20 ␮l) at a concentration of 215 ␮M were injected.
Alternatively VioA was pre-incubated in the presence of 500
␮M IEA for 3 h. Elution was monitored at 280 and 450 nm,
respectively.
Small-angle X-ray Scattering
Data Collection, Structure Determination, and Refinement
SAXS experiments were carried out on beamline BM29 (51)
of the European Synchrotron Radiation Facility (Grenoble,
France) to elucidate the oligomeric architecture of VioA. Sam-
ples of VioA (or of VioA in the presence of 1 mM IEA) at con-
centrations ranging from 10 to 265 ␮M in buffer 1 were loaded
by an automated sample changer to a quartz capillary mounted
in vacuum and exposed to an x-ray energy of 12.5 keV. Scatter-
ing images covering a momentum transfer range of 0.0025–5
nmϪ1 were recorded with a Dectris Pilatus 1M hybrid photon
counting detector. A total of 10 images were recorded per sam-
ple with an exposure time of 1 s per frame. To reduce radiation
damage, the protein solution was kept under steady flow while
being irradiated. The scattering functions derived from the
images were normalized for exposure time and sample trans-
mission and checked for effects of radiation damage in PRIMUS
(52). Data unaffected by radiation damage were averaged, cor-
rected for buffer scattering, normalized, and merged from the
different concentrations. The comparison of the determined
scattering data with the theoretical scattering curves of a VioA
monomer or two potential dimers (33) was performed with
CRYSOL (53). The radius of gyration (Rg) and the pair distance
distribution P(r) were derived with GNOM (54). Twenty mod-
els were computed from SAXS data with DAMMIF (55) and
used for the construction of a representative ab initio model
Diffraction experiments were performed on the beamline
X06DA, operated by the Paul Scherrer Institute at the Swiss
Light Source (Villigen, Switzerland), on beamline P11, operated
by DESY at PETRA III synchrotron (Hamburg, Germany) (58),
and on beamline 14.1, operated by the Helmholtz-Zentrum
Berlin at the BESSY II electron storage ring (Berlin-Adlershof,
Germany) (59). Data processing was carried out with XDS (60),
and the upper resolution limits were assessed through careful
observation of I/␴I and CC1/2 (61). The phase problem was
solved by a single-wavelength anomalous diffraction experi-
ment at ␭ ϭ 1.77 Å using the anomalous signal arising from the
proximity of samarium’s L-III absorption edge. ShelxC/D/E
(62) was employed to locate heavy metal sites, to compute a first
electron density map, and to build an initial structural model.
The other crystal forms were phased by molecular replacement,
with Phaser (63), using the initial model obtained from the Sm
derivative. With subsequent refinement in Phenix.refine (64)
and manual rebuilding in Coot (65), the models were finalized.
Data collection and refinement statistics are shown in Table 1.
Author Contributions—J. M., D. J., S. S., W. B., J. J. F., R. R., and J. K.
designed the study and wrote the paper. J. J. F., J. M., R. R., J. K.,
K. E. R., and N. P. D. conducted the experiments and analyzed the
results. R. R. synthesized inhibitors and artificial substrates. J. K.
using DAMAVER and SUPCOMP (56, 57). The models had a conducted crystal x-ray measurements and solved the structure. All
¯
authors analyzed the results and approved the final version of the
manuscript.
meannormalizedspatialdiscrepancy(NSD)of1.244andastan-
dard deviation of NSD (␴NSD) of 0.189. One of the 20 models
¯
showed an NSD of greater than 1.622 (ϭ NSD ϩ 2 ϫ ␴NSD)
Acknowledgments—We thank Jutta Pruss for experimental assistance
during mutagenesis and Beate Jaschok-Kentner for N-terminal pro-
tein sequencing.
and was thus omitted for the calculation of the final ab initio
model.
Micro Scale Thermophoresis
References
VioA-ligand/inhibitor interaction was measured on a Mono-
lith NT.115 instrument (NanoTemper Technologies, GmbH).
