Stereospecific synthesis of threo- and erythro-β-hydroxyglutamic acid during kutzneride biosynthesis

Article abstract of DOI:10.1021/ja9054417

The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one α-hydroxy acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found in nonribosomally produced natural products. They contain D-3-h

Full text of DOI:10.1021/ja9054417

Published on Web 09/01/2009
Stereospecific Synthesis of threo- and
erythro-ꢀ-Hydroxyglutamic Acid During Kutzneride
Biosynthesis
Matthias Strieker,†,‡ Elizabeth M. Nolan,‡ Christopher T. Walsh,*,‡ and
Mohamed A. Marahiel*,†
Department of Chemistry/Biochemistry, Philipps-UniVersity, Hans-Meerwein-Strasse, D-35032
Marburg, Germany, and Department of Biological Chemistry and Molecular Pharmacology,
HarVard Medical School, 240 Longwood AVenue, Boston, Massachusetts 02115
Received July 2, 2009; E-mail: Christopher_Walsh@hms.harvard.edu; Marahiel@staff.uni-marburg.de
Abstract: The antifungal and antimicrobial kutznerides, hexadepsipeptides composed of one R-hydroxy
acid and five nonproteinogenic amino acids, are remarkable examples of the structural diversity found
in nonribosomally produced natural products. They contain D-3-hydroxyglutamic acid, which is found
in the threo and erythro isomers in mature kutznerides. In this study, two putative nonheme iron
oxygenase enzymes, KtzO and KtzP, were recombinantly expressed, characterized biochemically in
vitro, and found to stereospecifically hydroxylate the ꢀ-position of glutamic acid. KtzO generates threo-
L-hydroxyglutamic acid and KtzP catalyzes the formation of the erythro-isomer bound to the peptidyl
carrier protein of the third module of the nonribosomal peptide synthetase KtzH. This module has a
truncated adenylation domain and is unable to activate and incorporate glutamic acid. The lack of a
functional adenylation domain in the third KtzH module is compensated in trans by the stand-alone
adenylation domain KtzN, which activates and transfers glutamic acid onto the carrier of KtzH in the
presence of the truncated adenylation domain and either KtzO or KtzP. A method that employs
nonhydrolyzable coenzyme A analogs was developed and used to determine the kinetic parameters
for KtzO- and KtzP-catalyzed hydroxylation of glutamic acid bound to the carrier protein. A detailed
mechanism for the in trans compensation of the truncated adenylation domain and the stereospecific
hydroxyglutamic acid generation and incorporation is presented. These insights may guide the use of
KtzO/KtzP and KtzN or other in trans modification/restoration tools in biocombinatorial engineering
approaches.
(CDA),8 halogenation (syringomycin E)9 and hydroxylation
(CDA,10 syringomycin E,11 viomycin12) may occur prior to,
Introduction
A defining characteristic of secondary metabolites, in par-
ticular those produced by large multimodular nonribosomal
peptide synthetases (NRPS), is their broad structural diversity.1
The origin of this diversity can be traced back to the large pool
of available building blocks, which are utilized during the
biosynthesis of these natural products. These building blocks
include proteinogenic amino acids, fatty acids, and nonprotei-
nogenic amino acids.2 Additional modifications,3 such as
glycosylation (vancomycin,4 balhimycin5), methylation (calcium-
dependent antibiotics (CDA),6,7 daptomycin7), phosphorylation
during or after assembly of the natural product and provide
further structural variations. To understand how this complexity
is generated, the enzymatic mechanisms underlying such
modification reactions must be investigated. Such mechanistic
insights will aid portability and reengineering efforts to incor-
porate tailoring enzymes into other NRPS assembly lines.
Extraordinary examples of structural diversity and the oc-
currence of nonproteinogenic amino acids are the antifungal and
antimicrobial kutznerides 1-9 (Figure 1).13,14 These cyclic
hexadepsipeptides, isolated from the soil actinomycete Kutzneria
† Philipps-University.
(8) Neary, J. M.; Powell, A.; Gordon, L.; Milne, C.; Flett, F.; Wilkinson,
B.; Smith, C. P.; Micklefield, J. Microbiology 2007, 153, 768–776.
(9) Vaillancourt, F. H.; Yin, J.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A.
2005, 102, 10111–10116.
‡ Harvard Medical School.
(1) Walsh, C. T. Science 2004, 303, 1805–1810.
(2) Strieker, M.; Marahiel, M. A. Chembiochem 2009, 10, 607–616.
(3) Samel, S. A.; Marahiel, M. A.; Essen, L. O. Mol. Biosyst. 2008, 4,
387–393.
(10) Strieker, M.; Kopp, F.; Mahlert, C.; Essen, L. O.; Marahiel, M. A.
ACS Chem. Biol. 2007, 2, 187–196.
(4) Hubbard, B. K.; Walsh, C. T. Angew. Chem., Int. Ed. 2003, 42, 730–
765.
