Structure and mechanism of ORF36, an amino sugar oxidizing enzyme in everninomicin biosynthesis

Article abstract of DOI:10.1021/bi101336u

Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro sugar, l-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)-l-epi-vancosamine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that ORF36 catalyzes oxidation of biogenic TDP-l-epi-vancosamine and to determine whether ORF36 exhibits catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitrososynthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine oxidation state. Performing the in vitro reactions in the presence of ¹⁸O₂ establishes that molecular oxygen, rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The 3.15 A resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3. However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase mechanism for amino sugar oxidation by ORF36.

Full text of DOI:10.1021/bi101336u

9306 Biochemistry 2010, 49, 9306–9317
DOI: 10.1021/bi101336u
Structure and Mechanism of ORF36, an Amino Sugar Oxidizing Enzyme
in Everninomicin Biosynthesis†,‡
Jessica L. Vey,§ Ahmad Al-Mestarihi,^ Yunfeng Hu,^,r Michael A. Funk,§,@ Brian O. Bachmann,*,^, and
T. M. Iverson*,§,
§Department of Pharmacology, and Department of Biochemistry, Vanderbilt University Medical Center, Nashville, Tennessee 37232,
United States, and ^Department of Chemistry, Vanderbilt University, Nashville, Tennessee 37235, United States.
rPresent address: Department of Chemistry, Brown University, Providence, RI 02912. @Present address:
Department of Chemistry, Massachusetts Institute of Technology, Cambridge, MA 02139.
Received August 18, 2010; Revised Manuscript Received September 20, 2010
ABSTRACT: Everninomicin is a highly modified octasaccharide that belongs to the orthosomycin family of
antibiotics and possesses potent Gram-positive antibiotic activity, including broad-spectrum efficacy against
multidrug resistant enterococci and Staphylococcus aureus. Among its distinctive structural features is a nitro
sugar,
nitrososynthase enzyme encoded by orf36 from Micromonospora carbonacea var. africana that mediates the
flavin-dependent double oxidation of synthetically generated thymidine diphosphate (TDP)- -epi-vancosa-
mine to the corresponding nitroso sugar. Herein, we utilize a five-enzyme in vitro pathway both to verify that
ORF36 catalyzes oxidation of biogenic TDP- -epi-vancosamine and to determine whether ORF36 exhibits
L
-evernitrose, analogues of which decorate a variety of natural products. Recently, we identified a
L
L
catalytic competence for any of its biosynthetic progenitors, which are candidate substrates for nitroso-
synthases in vivo. Progenitors solely undergo single-oxidation reactions and terminate in the hydroxylamine
oxidation state. Performing the in vitro reactions in the presence of 18O2 establishes that molecular oxygen,
rather than oxygen from water, is incorporated into ORF36-generated intermediates and products and
identifies an off-pathway product that correlates with the oxidation product of a progenitor substrate. The
˚
3.15 A resolution X-ray crystal structure of ORF36 reveals a tetrameric enzyme that shares a fold with acyl-
CoA dehydrogenases and class D flavin-containing monooxygenases, including the nitrososynthase KijD3.
However, ORF36 and KijD3 have unusually open active sites in comparison to these related enzymes. Taken
together, these studies map substrate determinants and allow the proposal of a minimal monooxygenase
mechanism for amino sugar oxidation by ORF36.
The orthosomycins are oligosaccharide antibiotics that possess
potent broad-spectrum antibacterial activity (1-3). These com-
pounds are thought to target protein translation in bacteria by
binding to a single site on the 50S ribosomal subunit (4). One
such orthosomycin is everninomicin [1, Ziracin (Figure 1)], which
harbors an unusual nitro sugar moiety that may be key for its
activity (5, 6). Everninomicin was developed through phase III
clinical trials that were eventually discontinued because of
apparent pharmacological complications (7). Because it is not
uncommon for related antibiotics to exhibit similar efficacy but
have altered pharmacological properties, the exploration of
compounds related to everninomicin may identify molecules
suitable for clinical use. Only limited chemical derivatization studies
of everninomicin have been performed (5, 6), likely because the
complexity of orthosomycins renders the chemical synthesis re-
quired for structure-activity and optimization studies challenging.
Enzymes in the natural biosynthetic pathway of everninomicin
could be used to complement chemical synthesis during rational
design of the antibiotic’s scaffold.
Using a comparative genomics approach, we recently identi-
fied and performed preliminary characterization of an enzyme
from the everninomicin producer Micromonospora carbonacea
var. africana (8). This enzyme, encoded by orf36 (also called
EvdC), catalyzes the two-step oxidation of the close substrate
analogue, TDP-L-epi-vancosamine 10, to its nitroso congener
14 (8) and as a result has been termed a nitrososynthase. The
nitrososynthases make up an expanding family enzymes that
catalyze nitroso sugar formation on oligosaccharide antibiotics,
including RubN8 of the rubradirin biosynthetic pathway (9),
whose product is TDP-
of the kijanimicin biosynthetic pathway, which yields TDP-
-kijanose [4 (Figure 1)]; and isobutylamine N-oxidase (12) of
D-rubranitrose [3(Figure1)];KijD3(10, 11)
D
the valanimicin pathway. Formation of the nitroso sugar by
nitrososynthases is consistent with previous studies that suggest
that the oxidation to the nitro oxidation state is the result of a
spontaneous photochemical reaction (9).
N-Oxidation of primary amines has been observed in a variety
of biological systems. For example, oxidation of amines to
nitroso and nitro groups via a hydroxylamine intermediate
can be catalyzed by Rieske N-oxygenases (13) and non-heme
†This work was supported by Office of Naval Research Grant
N000140610144 to B.O.B. and National Institutes of Health Grant
GM077189 to T.M.I.
‡The atomic coordinates and structure factors for ORF36 (entry
3MXL) have been deposited in the Protein Data Bank.
*To whom correspondence should be addressed. B.O.B.: fax,
(615) 322-8865; phone, (615) 322-8865; e-mail, brian.o.bachmann@
vanderbilt.edu. T.M.I.: fax, (615) 343-6532; phone, (615) 322-7817;
e-mail, tina.iverson@vanderbilt.edu.
r
pubs.acs.org/Biochemistry
Published on Web 09/24/2010
2010 American Chemical Society

Article
Biochemistry, Vol. 49, No. 43, 2010 9307
monooxygenase 4-hydroxyphenylacetate monooxygenase from
Acinetobacter baumannii (24).
Application of classical flavoenzyme methodologies to our
studies of ORF36 was impeded by several factors, including the
instability of the putative substrate (TDP-L-evernosamine), the
challenges related to its synthesis, and the low affinity of the
flavin cofactor, which normally provides a convenient readout
for monitoring the redox state of flavoenzymes throughout the
reaction coordinate. To address these issues, we have implemen-
ted a modified procedure to generate the close substrate analogue
TDP-L-epi-vancosamine (des-4-O-Me evernitrosamine) 10 via an
in vitro enzymatic synthetic pathway in yields sufficient for
studying enzyme turnover and confirming the activity of this
substrate analogue. Fortuitously, this synthetic strategy provides
access to progenitor substrates, which allows the assessment of
enzymatic competence on biological precursors on the presumed
substrate. We next performed 18O2 incorporation studies with
biogenic TDP-
monooxygenase mechanism and identified a new activity of
ORF36 with the substrate analogue TDP- -epi-vancosamine.