VioA (20 ␮M) was labeled using the Monolith NTTM protein
labeling kit (NT-647-Red-NHS amine reactive fluorescent dye,
NanoTemper Technologies, GmbH). The labeled protein was
supplemented with 0.05% (w/v) Tween 20 and different con-
centrations of IAA, IEA, and citrate (30 nM to 20 mM) were
titrated, respectively. Thermophoresis was measured in hydro-
philic capillaries (NanoTemper Technologies, GmbH) using a
laser intensity of 20% for 35 s.
1. Antônio, R. V., and Creczynski-Pasa, T. B. (2004) Genetic analysis of vi-
olacein biosynthesis by Chromobacterium violaceum. Genet. Mol. Res. 3,
85–91
2. Durán, N., and Menck, C. F. (2001) Chromobacterium violaceum: a review
of pharmacological and industrial perspectives. Crit. Rev. Microbiol. 27,
201–222
3. August, P. R., Grossman, T. H., Minor, C., Draper, M. P., MacNeil, I. A.,
Pemberton, J. M., Call, K. M., Holt, D., and Osburne, M. S. (2000) Se-
quence analysis and functional characterization of the violacein biosyn-
thetic pathway from Chromobacterium violaceum. J. Mol. Microbiol. Bio-
technol. 2, 513–519
20082 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Crystal Structure of VioA
4. Durán, N., Justo, G. Z., Ferreira, C. V., Melo, P. S., Cordi, L., and Martins,
D. (2007) Violacein: properties and biological activities. Biotechnol. Appl.
Biochem. 48, 127–133
dent substrate dehydrogenation. Proc. Natl. Acad. Sci. U.S.A. 97,
12463–12468
23. Matz, C., Webb, J. S., Schupp, P. J., Phang, S. Y., Penesyan, A., Egan, S.,
Steinberg, P., and Kjelleberg, S. (2008) Marine biofilm bacteria evade eu-
karyotic predation by targeted chemical defense. PLoS ONE 3, e2744
5. Tamaoki, T., Nomoto, H., Takahashi, I., Kato, Y., Morimoto, M., and
Tomita, F. (1986) Staurosporine, a potent inhibitor of phospholipid/Ca2ϩ
-
dependent protein kinase. Biochem. Biophys. Res. Commun. 135, 397–402 24. Macheroux, P. (1999) UV-visible spectroscopy as a tool to study flavopro-
6. Anizon, F., Belin, L., Moreau, P., Sancelme, M., Voldoire, A., Prudhomme,
teins. Methods Mol. Biol. 131, 1–7
M., Ollier, M., Sevère, D., Riou, J.-F., Bailly, C., Fabbro, D., and Meyer, T. 25. Faust, A., Niefind, K., Hummel, W., and Schomburg, D. (2007) The struc-
(1997) Syntheses and biological activities (topoisomerase inhibition and
antitumor and antimicrobial properties) of rebeccamycin analogues bear-
ing modified sugar moieties and substituted on the imide nitrogen with a
methyl group. J. Med. Chem. 40, 3456–3465
ture of a bacterial L-amino acid oxidase from Rhodococcus opacus gives
new evidence for the hydride mechanism for dehydrogenation. J. Mol.
Biol. 367, 234–248
26. Ida, K., Kurabayashi, M., Suguro, M., Hiruma, Y., Hikima, T., Yamomoto,
M., and Suzuki, H. (2008) Structural basis of proteolytic activation of
L-phenylalanine oxidase from Pseudomonas sp. P-501. J. Biol. Chem. 283,
16584–16590
7. Balibar, C. J., and Walsh, C. T. (2006) In vitro biosynthesis of violacein
from L-tryptophan by the enzymes VioA-E from Chromobacterium viola-
ceum. Biochemistry 45, 15444–15457
8. Nishizawa, T., Aldrich, C. C., and Sherman, D. H. (2005) Molecular anal-
ysis of the rebeccamycin L-amino acid oxidase from Lechevalieria aero-
colonigenes ATCC 39243. J. Bacteriol. 187, 2084–2092
27. Holzapfel, C. W., Bischofberger, K., and Olivier, J. (1994) A simple cy-
cloaddition approach to a racemate of the natural sweetener monatin.