(11) Singh, G. M.; Fortin, P. D.; Koglin, A.; Walsh, C. T. Biochemistry
2008, 47, 11310–11320.
(5) Sussmuth, R. D.; Wohlleben, W. Appl. Microbiol. Biotechnol. 2004,
63, 344–350.
(12) Helmetag, V.; Samel, S. A.; Thomas, M. G.; Marahiel, M. A.; Essen,
L. O. Febs J. 2009, 276, 3669–3682.
(6) Milne, C.; Powell, A.; Jim, J.; Al Nakeeb, M.; Smith, C. P.;
Micklefield, J. J. Am. Chem. Soc. 2006, 128, 11250–11259.
(7) Mahlert, C.; Kopp, F.; Thirlway, J.; Micklefield, J.; Marahiel, M. A.
J. Am. Chem. Soc. 2007, 129, 12011–12018.
(13) Broberg, A.; Menkis, A.; Vasiliauskas, R. J. Nat. Prod. 2006, 69, 97–
102.
(14) Pohanka, A.; Menkis, A.; Levenfors, J.; Broberg, A. J. Nat. Prod.
2006, 69, 1776–1781.
9
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J. AM. CHEM. SOC. 2009, 131, 13523–13530 13523

A R T I C L E S
Strieker et al.
Figure 1. Proposed biosynthesis and structures of kutznerides 1-9. Adenylation (A) domains are shown in red, peptidyl carrier protein (PCP) domains in
green, condensation (C) domains in gray and epimerization (E) domains in blue. The third A domain of KtzH (A*) is only 88-aa in size and nonfunctional.
The thioesterase (yellow) is proposed to cleave the mature kutzneride from the assembly line by cyclization and thereby yield kutznerides 1-9. The site of
glutamic acid hydroxylation is highlighted in gray. All known kutzneride species where found to contain D-ꢀ-hydroxylated glutamic acid either in the
erythro (1, 2, 7, 9) or threo (3-6, 8) form.
sp. 744, are comprised of five unusual nonproteinogenic amino
acids and one R-hydroxy acid, and differ in the extent of
substitution and stereochemistry of constituent residues.13,14 All
kutzneridescontaintheunusualtricyclicdihalogenated(2S,3aR,8aS)-
6,7,dichloro-3a-hydroxy-hexahydropyrrolo[2,3-b]indole-2-car-
boxylic acid (PIC), followed by 2-(1-methylcyclopropyl)-D-
glycine (D-MecPG), which is connected to the R-hydroxy acid
moiety of either (S)-2-hydroxy-3-methylbutyric acid or (S)-2-
hydroxy-3,3-dimethylbutyric acid. The hydroxy acid is followed
by a D-piperazic acid (Pip) moiety found in four different forms
(Figure 1). In addition, kutznerides contain O-methyl-L-serine
and either the threo or erythro isomer of 3-hydroxy-D-glutamic
acid (D-hGlu).
The organization of the kutzneride NRPS assembly line is
also unusual (Figure 1).15 Kutznerides are thought to be
assembled by three nonribosomal peptide synthetases, KtzE,
KtzG and KtzH. KtzE is predicted to activate MecPG and
catalyze its condensation with the hydroxy acid, which is
activated by the adenylation (A) domain of KtzG. KtzG was
shown to adenylate 2-keto-isovaleric acid (KIV) and it is
postulated that after in situ reduction of the keto function by
the ketoreductase domain (KR), a methyltransferase forms the
t-butyl group found in many kutznerides.15 Whether the me-
thylated hydroxy acid is transferred onto the second peptidyl
carrier protein (PCP) domain of KtzE prior to condensation is
unclear. KtzH contains the remaining four modules needed for
kutzneride assembly. The first module is predicted to activate
Pip and the second to activate Ser. The A domain of module
two of KtzH is also thought to be responsible for O-methylation
because it contains a methyltransferase. The third module of
KtzH deviates from standard NRPS logic. The A domain found
in module three, designated as A*, is only approximately 20%
of the size of typical A domains and thus likely to be
nonfunctional. The lack of a functional amino acid activating
domain in the third module may be compensated by the stand-
alone A domain KtzN, which was shown to activate glutamic
acid.15 Lastly, the fourth module of KtzH is predicted to
incorporate the differently substituted PICs prior to cyclization
by the thioesterase (TE) domain and peptide release. The
presence of three epimerization (E) domains in the NRPSs
agrees with the occurrence of three D-amino acids.