L-epi-vancosamine, which provided evidence of a
L
Finally, we determined the X-ray crystal structure of ORF36.
Along with the previously determined structure of the homologue
KijD3 (11), this structure reveals the chemical and physical
constraints of the protein scaffold that houses the double oxi-
dation of an amino sugar. To the best of our knowledge, ORF36
is the first structurally characterized enzyme with demonstrated
nitrososynthase activity. Moreover, these studies comprise the
first detailed structural and biochemical insights into the bio-
synthesis of the stage III clinical candidate everninomicin and its
precursor TDP-evernosamine. The assay of progenitor substrates
delineates the most likely timing of N-oxidation in the biosynth-
esis of TDP-evernitrose, and isotopic incorporation studies reveal
an additional reaction product and permit the assignment of
this new class of flavoenzyme as a monooxygenase.
F
proposed TDP-nitro sugar precursors to rubradirin and kijanimicin.
IGURE 1: Everninomicin, its precursor TDP-L-evernitrose, and related,
diiron monooxygenases (14, 15), while oxidation of substrate
nitrogen atoms to the corresponding hydroxylamine or oxide can
be accomplished by P450 enzymes (16, 17) and several single-
component flavin monooxygenases (18, 19). On the basis of
preliminary biochemical data (8), ORF36 and related nitroso-
synthases are likely flavin-containing monooxygenases. The
flavin monooxygenases make up a diverse set of enzymes that
catalyze various oxidation reactions, including hydroxylation,
halogenation,sulfoxidation,andBaeyer-Villigeroxidations(20).
The double oxidation of an amino sugar represents a new addi-
tion to this set of reactivities. Sequence and structural similarities
divide flavin-containing monooxygenases into subclasses (20),
and nitrososynthases may be best classified as class D flavin-
containing monooxygenases, which are related in sequence and
structure to the acyl-CoA dehydrogenases (11, 21). The protein
scaffold of this superfamily can house distinct reaction types,
with acyl-CoA dehydrogenases catalyzing dehydrogenation and
flavin-containing monooxygenases performing monooxygena-
tion (22, 23). Interestingly, ORF36 and other nitrososynthases
are more similar in sequence to the dehydrogenases (∼25%
identical) than to flavin-containing monooxygenases (∼15%
identical), seemingly at odds with the functional evidence sug-
gesting that ORF36 performs a monooxygenase reaction.
To date, the characterized class D flavin-containing mono-
oxygenases (24-27) participate in two-component flavin-depen-
dent monooxygenase systems (20, 28, 29) in which a first enzyme,
a flavin reductase, reduces oxidized flavin with NAD(P)H and a
second enzyme, the monooxygenase, preferentially binds reduced
flavin (24, 26, 29), activates molecular oxygen, and oxygenates
the substrate (28). Consistent with the classification as a class
D flavin-containing monooxygenase, previous biochemical char-
acterization of ORF36 confirmed that activity depends upon
flavin and NADPH, and is dramatically enhanced by the addition
of flavin reductase, but displays relatively weak, reversible binding
for oxidized flavin (8). Curiously, these in vitro assays of ORF36
activity suggested that the enzyme exhibits flexible flavin cofactor
utilization, with efficient amino sugar oxidation observed using
either reduced FAD or FMN as the cofactor (8). Similar
promiscuous cofactor usage was observed in the class D flavin
EXPERIMENTAL PROCEDURES
Overexpression and Purification of Enzymes. ORF36
from M. carbonacea var. africana (8), EvaA-E from Amycola-
topsis orientalis (30), and RfbB from Salmonella enterica (strain
LT2) (31, 32) were purified from freshly transformed Escherichia
coli BL21(DE3) by nickel affinity chromatography as previously
described with the exception of EvaE, which was isolated as an
insoluble preparation. EvaE preparations were generated from
3 L induced cultures, which were disrupted via a French pressure
cell. The insoluble fraction was isolated by centrifugation and
washed with 20 mM Tris-HCl (pH 7.5), resuspended in 10 mL of
20 mM Tris-HCl (pH 7.5) and 5% glycerol, and stored at -80 ꢀC
prior to use.
Preparation of TDP-6-deoxy-4-keto-D-glucose 6. Reac-
tion mixtures (1 mL) containing 8 mM thymidine 50-dipho-
sphate-glucose disodium salt (4.5 mg), 50 mM Tris-HCl (pH
8.0), 4 mM NADPþ, and 160 μM RfbB were incubated at 30 ꢀC
for 2 h. Proteins were removed by 10K molecular weight cutoff
filtration (Centricon, Millipore Corp.), and the product was
purified via SAX-HPLC (see below) followed by lyophilization.
The final yield was 4.0 mg of TDP-6-deoxy-4-keto-D-glucose 6
(88% yield). Compound 6 was analyzed by LC-ESI-MS, which
determined the m/z to be 563.20 ([M - H þ H2O]-, calcd for
C16H25N2O16P2 563.07).
Preparation of Other TDP-sugar Substrates and Inter-
mediates. Other TDP-sugars were biochemically synthesized

9308 Biochemistry, Vol. 49, No. 43, 2010
Vey et al.
from 6 using purified enzyme preparations and the general
procedures outlined by Chen et al. (30) with the following modi-
fications. Briefly, thymidine-50-diphosphate-3-amino-2,3,6-tri-
quenching the reactions via addition of 25 μL of acetone, and
storing samples at -80 ꢀC until they were analyzed by LC-ESI-
MS as described below.
deoxy-3-C-methyl-
D
-erythro-hexopyranos-4-ulose 8 was synthe-
LC-ESI-MS Method for ORF36 Substrates. The oxida-
tion of ORF36 substrates was analyzed using an Agilent 1100
HPLC system (Agilent, Palo Alto, CA) with a LCQ Quantum
Decca XP ion trap mass analyzer (ThermoFinnigan, San Jose, CA)
equipped with an API electrospray ionization source fitted
with a 50 μm inside diameter deactivated fused silica capillary.
Injections of 10 μL were separated using a Hypercarb column
(3 mm ꢀ 50 mm, Thermo Inc.). Mobile phases were (A) H2O with
50 mM NH4CH3COO and 0.1% (v/v) diethylamine and (B) a
H2O/acetonitrile mixture (5:95) with 50 mM NH4CH3COO and
0.1% (v/v) diethylamine. Gradient conditions were as follows:
15% B from 0 to 5 min, linear gradient to 35% B from 5 to 15 min,
linear gradient to 100% B from 15 to 16 min, 100% B from 16
to 21 min, linear gradient to 15% B from 21 to 22 min, and 15% B
from 22 to 30 min. The flow rate was maintained at 0.3 mL/min.
The mass spectrometer was operated in both the negative ion and
full scan profile modes, and the electrospray needle was main-
tained at 3400 V. The ion transfer tube was operated at -47.50 V
and 275 ꢀC. The tube lens voltage was set to -46 V. The collision
sized via tandem reaction with EvaA, EvaB, and EvaC and
purified as described below in a 10% overall yield in three steps.