Synth. Commun. 24, 3197–3211
9. Kameya, M., Onaka, H., and Asano, Y. (2013) Selective tryptophan deter- 28. Yu, C., Liu, B., and Hu, L. (2001) Efficient Baylis-Hillman reaction using
mination using tryptophan oxidases involved in bis-indole antibiotic bio-
stoichiometric base catalyst and an aqueous medium. J. Org. Chem. 66,
synthesis. Anal. Biochem. 438, 124–132
5413–5418
10. Howard-Jones, A. R., and Walsh, C. T. (2005) Enzymatic generation of the 29. Borrell, J. I., Teixidó, J., Martínez-Teipel, B., Matallana, J. L., Copete, M. T.,
chromopyrrolic acid scaffold of rebeccamycin by the tandem action of
RebO and RebD. Biochemistry 44, 15652–15663
Llimargas, A., and García, E. (1998) Synthesis and biological activity of
4-amino-7-oxo-substituted analogues of 5-deaza-5,6,7,8-tetrahydrofolic
acid and 5,10-dideaza-5, 6,7,8-tetrahydrofolic acid. J. Med. Chem. 41,
3539–3545
11. Asamizu, S., Kato, Y., Igarashi, Y., Furumai, T., and Onaka, H. (2006)
Direct formation of chromopyrrolic acid from indole-3-pyruvic acid by
StaD, a novel hemoprotein in indolocarbazole biosynthesis. Tetrahedron 30. Kwon Youn, I., Hwan Yon, G., and Siek Pak, C. (1986) Magnesium-meth-
Lett. 47, 473–475
anol as a simple convenient reducing agent for ␣,␤-unsaturated esters.
Tetrahedron Lett. 27, 2409–2410
12. Asamizu, S., Hirano, S., Onaka, H., Koshino, H., Shiro, Y., and Nagano, S.
(2012) Coupling reaction of indolepyruvic acid by StaD and its product: 31. Mori, K., and Kisida, H. (1986) Synthesis of both the enantiomers of the
implications for biosynthesis of indolocarbazole and violacein. ChemBio-
Chem 13, 2495–2500
heterocyclic pheromones isolated from the male swift moth Hepialus
hecta L. Tetrahedron 42, 5281–5290
13. Spolitak, T., and Ballou, D. P. (2015) Evidence for catalytic intermediates 32. Jerabek-Willemsen, M., Wienken, C. J., Braun, D., Baaske, P., and Duhr, S.
involved in generating the chromopyrrolic acid scaffold of rebeccamycin
by RebO and RebD. Arch. Biochem. Biophys. 573, 111–119
(2011) Molecular interaction studies using microscale thermophoresis.
Assay Drug Dev. Technol. 9, 342–353
14. Sánchez, C., Braña, A. F., Méndez, C., and Salas, J. A. (2006) Reevaluation
of the violacein biosynthetic pathway and its relationship to indolocarba-
zole biosynthesis. ChemBioChem 7, 1231–1240
33. Baskaran, K., Duarte, J. M., Biyani, N., Bliven, S., and Capitani, G. (2014) A
PDB-wide, evolution-based assessment of protein-protein interfaces.