The presence of the threo- and erythro isomers of 3-hydroxy-
glutamic acid, and the first occurrence of a putative in trans
compensation mechanism makes the hGlu-residue interesting
from the standpoints of biosynthesis and mechanism. Investiga-
tions regarding its generation should provide insights into NRPS
assembly logic extending beyond the classical linear modularity
principle. Furthermore, the stereospecific hydroxylation of amino
acids at the relatively unreactive ꢀ-position is a synthetic
challenge. In nature, this modification is usually catalyzed by
nonheme iron(II)- and R-ketoglutarate (RKG)-dependent
hydroxylases.16,17 Hydroxylation may occur during synthesis
of the amino acid precursor, as for 3-hydroxyarginine12 and
3-hydroxyasparagine10 during viomycin and CDA biosynthesis,
respectively, or once the amino acid is bound to a carrier protein
(PCP-S-amino acid), as in syringomycin E biosynthesis.11
Hydroxylase candidates capable of introducing the OH-group
at the ꢀ-position of the glutamic acid residue during kutzneride
assembly have been identified previously on the gene level.15
KtzO and KtzP, the proteins encoded by these genes, exhibit
the conserved HXD/E.. .H iron(II)-binding motif. They also
show high sequence homology (53% and 49%, Supporting
Information Table S1) to SyrP, the nonheme iron hydroxylase
from the syringomycin E synthesis cluster, which exclusively
(16) Hausinger, R. P. Crit. ReV. Biochem. Mol. Biol. 2004, 39, 21–68.
(17) Clifton, I. J.; McDonough, M. A.; Ehrismann, D.; Kershaw, N. J.;
Granatino, N.; Schofield, C. J. J. Inorg. Biochem. 2006, 100, 644–
669.
(15) Fujimori, D. G.; Hrvatin, S.; Neumann, C. S.; Strieker, M.; Marahiel,
M. A.; Walsh, C. T. Proc. Natl. Acad. Sci. U.S.A. 2007, 104, 16498–
16503.
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threo- and erythro-Hydroxyglutamate Biosynthesis
A R T I C L E S
Production of Recombinant Enzymes. The pQE30-plasmids
containing the genes or gene fragments of interest and the previously
described ktzN and cda-T9 expression plasmids10,15 were used to
transform E. coli BL21(DE3) Star (Invitrogen). For production of
recombinant KtzO, KtzP, A*PCP3 and PCP3 the transformed cells
were grown at 34 °C to an optical density of 0.6 (600 nm), induced
with isopropyl-ꢀ-D-thiogalactopyranoside (IPTG, final concentration
of 0.1 mM) and grown for an additional 5 h at 28 °C. The
recombinant proteins were purified by Ni-NTA (Qiagen) affinity
chromatography using a FPLC (Amersham Pharmacia Biotech).
The Ni column was equilibrated in 50 mM HEPES, 250 mM NaCl
pH 8.0 buffer and an increasing gradient of imidazole was employed
(7.5 mM to 250 mM in 30 min). Fractions containing the
recombinant protein were identified by SDS-PAGE analysis,
combined, and subjected to buffer exchange into 25 mM HEPES,
50 mM NaCl, pH 7.0 using HiTrap Desalting Columns (GE Health
Care). The protein concentrations were determined spectrophoto-
metrically using calculated extinction coefficients at 280 nm. The
proteins were flash-frozen in liquid nitrogen and stored at -80 °C
until use. KtzN and CDA-T9 were produced and purified as
previously described.10,15
acts on PCP-S-Asp. Thus, it is likely that KtzO and KtzP
catalyze the hydroxylation of glutamic acid tethered to the third
PCP domain of KtzH (PCP3-S-Glu). One or both enzymes could
introduce the hydroxyl group as a racemic mixture or act
stereospecifically such that one enzyme generates erythro-hGlu,
found in kutznerides 1, 2, 7, 9, and the other forms threo-hGlu,
found kutznerides 3-6 and 8.
In this study, the two putative nonheme iron oxygenases KtzO
and KtzP were cloned and recombinantly expressed in E. coli.
The enzymes were characterized biochemically in Vitro and were
found to catalyze the stereospecific hydroxylation of the
ꢀ-position of glutamic acid bound to the PCP of the third module
of KtzH. Direct kinetic measurements were determined by using
nonhydrolyzable coenzyme A analogs18 where glutamic acid
is bound to the PCP by an amide bond.
This study also details the first biochemical characterization
of a stand-alone adenylation domain that, in trans, reconstitutes
a nonfunctional NRPS assembly line. To provide mechanistic
insights, the ability of KtzN to activate and transfer Glu was
determined in the presence/absence of the truncated A* domain
of KtzH and in presence/absence of the hydroxylases KtzO and
KtzP. The detailed mechanism of hGlu incorporation into a
natural product on truncated modules of NRPS assembly lines
may aid the use of these and other modification/restoration
enzymes in biocombinatorial engineering approaches to small
molecule synthesis.
General Hydroxylation Assay. The recombinant hydroxylase
(KtzO or KtzP) (5 µM) was incubated for 2 h at 28 °C with the
cosubstrate RKG (2 mM), (NH4)2FeSO4 as source of the ferrous
iron cofactor (0.5 mM), and the substrate of interest (200 µM) in
100 µL of 25 mM HEPES, 50 mM NaCl (pH 7). Reactions were
stopped by addition of formic acid (final concentration of 4% v/v).