ESI mass spectra yielded m/z 560.27 ([M - H þ H2O]-, calcd for
C17H28N3O14P2 560.10). Thymidine-50-diphospho-3-amino-2,3,6-
trideoxy-3-C-methyl-L-arabinohexose (TDP-L-epi-vancosamine 10)
was generated from 8 (1 mM) in a 1 mL reaction mixture con-
taining 100 mM Tris-HCl (pH 7.5), 5 mM NADPH, 20 μM
EvaD, and 50 μL of the resuspended EvaE pellet. The reaction
mixture was incubated at 24 ꢀC for 2 h; proteins and cell debris
were removed using a 10K centrifugal filter (Centricon), and the
product was purified by SAX-HPLC (described below) followed
by lyophilization, which resulted in 300 μg (55% yield) of 10.
LC-ESI-MS confirmed the mass of 10 ([M - H]- calcd for
C17H28N3O13P2 544.11, found 544.27).
Purification of TDP-sugars. Preparative enzymatic reac-
tion progress was followed via HPLC using a series 600 Waters
HPLC system with a Waters 2996 diode array detector. Analy-
tical separations were performed on an Adsorbosphere strong
anion exchange (SAX) column (5 mm, 4.6 mm ꢀ 20 mm, Alltech
Associates) and a linear gradient from 50 to 500 mM NH4HCO2
(pH 3.5) at 1 mL/min for 45 min. Preparative HPLC separations
were performed using a semipreparative Adsorbosphere SAX
column (5 μm, 22 mm ꢀ 250 mm) using a similar protocol, but
at 10 mL/min. The pH values of the fractions containing TDP-
chromophores (267 nm) were immediately adjusted to ∼7 with
1 M NH4OH, and fractions containing reaction product were
pooled, lyophilized, and stored at -80 ꢀC until they were assayed.
Resuspended compound concentrations were determined by
measurement of the absorbance at 267 nm using a Nanodrop
spectrophotometer (Thermo, Inc.) and comparison to a standard
curve of dTDP.
ORF36 Enzymatic Reactions with 8-10. Reactions of
ORF36 with 8 and 10 were performed in a total volume of 50 μL
containing the corresponding substrates at 30 μM with 30 μM
FAD, 1 unit/mL catalase, 0.05 unit of flavin reductase, and
2.0 mM NADPH. Compound 9 was generated in situ by includ-
ing 20 μM EvaD in the reaction mixture with 8. All reactions
were initiated by addition of 12 μM ORF36, and 10 μL samples
were withdrawn at increasing time points (0-120 min), reac-
tions quenched with 10 μL of acetone, and mixtures stored at
-80 ꢀC until they were analyzed by LC-ESI-MS. Control assays
were performed without ORF36, FAD, or NADPH.
energy for all product ion scans was set at 30%. For TDP-L-epi-
vancosamine 10, full product and ion scans were set as follows:
544 f 155-548, 558 f 155-560, 560 f 155-562. For the assays
with 8 and 9, the scans were set as follows: 542 f 155-548, 560 f
155-562, and 558 f 155-560.
Data were acquired in profile mode. The following optimized
parameters were used for the detection: N2 sheath gas at 46 psi,
N2 auxiliary gas at 13 psi, spray voltage of 3.4 kV.
Crystallization of ORF36. Initial crystallization conditions
for ORF36 were identified by sparse matrix screening and were
improved using diffraction-based feedback on Life Sciences
Collaborative Access Team (LS-CAT) beamlines 21-ID-D,
21-ID-F, and 21-ID-G at the Advanced Photon Source (APS),
and beamline 9-1 at Stanford Synchrotron Radiation Light-
source (SSRL). Optimized crystals of ORF36 were grown using
the hanging drop vapor diffusion technique at 277 K from drops
containing 1 μL of protein solution [7 mg/mL ORF36 in 20 mM
Tris-HCl, 5% glycerol, and 1 mM DTT (pH 7.5)] and 1 μL of
crystallization solution [0.1 M Tris-HCl (pH 8.5), 0.2 M MgCl2,
and 10% polyethylene glycol 4000], equilibrated against 1 mL of
crystallization solution. Crystals belonging to hexagonal space
group P65 (Table 1) reached a maximal size of 0.1 mm ꢀ 0.1 mm ꢀ
0.4 mm in 5-6 days. The volume of the unit cell was consistent
with a tetramer in the asymmetric unit and 53% solvent content.
Crystals were cryoprotected by equilibration in a crystallization
solution supplemented with 20% glycerol or 20% ethylene glycol
and cryocooled in liquid nitrogen prior to data collection.
Native data set 1 was collected at 100 K at beamline 21-ID-D
of the Advanced Photon Source using a Rayonix MX 225
detector. Native data set 2 was collected at 100 K on beamline
9-2 of SSRL using a MarUSA MarMosaic-325 CCD detector.
Data integration and scaling were performed with the HKL2000
suite of programs (Table 1) (33).
18O2 Incorporation Assays. Reactions with 18O2 gas were
performed in a 5 mL round-bottomed flask fitted with a rubber
septum and a balloon (∼5 mL, connected via a syringe needle).
The reaction vessel at 4 ꢀC was connected via stainless steel cannula
to a closed gas cylinder (Cambridge Isotope Laboratories)
containing 1 L of 18O2 (22 psi) that was also fitted with a rubber
septum. A freshly prepared solution (200 μL) containing 20 mM
Tris-HCl (pH 7.5), 30 μM FAD, 1 unit/mL catalase, 0.05 unit/mL
flavin reductase, 2.0 mM NADPH, and 0.5 mg/mL ORF36 was
introduced into the flask, and the entire reaction system was
degassed in vacuo (25-50 mmHg) for 20 min. The 18O2 cylinder
was opened to release sufficient 18O2 gas to fill the flask and the
small balloon with ∼5-10 mL of gas. Reactions were initiated by
Structure Determination and Refinement. The structure of
ORF36 was determined by molecular replacement in Phaser (34)
using a polyalanine version of the human short branched chain
acyl-CoA dehydrogenase tetramer as the search model [Protein
Data Bank (PDB) entry 2JIF (35), 25% identical sequence].
Molecular replacement was only successful with native data set 1.
The molecular replacement solution was transferred into the
the addition of 30 μM vacuum-degassed TDP-L-epi-vancosamine
10 via a gastight syringe. We followed reactions for 2 h at 30 ꢀC by
withdrawing 25 μL aliquots via syringe at increasing time points,

Article
Biochemistry, Vol. 49, No. 43, 2010 9309
Table 1: Crystallographic Data Collection Statistics
native 1a
native 2a
˚
resolution (A)
50.0-3.60 (3.73-3.60)
P65
50-3.15 (3.26-3.15)
P65
space group
˚
unit cell dimensions (A)
a = b = 104.01, c = 294.36
6.3 (43.8)
a = b = 103.81, c = 295.95
11.6 (47.4)
b
Rsym
completeness (%)
Imean/σ
99.8 (100.0)
29.2 (5.4)
99.6 (99.5)
8.93 (2.50)
total no. of reflections
no. of unique reflections
redundancy
159943
127962
20697 (2046)
7.7 (7.7)
31158 (3113)
4.1 (3.5)
˚
wavelength (A)
1.12713
1.20000
beamline
APS 21-ID-D
APS 21-ID-D
P
P
P
P
b
aValues in parentheses refer to the high-resolution bin. Rsym
=
i|Ii(hkl) - I(hkl)|(100)/
iIi(hkl), where Ii(hkl) and I(hkl) are the ith and mean
hkl
hkl
measurements of the intensity of reflection hkl, respectively.