BMC Struct. Biol. 14, 22
15. Sánchez, C., Butovich, I. A., Braña, A. F., Rohr, J., Méndez, C., and Salas, 34. Dym, O., and Eisenberg, D. (2001) Sequence-structure analysis of FAD-
J. A. (2002) The biosynthetic gene cluster for the antitumor rebeccamycin:
characterization and generation of indolocarbazole derivatives. Chem. 35. Holm, L., and Rosenström, P. (2010) Dali server: conservation mapping in
Biol. 9, 519–531 3D. Nucleic Acids Res. 38, W545–W549
16. Onaka, H., Taniguchi, S., Igarashi, Y., and Furumai, T. (2003) Character- 36. Moustafa, I. M., Foster, S., Lyubimov, A. Y., and Vrielink, A. (2006) Crystal
containing proteins. Protein Sci. 10, 1712–1728
ization of the biosynthetic gene cluster of rebeccamycin from Lecheva-
lieria aerocolonigenes ATCC 39243. Biosci. Biotechnol. Biochem. 67,
127–138
structure of LAAO from Calloselasma rhodostoma with an L-phenylala-
nine substrate: insights into structure and mechanism. J. Mol. Biol. 364,
991–1002
17. Sievers, F., Wilm, A., Dineen, D., Gibson, T. J., Karplus, K., Li, W., Lopez,
R., McWilliam, H., Remmert, M., Söding, J., Thompson, J. D., and Higgins,
D. G. (2011) Fast, scalable generation of high-quality protein multiple
sequence alignments using Clustal Omega. Mol. Syst. Biol. 7, 539–539
18. Shinoda, K., Hasegawa, T., Sato, H., Shinozaki, M., Kuramoto, H., Taka-
37. Yano, J., Kern, J., Irrgang, K. D., Latimer, M. J., Bergmann, U., Glatzel, P.,
Pushkar, Y., Biesiadka, J., Loll, B., Sauer, K., Messinger, J., Zouni, A., and
Yachandra, V. K. (2005) X-ray damage to the Mn4Ca complex in single
crystals of photosystem II: a case study for metalloprotein crystallography.
Proc. Natl. Acad. Sci. U.S.A. 102, 12047–12052
miya, Y., Sato, T., Nikaidou, N., Watanabe, T., and Hoshino, T. (2007) 38. Pierce, L. T., Cahill, M. M., and McCarthy, F. O. (2011) Synthesis of novel
Biosynthesis of violacein: a genuine intermediate, protoviolaceinic acid,
produced by VioABDE, and insight into VioC function. Chem. Commun.
40, 4140–4142
3,4-diaryl-5-aminopyrazoles as potential kinase inhibitors. Tetrahedron
67, 4601–4611
39. Wartmann, T., and Lindel, T. (2013) L-Phototryptophan. Eur. J. Org.
Chem. 2013, 1649–1652
19. Blanchard, M., Green, D. E., Nocito, V., and Ratner, S. (1944) L-Amino
acid oxidase of animal tissue. J. Biochem. 155, 421–440
40. Henderson Pozzi, M., and Fitzpatrick, P. F. (2010) A lysine conserved in
the monoamine oxidase family is involved in oxidation of the reduced
flavin in mouse polyamine oxidase. Arch. Biochem. Biophys. 498, 83–88
41. Chen, H. S., Wang, Y. M., Huang, W. T., Huang, K. F., and Tsai, I. H. (2012)
Cloning, characterization and mutagenesis of Russell’s viper venom
L-amino acid oxidase: Insights into its catalytic mechanism. Biochimie 94,
335–344
20. Geueke, B., Hummel, W. (2002) A new bacterial l-amino acid oxidase with
a broad substrate specificity: purification and characterization. Enzyme
Microb. Technol. 31, 77–87
21. Pawelek, P. D., Cheah, J., Coulombe, R., Macheroux, P., Ghisla, S., and
Vrielink, A. (2000) The structure of L-amino acid oxidase reveals the sub-
strate trajectory into an enantiomerically conserved active site. EMBO J.