Control reactions were carried out by excluding either the hydroxy-
lase or RKG.
Hydroxylation Assay with Free Amino Acids. The assays were
carried out as described above except that the reaction was stopped
by addition of nonafluoropentanoic acid (final concentration of 4%
v/v). The mixture was then analyzed for the presence of hydroxy-
lated products by reversed-phase HPLC-MS on an ESI-Quad
1100(A) Series MSD mass spectrometer (Agilent) equipped with
a Hypercarb column (Thermo Electron Corporation, 100% carbon,
pore diameter of 250 Å, particle size of 5 µm). Solvent A was
aqueous nonafluoropentanoic acid (20 mM) solution, and solvent
B was acetonitrile. Gradient elution was applied starting with 0%
B for 5 min, followed by 0-30% B in 25 min, with a flow rate of
0.2 mL/min at 15 °C.
Hydroxylation Assays with PCP-S-Amino Acid, PCP-HN-Glu
and Kinetics. The recombinant PCP, PCP3 (200 µM), or the
recombinant PCP with the incomplete A domain, A*PCP3 (200
µM), were artificially loaded with either coenzyme A (CoA)
coupled to an amino acid (mainly CoA-Glu, 1 mM) or with
amino-coenzyme A (NH2-CoA) coupled to glutamic acid (1
mM) by using the promiscuous phosphopantetheinyl (ppant)
transferase Sfp19,20 (2 µM). The loading reaction was incubated
at 28 °C for 15 min in buffered aqueous solution (pH 7, 25 mM
HEPES, 50 mM NaCl, 1 mM MgCl2). The synthesis of the CoA-
amino acids and a detailed description, based on previously
published work,18,21,22 of NH2-CoA synthesis and its coupling
to an amino acid are included in the Supporting Information
section. To ensure that complete conversion of apo-PCP to PCP-
S-amino acid or PCP-HN-Glu was achieved, the loading assays
were stopped by addition of formic acid (final concentration of
4% v/v) and the reaction mixture was analyzed by reversed phase
HPLC-MS using a QTOF-MS QStar Pulsar i (Applied Biosys-
Experimental Section
Strains, Culture, Media and General Methods. The E. coli
strains were grown in Luria-Bertani medium, supplemented with
100 µg/mL ampicillin (final concentration). All chemicals were
purchased from Sigma-Aldrich unless stated otherwise. Oligonucle-
otides were purchased from Integrated DNA Technologies. DNA
dideoxy sequencing confirmed the identity of all constructed
plasmids (Dana-Farber Cancer Institute).
Cloning of KtzN, KtzO, KtzP, A*PCP3, PCP3 and CDA-T9. The
genes coding for ktzO and ktzP as well as the A*PCP3 and the
PCP3 gene fragments were amplified by PCR from fosmid DNA
containing the kutzneride biosynthetic cluster15 using the Phusion
High-Fidelity PCR Master Mix with GC buffer (New England
Biosciences) according to the manufacturer’s protocol. The ampli-
fication of ktzO was carried out using the oligonucleotides 5′-ktzO
(5′-AAA AAA GGA TCC ATG ACG AAC TCG ACG GAC GA)
and 3′-ktzO (5′-AAA AAA GCG GCC GCT CAG CGG TCG CGG
TGG ACG G). The ktzP gene was amplified using the primer
combination of 5′-ktzP (5′-AAA AAA GGA TCC GTG ACT GTG
GAA CCC AGG CG) and 3′-ktzP (5′-AAA AAA GCG GCC GCT
CAG CGG GAA TCG AGA GTC G). The gene fragment A*PCP3
coding for the A domain fragment A* and the PCP domain of the
third module of ktzH, was amplified using the oligonucleotides 5′-
A*PCP3 (5′-AAA AAA GGA TCC GAC CTC GCG CTG GTC
GAG GC) and 3′-A*PCP3 (5′-AAA AAA GCG GCC GCT CAG
TCG GCC ACC CGT GCG GCC A). For amplification of the gene
fragment PCP3, coding for the third PCP domain of ktzH, the
oligonucleotides 5′-PCP3 (5′-AAA AAA GGA TCC ATC AGG
GCC CCG CGG ACG GA) and 3′-A*PCP3 were used, as both
gene fragments have the same 3′-end. The sites where the restriction
endonucleases cut are underlined in the sequences. After purifica-
tion, the PCR products were digested with BamHI and NotI, and
the genes of interest (ktzO, ktzP and the gene fragments A*PCP3
and PCP3) were each ligated into a BamHI- and NotI-digested
pQE30-derived expression vector (Qiagen). The genes coding for
ktzN and CDA-T9 were cloned previously10,15 and the resulting
expression vectors used to transform E. coli expression strains.
(19) Lambalot, R. H.; Gehring, A. M.; Flugel, R. S.; Zuber, P.; LaCelle,
M.; Marahiel, M. A.; Reid, R.; Khosla, C.; Walsh, C. T. Chem. Biol.