A model for TDP-L-evernosamine was constructed by manual
combination of deoxythymidine diphosphate (ligand name TYD)
and the epi-vancosaminyl derivative of vancomycin (ligand name
VAX), both available via the HIC-Up server (41). The TDP-L-
Table 2: Crystallographic Refinement Statistics for Native Data Set 2
˚
resolution limits (A)
50-3.15 (3.26-3.15)a
b
Rcryst
0.242
0.281
0.009
1.3
b
Rfree
˚
evernosamine was manually positioned within the active site such
bond length deviation (A)
bond angle deviation (deg)
˚
that the amino sugar was within 5.5 A of the expected position of
b
aValues in parentheses refer to the high-resolution bin. Rcryst and Rfree
the C4a of the isoalloxazine ring, but the location of the TDP
varied. The docking algorithm in MOE was used to improve each
manual starting point. Docking was performed using a radius of
P
P
equal hkl||Fo| - k|Fc||/ hkl|Fo|, where Fo and Fc are the observed and
calculated structure factors for reflection hkl, respectively, and k is a
weighting factor. Rfree is calculated on only test reflections.
˚
10 A around the initial position of the ligand, the triangle matcher
algorithm for placement, London dG rescoring, and force field
refinement. Between 30 and 100 positions were retained from
each trial and validated manually by evaluating the orientation of
the sugar moiety in the active site, and the distance of the amino
group from the expected position of C4a of the isoalloxazine ring.
higher-resolution native data set 2 (Table 1) and refined using
alternating rounds of manual model building in XtalView (36)
and refinement in CNS (37) with strict noncrystallographic sym-
metry (NCS) constraints. Once Rcryst reached 26%, each chain
was examined for main chain differences, which were observed
within the central β-domain between the ORF36 monomers. As a
result, during the final four cycles of refinement, each monomer
was refined with NCS restraints applied to the N- and C-terminal
domains, but deviations from NCS were allowed in the central
β-domain.
The final model of ORF36 comprises residues 1-395 (of 412
total residues), with several breaks observed in chains B-D. In
chain B, electron density for residues 143-151, 177-184, and
229-235 is not observed; in chain C, electron density for residues
144-156, 176-182, 218-221, and 235 is not observed, and in
chain D, electron density for residues 226, 227, and 316 is not
observed. The test set of reflections for the Rfree consisted of 9.1%
of the data, totaling 2816 reflections. The final model has an Rcryst
of 24.2%, an Rfree of 28.1%, 88.5% of the residues in the most
favored regions of the Ramachandran diagram, 10.9% in the
additionally allowed regions, 0.6% in the generously allowed
regions, and 0.0% in the disallowed regions (Table 2). Figures 5
and 8 and Figures S2, S6, and S7 of the Supporting Information
were prepared with PyMOL (38). Figure 7 was prepared using
MOLSCRIPT (39) and Raster3D (40).
The optimized position for TDP-L-evernosamine had a loca-
tion of the TDP similar to that experimentally observed for
dTDP-phenol crystallized in a complex with the nitrososynthase
KijD3 (11).
Phylogenetic Analysis. The evolutionary history was in-
ferred using the Neighbor-Joining method (42). The bootstrap
consensus tree inferred from 1000 replicates (43) is taken to
represent the evolutionary history of the taxa analyzed (43). The
evolutionary distances were computed using the Poisson correc-
tion method (44) and are in the units of the number of amino acid
substitutions per site. All positions containing gaps and missing
data were eliminated from the data set (Complete deletion
option). There was a total of 333 positions in the final data set.
Phylogenetic analyses were conducted in MEGA4 (45).
RESULTS
Substrate Determinants of Activity. NCBI BLAST anal-
ysis of sequenced microbial genomes contained in GenBank (46)
reveals an ever-increasing number of nitrososynthase homolo-
gues, which corresponds with the observation that nitro sugars
decorate a wide variety of known bioactive natural products with
distinct scaffolds. The hypothetical TDP-sugar precursors of the
known nitro sugar-containing natural products (Figure 1) pos-
sess conserved structural features, with notable functional varia-
bility evident at the C-4 position (found as a hydroxyl, O-methyl,
or N-carbamoyl group) and C-5 position (which can possess
Modeling of the Substrate and Cofactor. FAD was
modeled into the active site by superpositioning the ORF36
structure with that of human short branched chain acyl-CoA
dehydrogenase [PDB entry 2JIF (35)] and using the position of
bound FAD from 2JIF as a starting point. Attempts to improve
the position of FAD using the molecular docking program
Molecular Operating Environment (MOE, Chemical Computing
Group, Montreal, QC) did not result in a solution that both
alleviated steric clashes between the FAD and protein and
retained a reasonable position for the isoalloxazine ring.
either the
D or L configuration). This structural diversity in the
substrates is in striking contrast with the 60-65% sequence
identity between proposed nitrososynthases (11), prompting us to
investigate the possibility that the reported nitrososynthases act

9310 Biochemistry, Vol. 49, No. 43, 2010
Vey et al.
on a common intermediate biochemically upstream of their
amino congeners in the corresponding pathways (47).
was competent for the double oxidation that results in the nitroso
congener 14 (Figure 3). The precursor of TDP-L-epi-vancosamine
To address this possibility and to further delineate the timing
of amine oxidation in broader nitro sugar biosynthesis, we
reconstituted the biosynthetic pathway for TDP-L-epi-vancosa-
10, keto sugar 9, was assayed by including a catalytic excess of the
C-5 epimerase (EvaD) in the nitrososynthase oxidation reaction.
Compound 9 was rapidly oxidized to the hydroxylamino con-
gener 12 but did not further oxidize to other species under these
mine 10 using enzymes obtained by the Walsh group for the
chloroereomycin pathway (Figure 2) (30) and assayed for the
biochemical competence of amino functional keto sugar precur-
sors 8 and 9 (Figure 3). These TDP-sugars were enzymatically
biosynthesized and purified using ion exchange chromatography.