19, 4204–4215
42. Wellner, D., and Meister, A. (1961) Studies on the mechanism of action of
L-amino acid oxidase. J. Biol. Chem. 236, 2357–2364
22. Umhau, S., Pollegioni, L., Molla, G., Diederichs, K., Welte, W., Pilone,
M. S., and Ghisla, S. (2000) The x-ray structure of D-amino acid oxidase at 43. Sanchez, C., Salas, A. P., Brana, A. F., Palomino, M., Pineda-Lucena, A.,
very high resolution identifies the chemical mechanism of flavin-depen-
Carbajo, R. J., Mendez, C., Moris, F., and Salas, J. A. (2009) Generation of
SEPTEMBER 16, 2016•VOLUME 291•NUMBER 38
JOURNAL OF BIOLOGICAL CHEMISTRY 20083

Crystal Structure of VioA
potent and selective kinase inhibitors by combinatorial biosynthesis of 55. Franke, D., and Svergun, D. I. (2009) DAMMIF, a program for rapid ab-
glycosylated indolocarbazoles. Chem. Commun. 27, 4118–4120
44. Cuenca-López, M. D., Serrano-Heras, G., Montero, J. C., Corrales-Sán-
chez, V., Gomez-Juarez, M., Gascón-Escribano, M. J., Morales, J. C., Voi-
sin, V., Núñez, L. E., Morís, F., Bader, G. D., Pandiella, A., and Ocaña, A.
(2015) Antitumor activity of the novel multi-kinase inhibitor EC-70124 in
triple negative breast cancer. Oncotarget 6, 27923–27937
initio shape determination in small-angle scattering. J. Appl. Crystallogr.
42, 342–346
56. Volkov, V. V., and Svergun, D. I. (2003) Uniqueness of ab initio shape
determination in small-angle scattering. J. Appl. Crystallogr. 36, 860–864
57. Kozin, M. B., and Svergun, D. I. (2001) Automated matching of high- and
low-resolution structural models. J. Appl. Crystallogr. 34, 33–41
58. Burkhardt, A., Pakendorf, T., Reime, B., Meyer, J., Fischer, P., Stübe, N.,
Panneerselvam, S., Lorbeer, O., Stachnik, K., Warmer, M., Rödig, P.,
Göries, D., and Meents, A. (2016) Status of the crystallography beamlines
at PETRA III. Eur. Phys. J. Plus 131, 56
45. Tilocca, A., Gamba, A., Vanoni, M. A., and Fois, E. (2002) First-principles
molecular dynamics investigation of the D-amino acid oxidative half-re-
action catalyzed by the flavoenzyme D-amino acid oxidase. Biochemistry
41, 14111–14121
46. Kachalova, G., Decker, K., Holt, A., and Bartunik, H. D. (2011) Crystallo-
graphic snapshots of the complete reaction cycle of nicotine degradation
by an amine oxidase of the monoamine oxidase (MAO) family. Proc. Natl.
Acad. Sci. U.S.A. 108, 4800–4805
59. Mueller, U., Förster, R., Hellmig, M., Huschmann, F. U., Kastner, A., Ma-
¨
lecki, P., Pühringer, S., Röwer, M., Sparta, K., Steffien, M., Uhlein, M.,
Wilk, P., and Weiss, M. S. (2015) The macromolecular crystallography
beamlines at BESSY II of the Helmholtz-Zentrum Berlin: Current status
and perspectives. EPJ Plus 130, 1–10
47. Edman, P. (1950) Method for determination of the amino acid sequence in
peptides. Acta Chem. Scand. 4, 283–293
60. Kabsch, W. (2010) XDS. Acta Crystallogr. D Biol. Crystallogr. 66, 125–132
61. Karplus, P. A., and Diederichs, K. (2012) Linking crystallographic model
and data quality. Science 336, 1030–1033
48. Edman, P., and Begg, G. (1967) A protein sequenator. Eur. J. Biochem. 1,
80–91
49. Niall, H. D. (1973) Automated Edman degradation: the protein sequena-
tor. Methods Enzymol. 27, 942–1010
62. Sheldrick, G. M. (2008) A short history of SHELX. Acta Crystallogr. A 64,
112–122
50. Pantoliano, M. W., Petrella, E. C., Kwasnoski, J. D., Lobanov, V. S., Myslik,
J., Graf, E., Carver, T., Asel, E., Springer, B. A., Lane, P., and Salemme, F. R.