1996, 3, 923–936.
(20) Reuter, K.; Mofid, M. R.; Marahiel, M. A.; Ficner, R. Embo J. 1999,
18, 6823–6831.
(21) Koglin, A.; Lohr, F.; Bernhard, F.; Rogov, V. V.; Frueh, D. P.; Strieter,
E. R.; Mofid, M. R.; Guntert, P.; Wagner, G.; Walsh, C. T.; Marahiel,
M. A.; Dotsch, V. Nature 2008, 454, 907–911.
(22) Meier, J. L.; Mercer, A. C.; Rivera, H., Jr.; Burkart, M. D. J. Am.
Chem. Soc. 2006, 128, 12174–12184.
(18) Liu, Y.; Bruner, S. D. Chembiochem 2007, 8, 617–621.
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A R T I C L E S
Strieker et al.
tems) coupled to a HP 1100 HPLC (Agilent) equipped with a
C-4 Nucleosil guard column (Macherey and Nagel, 10 × 3 mm,
pore diameter of 300 Å, particle size 5 µm) with the following
conditions: solvent A (water/0.45% formic acid), solvent B
(acetonitrile/0.45% formic acid), flow rate 0.2 mL/min, temper-
ature 45 °C with a gradient of 10-95% solvent B in 10 min,
the gradient was then held for 7 min. Protein mass reconstruction
was calculated using Analyst Software v1.5 (Applied Bio-
sciences). After complete loading of the PCP domain was
confirmed to occur within 15 min, the reaction was repeated
and after 15 min of preincubation for complete loading of the
PCP domain, the hydroxylase (either KtzO or KtzP), RKG and
iron(II) were added and the assay conducted as described above
for the general hydroxylation assay. For kinetic measurements
with A*PCP3-HN-Glu and noncognate CDA-T9-HN-Glu, the
hydroxylation assays were setup as described above and stopped
after 30 min, which was determined to be the optimal reaction
time for being in the linear conversion range. Substrate
concentrations were varied between 10 µM and 450 µM. Kinetic
parameters were determined using the calculated starting velocity
and the Enzyme Kinetic Module for Sigma Plot 8.0 (SPSS).
Enzymatic Synthesis of hGlu. The general hydroxylation assay
was carried out as described above except that the final reaction
volume was 200 µL. The proteins were precipitated by addition of
1 mL 10% TCA, and the resulting mixture centrifuged (45 min,
13,000 rpm, 4 °C). The supernatant was discarded and the pellet
was washed with 200 µL diethyl ether/absolute ethanol (3:1) twice
and with 200 µL diethyl ether once. The washed pellet was dried
for 5 min at 37 °C and 100 µL of 0.1 M aqueous potassium
hydroxide solution was added to cleave the thioester bond and
release the hydroxylated glutamic acid from the PCP domain. The
pellet was resuspended in the KOH(aq) solution and the resulting
mixture was incubated at 70 °C for 20 min at 1400 rpm in a
Thermomixer (Eppendorf). After thioester cleavage, methanol (1
mL) was added, and the mixture was incubated overnight at -20
°C. Precipitated protein debris was separated from the hGlu-
containing supernatant by centrifugation (13,000 rpm, 30 min, 4
°C). The supernatant was transferred into a new tube and the
solvents were removed under reduced pressure by using a Speed-
Vac-manifold (13 000 rpm, 2 mbar, 30 °C). The pellets were stored
at -20 °C or derivatized immediately for HPLC analysis (see
below).
Derivatization and Determination of Configuration of
Enzymatically-Synthesized hGlu by HPLC. 3-Hydroxyglutamic
acid standards were synthesized as previously described.13 The
dabsyl derivatization of both enzymatically prepared and synthetic
hGlu was based on published protocols.23,24 The pellets of hGlu
were dissolved in buffered aqueous solution (100 µL, 50 mM TRIS,
pH 9) and a saturated solution of dabsyl chloride in acetone (100
µL) was added. The mixture was heated at 70 °C for 15 min at
1400 rpm in a Thermomixer and subsequently diluted with absolute
ethanol (1 mL). The resulting mixture was centrifuged at 8000 rpm
for 5 min, and 100 µL of the supernatant was analyzed by reversed-
phase HPLC-QTOF-MS. Gradient elution was carried out using a
Synergi Fusion-RP column (Phenomenex, polar embedded C-18
column, 250 × 3 mm, pore diameter of 80 Å, particle size 4 µm)
and the following conditions: solvent A was water/0.45% formic
acid; solvent B was acetonitrile/0.45% formic acid. The flow rate
was 0.3 mL/min with a gradient from 50% B to 75% B over 20
min and then increasing to 95% B over the next min. The gradient
was then held for a further 5 min.