While we were unable to isolate sufficient quantities of the
substrate to perform Michaelis-Menten kinetics (and the in-
herent storage instability of these glycosyl 1-phosphates likely
precludes their accurate quantitation), we were able to compare
turnover of substrates at identical, titrated substrate concentra-
tions (30 μM).
conditions. The penultimate precursor to TDP-L-epi-vancosa-
mine 10 is the C-5 epimeric keto sugar 8, which was also oxidized
to the hydroxylamino oxidation state 11, but at a substantially
slower apparent rate than when the epimerase was added to
reaction mixtures. This is in accord with the recently reported
partial oxidation of 8 by KijD3 (11), which likewise only evi-
denced the formation of hydroxylamine. Unlike these studies,
however, we did not observe an additional peak at m/z 527. While
the formation of the peak at m/z 527 was rationalized by the
authors as a decomposition product of 11 (proposed as dTDP-
As analyzed by LC-ESI-MS, all potential substrates dis-
played a degree of chemical competence for single oxidation to
hydroxylamine (Figure 3), but only TDP-L-epi-vancosamine 10
2,3,6-trideoxy-4-keto-3-methyl-D-glucose), this structure corre-
sponds to that of a reduction product of the nitroso sugar, not a
fragment or likely decomposition product, and the identity of this
compound remains undetermined. In comparison, ORF36 assays
with compounds 8 and 9 produced species corresponding only to
the hydroxylamino compounds 11 and 12 within the expected
mass ranges, and no other detectable TDP-sugar decomposition
products were apparent after MS or MS/MS analysis. The
oxidation of biogenic TDP-L-epi-vancosamine 10 confirms our
previous results using the chemically synthesized compound (8)
and, importantly, also validates the successful biocatalytic gen-
eration of its biosynthetic progenitors 8 and 9. Taken together,
these data indicate that catalytic competence of ORF36 improves
FIGURE 2: Pathway utilized for biochemical synthesis of TDP-L-epi-
vancosamine 10 and keto amine precursors 8 and 9.
in substrates more closely related to TDP-L-epi-vancosamine 10
F
IGURE 3: ORF36 catalysis with potential substrates. HPLC-ESI-MS traces of ORF36 reactions with three potential substrates. (A) TDP-L-epi-
vancosamine 10 (m/z 544) at 0 min (blue) and 100 min (green), hydroxylamine intermediate 13 (m/z 560) at 15 min (red), and nitroso product 14
(m/z 558) at 100 min (purple). (B) Keto amine 9 (m/z 542) at 0 min (blue) and 100 min (green) and hydroxylamine intermediate 12 (m/z 558) at 100
min (purple). (C) Keto amine 8 (m/z 542) at 0 min (blue) and 100 min (green) and hydroxylamine intermediate 11 (m/z 558) at 100 min (purple).

Article
Biochemistry, Vol. 49, No. 43, 2010 9311
F
IGURE 4: 18O2 incorporation studies. (A) Summary of proposed species observed in 18O2 incorporation experiments. (B-D) 18O2 incorporation
with ORF36 and TDP-L-epi-vancosamine 10 incubated with 16O2 (green) and 18O2 (red). Masses shown under structures correspond to unlabeled
species. HPLC-MS and MS/MS data were collected at a 15 min reaction time, when the hydroxylamine intermediate was most abundant.
(B) TDP-L-epi-vancosamine10(tR = 4.5 min). (C) Hydroxylamine 13(m/z 560, and tR = 5.3 min). (D) PeaksattR valuesof7-9 min are proposed
to correspond to the nitroso compound 14 (tR = 8.8 min) and an additional oxidation product at m/z 576 (tR = 7.8 min). This new mass has a
retention time distinct from that of nitroso sugar 14, and on the basis of MS/MS analysis, the additional mass of the m/z 576 ion is constrained to
the pyranose ring. One possible structure is the 4-keto-3-hydroxylamino sugar 12, which is detected as a hydrate 15 under ESI conditions. MS/MS
analysis supports this hypothesis in that the labeled ion at m/z 578 readily fragments to an ion at m/z 560.09, and this fragmentation pattern is
identical to the reaction product of 9 shifted by 2 mass units.
and that an abortive reaction occurs with the biochemical pro-
genitors. While the effect of C-4 O-methylation remains to be deter-
mined, the observation of both O-methyl and hydroxyl function-
ality in everninomicin congeners isolated from M. carbonacea var.
africana suggests that O-methyl substitution at this position is not
essential for nitrososynthase reactivity and may occur prior or
subsequent to amine oxidation reactions and/or glycosylation (3).
18O2 Incorporation Experiments. We next performed the
reactions under an 18O2 atmosphere to determine whether molec-
ular oxygen, as opposed to oxygen from H2O, was incorporated
into the product. Reaction mixtures containing NADPH, FAD,
flavin reductase, and ORF36 were incubated with a catalytic
excess of catalase under an atmosphere of either 18O2 or 16O2
(Figure 4). We reproducibly observed a high level of incor-
poration of 18O into hydroxylamine 13 (m/z 560) and nitroso
congeners 14 (m/z 558), which demonstrated enrichment of
the þ2 m/z shift at 86 and 87% incorporation, respectively.
As well as confirming the incorporation of molecular oxygen
into the expected products, these experiments identified a new
reaction product. In addition to the labeling of the nitroso sugar
14, we also observed a þ2 m/z shift in what we have previously
assigned to be the electrospray-induced hydrate of the nitroso
moiety [14 þ H2O at m/z 576 (8)]. However, the single incorpora-
tion of oxygen into this species and the lack of demonstrable
back-exchange of labeled nitroso 14 with H2O rule out the pro-
posed dihydroxylamino species. Moreover, we discovered that
subsequent to isolation of 14 by ion exchange HPLC and
reanalysis, the nitroso group did not form the hydrate under
our standard HPLC-MS conditions (data not shown). Indeed,
closer examination of the chromatograms indicated that within
the broad peak encompassing this region, the nitroso sugar 14
and the m/z 576 species possess different retention times (8.8 and
7.8 min, respectively). Both species contain fragmentation peaks
diagnostic of a TDP-sugar, including the loss of TDP (m/z 401)
and TMP (m/z 321), but the unknown m/z 576 compound
demonstrated an additional major fragment at m/z 558, indicat-
ing loss of water. We have thus far been unable to isolate the m/z
576 species from reactions generating the nitroso sugar for
further characterization. However, on the basis of MS/MS
analysis, which demonstrated that oxidation is constrained to the
pyranose ring, and considering the likelihood that oxidation is
occurring on the C-3/4 heteroatoms, we propose that the m/z 576
peak corresponds to a product oxidized at C-4, likely the C-4
ketone 12. Notably, this putative structure is advantageously
identical to 12 generated as a reaction product of substrate
analogue 9 (Figure 3), which possesses a fragmentation pattern
and a retention time identical to those of the m/z 576 peak. It
should also be noted, however, that other structures are possible,
including a possible electrocyclic rearrangement of the R-hydro-
xyketone (not shown) (48).
X-ray Crystal Structure of ORF36. The structure of ORF-
˚
36 (Figure 5) was determined to 3.15 A resolution by molecular
replacement using a polyalanine model of human short branched
chain acyl-CoA dehydrogenase (PDB entry 2JIF) (35) as the

9312 Biochemistry, Vol. 49, No. 43, 2010
Vey et al.
F
IGURE 5: Structure ofORF36. (A) The ORF36tetramer isshown asacartoon representationwitha transparentsurface, colored bydomain. The
N-terminal domains (residues 1-129) are colored gray, the central β-sheet domains (residues 130-235) green, and the C-terminal domains
(residues 235-398) purple. (B) ORF36 monomer shown in cartoon representation with modeled FAD and TDP-L-evernosamine shown as
transparent sticks. Colors are as follows: yellow for FAD carbons, magenta for TDP-L-evernosamine carbons, red for oxygen, blue for nitrogen,
and orange for phosphate. The view in panel B is rotated 45ꢀ about a vertical axis from the bottom monomer shown boxed in panel A.
search model. ORF36 forms a tetramer (Figure 5A) both in
solution (Figure S1 of the Supporting Information) and in the
crystal with 3500 A of surface area buried per monomer. Each
monooxygenases in complex with their substrates was used
to identify ideal cofactor-substrate distances and geometry
(24, 50-52).