(2001) High-density miniaturized thermal shift assays as a general strategy
for drug discovery. J. Biomol. Screen. 6, 429–440
63. McCoy, A. J., Grosse-Kunstleve, R. W., Adams, P. D., Winn, M. D., Sto-
roni, L. C., and Read, R. J. (2007) Phaser crystallographic software. J. Appl.
Crystallogr. 40, 658–674
64. Adams, P. D., Afonine, P. V., Bunkóczi, G., Chen, V. B., Davis, I. W.,
Echols, N., Headd, J. J., Hung, L. W., Kapral, G. J., Grosse-Kunstleve,
R. W., McCoy, A. J., Moriarty, N. W., Oeffner, R., Read, R. J., Richard-
son, D. C., et al. (2010) PHENIX: a comprehensive Python-based sys-
tem for macromolecular structure solution. Acta Crystallogr. D Biol.
Crystallogr. 66, 213–221
51. Pernot, P., Round, A., Barrett, R., De Maria Antolinos, A., Gobbo, A.,
Gordon, E., Huet, J., Kieffer, J., Lentini, M., Mattenet, M., Morawe, C.,
Mueller-Dieckmann, C., Ohlsson, S., Schmid, W., Surr, J., et al. (2013)
Upgraded ESRF BM29 beamline for SAXS on macromolecules in solution.
J. Synchrotron. Radiat. 20, 660–664
52. Konarev, P. V., Volkov, V. V., Sokolova, A. V., Koch, M. H. J., and Svergun,
D. I. (2003) PRIMUS: a Windows PC-based system for small-angle scat-
tering data analysis. J. Appl. Crystallogr. 36, 1277–1282
65. Emsley, P., Lohkamp, B., Scott, W. G., and Cowtan, K. (2010) Features and
development of Coot. Acta Crystallogr. D Biol. Crystallogr. 66, 486–501
66. Laskowski, R. A., and Swindells, M. B. (2011) LigPlotϩ: multiple ligand-
protein interaction diagrams for drug discovery. J. Chem. Inf. Model. 51,
2778–2786
67. Hirano, S., Asamizu, S., Onaka, H., Shiro, Y., and Nagano, S. (2008) Crystal
structure of VioE, a key player in the construction of the molecular skel-
eton of violacein. J. Biol. Chem. 283, 6459–6466
53. Svergun, D., Barberato, C., and Koch, M. H. J. (1995) CRYSOL–a program
to evaluate x-ray solution scattering of biological macromolecules from
atomic coordinates. J. Appl. Crystallogr. 28, 768–773
54. Svergun, D. (1992) Determination of the regularization parameter in in-
direct-transform methods using perceptual criteria. J. Appl. Crystallogr.
25, 495–503
20084 JOURNAL OF BIOLOGICAL CHEMISTRY
VOLUME 291•NUMBER 38•SEPTEMBER 16, 2016

Biosynthesis of Violacein, Structure and Function of l-Tryptophan Oxidase VioA
from Chromobacterium violaceum
Janis J. Füller, René Röpke, Joern Krausze, Kim E. Rennhack, Nils P. Daniel, Wulf
Blankenfeldt, Stefan Schulz, Dieter Jahn and Jürgen Moser
J. Biol. Chem. 2016, 291:20068-20084.
doi: 10.1074/jbc.M116.741561 originally published online July 27, 2016
Access the most updated version of this article at doi: 10.1074/jbc.M116.741561
Alerts:
• When this article is cited
• When a correction for this article is posted
Click here to choose from all of JBC's e-mail alerts
This article cites 67 references, 12 of which can be accessed free at
http://www.jbc.org/content/291/38/20068.full.html#ref-list-1