(complete conversion validated by QTOF-MS). The coupled assays
were also conducted in the presence of KtzO or KtzP (5 µM), RKG
(2 mM) and ferrous iron (0.5 mM). All reactions were incubated
in buffered aqueous solution (25 mM HEPES, 50 mM NaCl, pH
7), stopped after two hours by addition of formic acid (final
concentration of 4% v/v) and then analyzed by HPLC-QTOF-MS
as described above.
Results and Discussion
Expression and Purification of KtzO, KtzP, A*PCP3,
PCP3. The genes ktzO and ktzP, and the gene fragments of
A*PCP3 and PCP3, were amplified and cloned into expression
vectors and the corresponding recombinant proteins were
overproduced in E. coli as His7-tagged fusions (KtzP, 40.1 kDa;
KtzO, 39.5 kDa; A*PCP3, 22.3 kDa; PCP3, 11.3 kDa) and
purified using Ni-NTA affinity chromatography. SDS-PAGE
analysis indicated that each protein was isolated in high purity
(Supporting Information, Figure S1). Protein identity was
verified by mass spectrometry and the following protein yields
were obtained from 1 L of bacterial culture: KtzP, 2.2 mg; KtzO,
3.1 mg; A*PCP3, 5.7 mg; PCP3, 0.2 mg.
Hydroxylation of Carrier Protein-Bound Glutamic Acid.
Sequence alignments of KtzO and KtzP reveal a putative ferrous
iron binding motif HXD/E.. .H and similarity to the prototype
nonheme iron(II) and RKG-dependent taurine dioxygenase
TauD25 and other oxygenases (Supporting Information, Table
S1). KtzO and KtzP also share high sequence similarity with
SyrP, a hydroxylase acting on the ꢀ-position of aspartic acid
bound to the phosphopantetheinyl (ppant) cofactor of the eighth
peptidyl carrier protein of the syringomycin E megasynthetase
(SyrE-T8-S-Asp).11 Therefore, KtzO and KtzP were predicted
to hydroxylate glutamic acid at the ꢀ-position during kutzneride
biosynthesis and to act on glutamic acid tethered to the third
PCP of KtzH (Figure 1). One can speculate that either one
enzyme hydroxylates PCP-bound glutamic acid and generates
both isomers of L-3-hydroxyglutamic acid (threo- and erythro-
hGlu), and that the other is not involved in hGlu biosynthesis,
or that both work on PCP-tethered Glu. In the latter case, it is
possible that the enzymes act stereospecifically to each generate
one hGlu isomer.
To determine the biochemical origin of the hGlu residues
found in mature kutznerides, KtzO or KtzP was incubated with
the glutamic acid ppant thioesters of the truncated A* domain-
PCP (A*PCP3-S-Glu), as A* might play a role in hydroxylase-
PCP interaction. Preincubation of A*PCP3 with synthetic CoA-
S-Glu and the promiscuous ppant-transferase Sfp19,20 generated
the loaded carrier A*PCP3-S-Glu, which was subsequently
incubated with RKG, Fe(II) and either KtzO or KtzP. QTOF-
MS revealed that the apo-form of A*PCP3 (Figure 2A) with an
apparent mass of 22,343 Da was loaded with ppant-S-Glu to
generate A*PCP3-S-Glu (Figure 2B), indicated by the mass
increase of 470 Da. Incubations with KtzO (Figure 2C) and
KtzP (Figure 2D) resulted in the formation of hydroxylated
A*PCP3-bound L-glutamic acid (A*PCP3-S-hGlu), shown by the
mass difference of 16 Da. Hydrolysis of A*PCP3-S-Glu occurred
at neutral pH (Figure 2B, 2C, mass: 22,684 Da) after 2 h. The
control reaction lacking RKG only showed the mass of
A*PCP3-S-Glu and holo-A*PCP3 (Figure 2B), providing no
evidence for hydroxylase activity. Reactions conducted with the
single PCP3 (PCP3-S-Glu), also revealed a mass shift of 16 Da,
indicating that hydroxylation occurred (Supporting Information,
Coupled KtzN/PCP/Hydroxylase Assays. The A domain KtzN
(2 µM) was incubated with ATP (1 mM), L-glutamic acid (5 mM),
DTT (0.5 mM), MgCl2 (1 mM) holo-A*PCP3 (100 µM) or holo
PCP3 (200 µM). Holo-PCPs were generated by preincubation of
the PCPs with CoA (1 mM) and Sfp (2 µM) at 28 °C for 15 min
(23) Kim, T. Y.; Kim, H. J. J. Chromatogr. A 2001, 933, 99–106.
(24) Vendrell, J.; Aviles, F. X. J. Chromatogr. 1986, 358, 401–413.
(25) Eichhorn, E.; van der Ploeg, J. R.; Kertesz, M. A.; Leisinger, T. J. Biol.
Chem. 1997, 272, 23031–23036.