2
˚
ORF36 monomer adopts a three-domain R/β/R fold (Figure 5B)
with the N- and C-terminal R-helical domains separated by a
β-sheet domain. Interactions between the adjacent C-terminal helical
domains predominate the contacts mediating tetramerization.
A search for structural homologues of ORF36 using the
EMBL Dali server (49) identified the recently released nitroso-
synthase KijD3 (11) as the most similar structural homologue.
This pair of nitrososynthases shares structural similarity with
members of the acyl-CoA dehydrogenase superfamily of pro-
teins (21), which contains dehydrogenases, oxidases, and flavin-
containing monooxygenases (Table S1 of the Supporting In-
formation). Intriguingly, within this superfamily, ORF36 and
KijD3 are markedly more similar in both sequence and structure
to the dehydrogenases than to the nitrososynthase functional
homologues, the monooxygenases. From this, we hypothesize
that nitrososynthases independently evolved monooxygenase
activity from acyl-CoA dehydrogenases, while most other class
D flavin monooxygenases and acyl-CoA dehydrogenases under-
went a separate evolutionary divergence. This proposal is corro-
borated by phylogenic analysis of these structural relatives
(Figure 6).
Comparison of ORF36 to acyl-CoA dehydrogenses and
flavin-containing monooxygenases allowed us to identify pre-
formed pockets in the active site of ORF36 that likely bind the
isoalloxazine ring of the flavin and the TDP of TDP-L-ever-
nosamine. Residues near the predicted isoalloxazine binding
pocket are highly conserved in nitrososynthases and are similar
to flavin-containing monooxygenases and acyl-CoA dehydro-
genases (Figures S4-S6 of the Supporting Information), parti-
cularly at loops L3, L5, and L9. In contrast, the predicted binding
location for the adenine nucleotide of the flavin is in a region of
the active site where the loops are splayed open. In both acyl-CoA
dehydrogenases and flavin-containing monooxygenases, loop L7
(Figure 7A) forms specific contacts between the flavin and
protein, is shorter in enzymes that preferentially utilize FAD
and longer in enzymes that preferentially use FMN, and can
undergo modest conformational adjustment following flavin
binding to optimize contacts with the nucleotide. Despite the in
vitro ability of ORF36 to use either FMN or FAD as a cofactor,
loop L7 resembles loops from members of the acyl-CoA dehy-
drogenase superfamily that use FAD. In this structure, it adopts a
conformation that suggests an adjustment of the main chain will
accompany flavin binding.
Active Site. Residues contributing to the presumed active site
emanate predominantly from the N-terminal helical domain and
the β-sheet domain but include residues from the C-terminal
domain of an adjacent monomer (Figure S2 of the Supporting
Information) (21). The active site is built along the length of helix
R4, which forms the base of the active site cleft. Side chains from
R4, β1, R6, and R8 likely contribute to flavin binding, while side
chains from R4 and R6-R8 could contribute to substrate binding
(Figure S2B of the Supporting Information). Importantly, the
loops between the secondary structural elements surrounding the
Cofactor and Substrate Modeling. Extensive cocrystalliza-
tion and crystal soaking experiments of ORF36 with various
concentrations and combinations of FAD, FMN, the isolated
isoalloxazine riboflavin (each in oxidized and reduced states),
and the substrate TDP-L-epi-vancosamine were performed. None
of these resulted in the appearance of new electron density for a
cofactor or substrate within the active site. Instead, FAD and
TDP-L-evernosamine were modeled into the active site using a
combination of manual and automated methods (Figure 7A and
Figures S2 and S3 of the Supporting Information). Information
from an examination of the costructures of flavin-containing

Article
Biochemistry, Vol. 49, No. 43, 2010 9313
ORF36 are identical in loop L10 (Figure S5 of the Supporting
Information), and the structure of KijD3 also contains a tandem
cis peptide bond in this location (11).
In both ORF36 and KijD3, the temperature factors of the
residues of the loops surrounding the active site are significantly
elevated as compared to the temperature factors in the remainder
of the protein (Figure 8). Crystallographic temperature factors,
also known as atomic displacement parameters, can reflect the
thermal motion of atoms and hint at regions of increased relative
mobility in a given structure.
DISCUSSION
F
IGURE 6: Evolutionary relationships of 13 taxa including nitroso-
Substrate Selectivity of ORF36. Data presented in this
synthases, acyl-CoA dehydrogenases, and flavin-containing mono-
oxygenases. The nitrososynthases ORF36 and KijD3 are highlighted
inbluetext, the class D flavin-containingmonooxygenasesinred text,
and the acyl-CoA dehydrogenases and nitroalkane oxidase in black
text. Proteinsincludedhere(which are alsoincluded inTable S1ofthe
Supporting Information) are named as follows: ACAD1, short branched-
chain acyl-CoA dehydrogenase (H. sapiens), 2JIF (35); ACAD2,
butyryl-CoA dehydrogenase (Megasphaera elsedenii), PDB entry
1BUC (58); ACAD3, medium-chain acyl-CoA dehydrogenase
(Sus scrofa), PDB entry 3MDE (59); ACAD4, medium-chain acyl-
CoA dehydrogenase (S. scrofa), PDB entry 1UDY (60); ACAD5,
short-chain acyl-CoA dehydrogenase (Rattus norvegicus), PDB
entry 1JQI (61); HpaB1, 4-hydroxyphenylacetate monooxygenase
(A. baumannii), PDB entry 2JBT (24); monoox1, 3-hydroxy-9,10-
seco-nandrost-1,3,5(10)-triene-9,17-dione hydroxylase (Rhodococcus
sp. Rha1), PDB entry 2RFQ (62); monoox2, putative hydroxylase
(Rhodococcus sp. Rha1), PDB entry 2OR0 (63); NAO, nitroalkane
oxidase (Fusarium oxysporum), PDB entry 2C0U (64); HpaB2,
4-hydroxyphenylacetate monooxygenase (Thermus thermophilus),
PDB entry 2YYI (25); TftD, chlorophenol-4-monooxygenase (Burk-
holderia cepacia), PDB entry 3HWC (26).
work verify that TDP-L-epi-vancosamine 10 is a substrate for the
nitrososynthase ORF36. This was an expected outcome, as the
A-ring des-4-O-Me congener of everninomicin is a naturally
occurring variant in M. carbonacea var. africana (5). That the bio-
synthetic progenitors of TDP-L-epi-vancosamine 10 were not
fully oxidized upon incubation with ORF36 suggests that related
enzymes in kijanimicin and rubradirin biosynthesis act not on a
common precursor but on substrates at least closely related, if not
identical, to the amino congeners of the final nitro sugars in these
molecules. While other possibilities are conceivable, the apparent
abortive oxidation of the ketoamine substrate analogue 9 also
serendipitously aided in the potential deconvolution of a major
shunt reaction product, the C-4 ketone 12, in which the 4-hydroxy
position of TDP-L-epi-vancosamine is oxidized, perhaps via
dehydrogenation or hydroperoxylation of the C-4 hydroxyl to
the ketone oxidation state. While we have yet to be able to
generate the authentic substrate TDP-L-evernosamine, we expect
that reactions of this substrate will reveal that blocking of the C-4
hydroxyl group at its methyl ether will protect this sugar from
spurious oxidation. The potential C-4 oxidation reaction is
unprecedented and is interesting in the context of acyl-CoA
dehydrogenase homologues found associated with other second-
ary metabolic gene clusters in actinomycetes that apparently do
not contain amino sugar biosynthetic genes, expanding the
potential scope of this enzyme to other oxidation reactions.