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threo- and erythro-Hydroxyglutamate Biosynthesis
A R T I C L E S
Figure 3. Stereospecificity of KtzO and KtzP. KtzO and KtzP products
were cleaved off the PCP, derivatized with dabsyl-Cl, analyzed via HPLC-
MS and compared with synthetic standards of the dabsylated L-ꢀ-
hydroxyglutamic acids. The KtzP product coelutes with the Dab-L-erythro-
hGlu and the KtzO product with the threo form. High resolution mass
spectrometric analyses of the peaks (observed [M + H]+) 451.1287 m/z)
are in accordance with the theoretical masses (451.1282 m/z).
Transfer of Glutamic Acid on A*PCP3 by KtzN. Another
intriguing factor regarding tailoring of hGlu and the kutzneride
assembly line is the mechanism of activation and transfer of
the glutamic acid onto the third PCP domain of KtzH. It was
postulated that the stand-alone adenylation domain KtzN (Figure
1) acts as an in trans substitute domain for the truncated A*
domain in the third module of HtzH by activating glutamic acid
as an amino acyl adenylate, and transferring it to the third
module. KtzN was shown to activate glutamic acid in previous
studies, but the transfer event and factors that may mediated
this reaction, such as protein-protein interaction, the role of
the hydroxylases or A*, have not been investigated.15 KtzN is
predicted to be the first example for specific NRPS assembly
line in trans recovery, which most likely involves specific
protein-protein interactions between the third module of KtzH
and KtzN, Without such specific protein-protein interactions,
it is likely that KtzN would load all four PCPs of KtzH with
glutamic acid.
To evaluate what protein-protein interactions are required
for Glu transfer by KtzN and gain more mechanistic insight
into this unusual NRPS recovery strategy, Glu transfer was
monitored in an assay containing recombinant KtzN (Glu A
domain), ATP, L-Glu, holo-A*PCP3 and in the absence or
presence of one hydroxylase (KtzO or KtzP), RKG and ferrous
iron. Analysis of the assay of KtzN with holo-A*PCP3 in the
absence of hydroxylase revealed that no glutamic acid was
transferred (Figure 4A, blue trace). Upon addition of either KtzO
(Figure 4A, dark red trace) or KtzP (Figure 4A, green trace),
cosubstrate and cofactor, glutamic acid was transferred onto
A*PCP3 and subsequently hydroxylated. The reaction was either
not complete or hydrolysis of the PCP-S-hGlu thioester occurred
prior to analysis, indicated by the prominent holo-A*PCP3 peak
(Figure 4A). To evaluate the role of the truncated adenylation
Figure 2. QTOF-MS analysis of hydroxylation activity of KtzO and KtzP
on PCP-tethered glutamic acid. (A) apo-A*PCP3 with an apparent mass of
22343 Da. (B) Control reaction: A*PCP3 loaded with synthetic coenzyme
A glutamic acid by Sfp and incubated for 2 h. The mass shift of 470 Da
shows that ppant-S-Glu was transferred on the PCP domain. (C) A*PCP3
after incubation with RKG, (NH4)2Fe(SO4)2 and KtzO, preceded by
incubation with Sfp and CoA-S-Glu. The mass difference of 16 Da compared
with B indicates hydroxylation of the PCP-bound glutamic acid. (D) Same
as C, but after incubation with KtzP. Hydrolysis is observed for B and C
and D, and the mass of 22 684 Da corresponds to the holo-A*PCP3.
Figure S2). These findings demonstrate that both KtzO and KtzP
are indeed nonheme iron(II)- and RKG-dependent oxygenases
that catalyze the hydroxylation of PCP-tethered glutamic acid
in Vitro. The truncated A domain observed in module three of
KtzH is not required for hydroxylation of PCP-bound substrate.
ꢀ-Hydroxylation and Stereospecificity. In order to confirm
the Glu-ꢀ-hydroxylation and to ascertain the stereospecificity
of KtzO and KtzP, the threo and the erythro isomers of ꢀ-OH-
Glu were synthesized as previously described.13 The enantio-
merically pure threo-L-hydroxyglutamic acid and the erythro-
L-glutamic acid were dabsylated to facilitate separation and
identification by reversed phase HPLC-MS.23,24 Coelution
analyses employing synthetic and dabsylated hGlu standards
revealed that KtzO and KtzP are stereospecific hydroxylases
acting on the ꢀ-position of Glu. KtzO generates threo-L-
hydroxyglutamic acid and KtzP catalyzes the formation of
erythro-hGlu (Figure 3). This system is the first example for a
NRPS where products with two isomers of a Cꢀ modified amino
acid are generated by two modifying enzymes that catalyze the
very same reaction but with opposite stereospecificity.