Structural Comparison of ORF36 and KijD3. ORF36 is
65% identical to KijD3, the nitrososynthase homologue from
the kijanimicin biosynthetic pathway. As expected, their overall
structures are similar, with a root-mean-square deviation of
active site [loops L1-L11 (Figure 7A-D and Figure S3 of the
Supporting Information)] likely play an important role in sub-
strate binding and catalysis. In striking contrast to the main
scaffold of the protein, loops L1, L2, L4-L8, and L11 substan-
tially differ in structure among related enzymes (Figure 7A-C
and Figure S3 and Movie 1 of the Supporting Information), sug-
gesting that they may be important for conferring substrate and
cofactor specificity.
In both ORF36 and the nitrososynthase KijD3, the active site
˚
˚
measures 30 A wide by 20 A deep and appears to be solvent-
exposed as compared to acyl-CoA dehydrogenases and flavin-
containing monooxygenases (Figure 8). The unusually expansive
active site portico physically originates from the conformations
of active site loops L1, L2, L4, L6, L7, and L11 (Figure 7A),
which appear to be splayed in ORF36 and KijD3 as compared to
the acyl-CoA dehydrogenases and flavin-containing monooxy-
genases (Figure 7 A-C). With the exception of L7, these loops
are located in the region of the active site known to bind the
substrate in the acyl-CoA dehydrogenase superfamily, suggesting
that significant main chain adjustments could accompany sub-
strate binding. Of particular note is loop L10 in ORF36, which is
located on the floor of the active site cleft. This tight turn contains
tandem cis peptide bonds between Gln 376 and Pro 377 and
between Pro 377 and Tyr 378 (Figure 7D and Figure S7 of the
Supporting Information). While it is somewhat speculative to
assign a cis peptide bond at the resolution of this structure,
modeling the loop with trans peptide bonds or with only a single cis
proline resulted in disallowed Ramachandran angles for Tyr 378
and poor agreement of the main chain with the electron density.
Corroborating our interpretation, the sequences of KijD3 and
˚
0.76 A between the CR atoms of ORF36 and KijD3 tetramers.
The greatest differences in both sequence and structure between
these related enzymes are located within the regions that map to
the active site cleft and may be related to differences in function
and substrate binding. For example, while residues lining the pre-
dicted isoalloxazine and TDP binding sites are almost completely
conserved between ORF36 and KijD3 (Figure S8 of the Support-
ing Information), the interface of those two binding sites, where
the nitro sugar is presumed to bind, has greater sequence varia-
tion and may reflect differences in the preferred substrate. The
main structural differences between ORF36 and KijD3 occur in
active site loops L2, L4, and L6 located within the central β-sheet
domain, and L7 and L8 of the C-terminal domain. These regions
also display increased crystallographic temperature factors in
both structures, which may reflect flexibility within these active
site loops.
Active Site. Examination of the ORF36 and KijD3 (11)
chemical reactions and active sites identifies common features
that suggest large conformational adjustments could accompany

9314 Biochemistry, Vol. 49, No. 43, 2010
Vey et al.
F
IGURE 7: Activesite loops. The loops that form the activesite are asfollows:the loops between R3 and R4 (loopL1, residues 106-109), β1 and β2
(loop L2, residues 134-142), β3 and β4 (loop L3, residues 158-164), β4 and β5 (loop L4, residues 178-180), β6 and β7 (loop L5, residues
201-213), β9 and R6 (loop L6, residues 248-253), R60 and R70 (where the prime indicates this is from an adjacent monomer, loop L7, residues
272-280), R7 and R8 (loop L8, residues 310-317), R80 and R90 (loop L9, residues 351-353), and R9 and R10 (loop L10, residues 375-379) and at
the C-terminus (loop L11, residues 390-412). (A) Ribbon diagram of ORF36 shown with modeled flavin (yellow carbons) and TDP-
L-evernosamine (magenta carbons) as sticks. Active site loops L3, L5, L7, and L9 are predicted to interact with flavin and are colored orange;
active site loops L1, L4, L6, L8, and L11 are predicted to interact with substrate and are colored purple, and active site loops L2 and L10 are
predicted to interact with both the substrate and cofactor and are colored teal. (B) Ribbon diagram of human short branched chain acyl Co-A
dehydrogenase (PDB entry 2JIF) (35), highlighting loops L1-L11 colored as described for panel A. (C) Ribbon diagram of A. baumannii
4-hydroxyphenylacetate monooxygenase (PDB entry 2JBT) (24) highlighting loops L1-L11 colored as described for panel A. (D) Stereoview of
ORF36 loop L10 containing a tandem cis peptide. Loop L10 is shown as sticks with teal carbons. The Q376--P377 and P377-Y378 bonds both
adopt a cis conformation. Modeled flavin and substrate are displayed as in panel A.
productive cofactor and substrate binding (Figures 7 and 8).
Both nitrososynthase active sites are wide and highly solvent ac-
cessible, surrounded by loops that adopt splayed conformations
with elevated temperature factors as compared to those of the
remainder of the protein. In addition, both enzymes contain a
tandem cis peptide bond within active site loop L10 (Figure 7D
and Figure S7 of the Supporting Information), which could
undergo isomerization to initiate a conformational change.
Finally, the chemistry catalyzed by both these nitrososynthases
presumably requires exclusion of solvent from the active site; in
the acyl-coA dehydrogenases and class D flavin-containing
monooxygenases, this is achieved by the conformations of loops
L2, L4, L6-L8, and L11 (Figures 7A-C and 8). Taken together,
these observations suggest that it is reasonable, while still some-
what speculative, to expect a rearrangement of the active site
loops following cofactor and substrate binding.
Many enzymes undergo conformational adjustments upon
substrate binding that influence catalysis (53, 54), suggesting
that the open active site architecture could be physiologically
relevant in both ORF36 and KijD3 (11). One possible role for
the open loop positions may be to allow entry of large ligands
into the active site. Another prospect is that the active site
loops in these structures may adopt a nonproductive con-
formation to prevent unwanted side reactions or futile cofac-
tor cycling in the absence of substrate. These intriguing
possibilities clearly can be addressed only with further experi-
mentation.
reaction sequence, the enzyme first oxidizes TDP-L-epi-vancosa-
mine 10 to the diffusible hydroxylamine intermediate 13, which is
then further oxidized to nitroso sugar 14. Moreover, we have
demonstrated that the oxygen atom transfer reaction and the
successive oxidative dehydrogenation result in a single incorpora-
tion of oxygen from 18O2. Correspondingly, the general mecha-
nism we envision for this transformation (Figure 9), and for
oxidation of the biologically relevant substrate TDP-L-evernosa-
mine, centers on an oxygen atom transfer reaction in which
flavin-C4a-hydroperoxide serves as the electrophile in a nucleo-
philic substitution reaction (23, 55).
In overview, ORF36 likely first binds reduced flavin possibly
provided by an unidentified external reductase (several candidate
reductases exist in the genome of M. carbonacea var. africana,
GenBank accession number ACES00000000.1 or NCBI Refer-
ence Sequence NZ_ACES00000000.1) and promotes reaction
with molecular oxygen, yielding a flavin-C4a-hydroperoxide, a
well-known process in flavin monooxygenases (56, 57). Substrate
then binds and undergoes oxidation, after which the hydroxyla-
mine 13 and oxidized flavin are released to allow recycling of the
cofactor, as evidenced by the accumulation of the hydroxylamino
sugar 13 in reactions and everninomicin D analogues. The second
oxidative dehydrogenation sequence may result from an iterative
monooxygenase reaction followed by dehydration or, alterna-
tively, via a dehydrogenase mechanism akin to that catalyzed by
the homologous acyl-CoA dehydrogenases. On the basis of the
indirect observation that hydroxylamine 13 does not react with
ORF36 and oxidized flavin (generated by limiting NADPH in the
reaction) (8), we favor a monooxygenase mechanism in which a
Proposed Minimal Mechanism of ORF36. Results from
our biochemical investigations of ORF36 indicate that during the

Article
Biochemistry, Vol. 49, No. 43, 2010 9315
F
IGURE 8: Surface representation of the active site clefts of nitrososynthases, acyl-CoA dehydrogenases, and flavin-containing monooxygenases
colored by B factor. Active site loops are labeled L1-L11. Surfaces of each enzyme are shown colored using a rainbow color ramp in which red
corresponds to the highest B factor and blue corresponds to the lowest B factor. Areas of the surface that form the base of the active site cleft are
colored gray. Substrates and cofactors are shown as sticks with atoms colored as follows: FAD, yellow for carbon, red for oxygen, blue for
nitrogen, and orange for phosphate; TDP-L-evernosamine, dTDP, coenzyme A persulfide, magenta for carbon, red for oxygen, blue for nitrogen,
and orange for phosphate. All panels are shown in an orientation rotated by 45ꢀ about a horizontal axis with respect to Figure 3A. (A) ORF36.
Modeled FAD and TDP-L-evernosamine are displayed as sticks. In this orientation, loop L9 is concealed. The B factor color ramp gradient
2
2
˚
˚
minimum and maximum values are 70 and 180 A , respectively, and for this structure, the Wilson B factor is 111.2 A , with an average protein
2
B factor of 115.3 A . (B) KijD3 (PDB entry 3M9V) (11) with the dTDP of dTDP-phenol displayed as sticks. As in panel A, loop L9 is concealed.
The B factor color ramp minimum and maximum values for this panel are 10 and 80 A , respectively, and the average protein B value is 27.6 A .
The Wilson B factor was not reported. (C) Human short branched chain acyl-CoA dehydrogenase (PDB entry 2JIF) (35). The FAD molecule is
concealed by the protein surface in this orientation, and coenzyme A persulfide is shown in stick representation. Loops L9 and L10 are concealed
˚
2
2
˚
˚
2
˚
in this orientation. The B factor color ramp gradient has minimum and maximum values of 14 and 60 A , respectively; the Wilson B factor is
˚
2
2
˚
25.84 A , and the average protein B factor is 22.76 A . (D) A. baumannii 4-hydroxyphenylacetate monooxygenase (PDB entry 2JBR) (24). FMN,
4-hydroxyphenylacetate, and loops L3, L9, and L10 are concealed in this orientation. The B factor color ramp gradient minimum and maximum
2
2
values are 40 and 90 A , respectively, and the average protein B value is 56.77 A . The Wilson B factor was not reported.
˚
˚
second round of oxidation generates nitroso sugar 14 (Figure 9B)
perhaps via a dihydroxylamino or aminohydroperoxide inter-
mediate (not shown). This mechanism is supported by the
observed incorporation of oxygen from 18O2, but not H218O
(data not shown), into the final product (Figure 4). The
consistent stoichiometry of 18O2 incorporation may reflect
the exquisite ability of the acyl-CoA dehydrogenase fold to
control the access of O2 to the active site.
synthase enzyme. These insights are particularly relevant given
the known species targeting effects of the N-oxidation state at C-3
in the evernitrose ring (5) in this late stage III antibacterial clinical
candidate. Nitrososynthase articulates a new class of enzymes
revealing increasingly diverse flavin-dependent monooxygenase
reactivity in enzymes with an acyl-CoA dehydrogenase fold. The
results presented here have implications for the growing number
of enzymes in secondary metabolic gene clusters that have been
automatically annotated as acyl-CoA dehydrogenase-like en-
zymes but may instead be performing oxidation reactions with
little resemblance to the dehydrogenase paradigm. Future studies
of everninomicin biosynthesis will lead to a better understanding
of the effects of variability of C-4 O-methylation and C-5 stereo-
chemistry on enzyme oxidation cycles and the protein structural
determinants controlling selectivity and catalysis by this new
family of enzymes. The delineation of this minimal mechanism,
based on biochemical and structural studies, forms the basis for
future more detailed mechanistic studies.
Summary and Conclusions. This study has characterized the
conversion of the TDP-L-epi-vancosamine to the corresponding
nitroso sugar by nitrososynthase ORF36, which we propose
undergoes conformational adjustments upon substrate binding
prior to catalyzing monooxygenation of substrates specific to the
everninomicin biosynthetic pathway. Here, we conclusively show
a monooxygenase mechanism and identify the architectural
constraints of the enzyme housing this reaction. To the best of
our knowledge, these results comprise the first detailed biochem-
ical characterization of a functionally demonstrated nitroso-

9316 Biochemistry, Vol. 49, No. 43, 2010
Vey et al.
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ACKNOWLEDGMENT
We thank Prof. Michael Burkart (University of California,
San Diego, La Jolla, CA) and Prof. Hung-wen Liu (The University
of Texas, Austin, TX) for generously providing EvaA-E and
RfbB expression constructs, respectively. Use of the Advanced
Photon Source was supported by the U.S. Department of Energy,
Office of Science, Office of Basic Energy Sciences, under Con-
tract DE-AC02-06CH11357. Use of LS-CAT Sector 21 was sup-
ported by the Michigan Economic Development Corp. and the
Michigan Technology Tri-Corridor (Grant 085P1000817). Use of
the Stanford Synchrotron Radiation Lightsource was supported
by Stanford University and the U.S. Department of Energy.
We thank Gary Cecchini, Bill McIntire, and Carmello Rizzo for
critical reading.
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of the acyl-CoA dehydrogenases. FASEB J. 9, 718–725.
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catalyzed reactions. Eur. J. Biochem. 181, 1–17.
23. Palfey, B. A., and McDonald, C. A. (2010) Control of catalysis in
flavin-dependent monooxygenases. Arch. Biochem. Biophys. 493,
26–36.
SUPPORTING INFORMATION AVAILABLE
One movie, eight figures, and one table. This material is
available free of charge via the Internet at http://pubs.acs.org.
24. Alfieri, A., Fersini, F., Ruangchan, N., Prongjit, M., Chaiyen, P., and
Mattevi, A. (2007) Structure of the monooxygenase component of a
two-component flavoprotein monooxygenase. Proc. Natl. Acad. Sci.
U.S.A. 104, 1177–1182.
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