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A R T I C L E S
Strieker et al.
coenzyme A coupled to glutamic acid as detailed in Supporting
Information. Instead of starting the synthesis with trityl-
diaminoethane resin, which was used in previous studies,18 free
pantothenic acid was used. The diol was protected and the
pantothenic acid was coupled to Boc-diaminoethane to yield
fully protected aminopantetheine. After deprotection amino-
pantetheine was incubated with the three coenzyme A biosyn-
thesis27 enzymes PanK, PPAT, DPCK, which were recombi-
nantly expressed in E. coli,18,28 and ATP to yield the coenzyme
A analog, in which the thiol group is replaced by an amine
group (Supporting Information, Scheme S1). The amino-CoA
was then coupled to the Boc-protected glutamic acid using
standard amide coupling reagents. Upon acidic in situ depro-
tection, CoA-HN-Glu was obtained with an overall yield of 4%.
A*PCP3 was loaded with CoA-HN-Glu by Sfp to yield the
A*PCP3-HN-Glu and MS analysis revealed complete conver-
sion of apo-A*PCP3 to A*PCP3-HN-Glu (Figure 5A). After
incubation with either KtzO or KtzP, RKG and ferrous iron, a
mass shift of 16 Da was observed (KtzO, Figure 5B, KtzP,
Figure 5C), demonstrating that KtzO and KtzP catalyzed the
hydroxylation of A*PCP3-HN-Glu. The additional oxygen
introduced by the hydroxylation reaction is of no consequence
for the ionization properties of examined protein. Thus, the mass
spectrometer counts for the loaded A*PCP3-HN-Glu and its
hydroxylated form were used to determine the percentage of
the hydroxylated protein species relative to the nonhydroxylated
one. By variation of the A*PCP3-HN-Glu concentration (10
to 450 µM), it was possible to measure the starting velocities
and therefore to determine Michaelis-Menten kinetic param-
eters (Supporting Information, Figure S3), which are listed in
Table 1. One characteristic of these hydroxylases is the high
affinity to their substrate, which is shown by the relatively low
KM values (KtzO: 121.5 µM, KtzP: 168.9 µM) compared with
free amino acids modifying enzymes (AsnO: 479 µM, VioC:
3400 µM)10,12 (Table 1). Other features are their low turnover
numbers (KtzO: 0.34 min-1, KtzP: 0.44 min-1) compared with
free amino acid hydroxylases (AsnO: 299 min-1, VioC: 2611
min-1) and the low catalytic efficiencies (Table 1). However,
the catalytic efficiencies of KtzO and KtzP are identical, which
explains why both isomers of the hydroxylated glutamic are
found at equal amounts in the mature kutznerides, and only
differ in the grade of substitution of other residues (Figure 1).
A*PCP3 was chosen for kinetic measurements over the single
PCP as its production level and its ionization properties are much
better. However, the incubation of the single PCP with KtzO
or KtzP yielded the same conversion percentage (data not
shown), indicating that that similar kinetic properties should
be expected.
Figure 4. MS analysis of coupled glutamic acid transfer assays. (A)
Incubation of KtzN with holo A*PCP3, KtzO (red trace) or KtzP (green
trace) and their cosubstrates yielded the transfer of glutamic acid and
subsequent hydroxylation by KtzO and KtzP. No transfer of glutamic acid
by KtzN was observed when a hydroxylase was not added (blue trace). (B)
Role of A*: The KtzN coupled assay was carried out as in A, but the single
PCP was used instead of A*PCP of the third module of KtzH. In all cases,
only holo-PCP was detected after the coupled assays, indicating that one
hydroxylase and the A* domain is needed to allow KtzN-mediated transfer
of glutamic acid on the PCP domain.
domain A*, the assay was carried out using the single PCP
domain instead of A*PCP. MS analyses showed that no glutamic
acid was transferred regardless of the presence or absence of
any hydroxylase (Figure 4B). These results demonstrate that
two factors are critical for KtzN-mediated transfer of glutamic
acid onto the third PCP domain of KtzH in Vitro: the presence
of one of the two hydroxylases and the presence of A*, the
truncated A domain in KtzH. These requirements explain the
selective transfer of Glu to the third module of KtzH by KtzN
during the in trans compensation mechanism. KtzN is indeed
able to restore the assembly line by acting in trans and is the
first example for an in trans acting NRPS adenylation domain
recovery protein.
Kinetic Parameter Determination Using CoA Analogs. The
unstable nature of the thioester bond between glutamic acid and
PCP, as observed in the assays stated above (Figure 2B, C,
Figure 4A), makes it difficult to collect accurate biochemical
data for determining the kinetic parameters for KtzO- and KtzP-
catalyzed reactions. Although different strategies have been
developed to circumvent thioester hydrolysis, for example,
amide ligation,26 basic cleavage or thioesterase type II mediated
cleavage,9 a convincing method for direct kinetic parameter
determination of carrier protein ppant-thioesters has not been
established to date. The reported methods involve thioester
cleavage, and thus the amount of substrate hydrolyzed prior to
cleavage is impossible to quantify.
Substrate Specificity. To further characterize KtzO and
KtzP, their substrate specificities were evaluated. Free amino
acids (L-Glu, D-Glu), different PCP-bound amino acids,
especially aspartic acid because of its observed activity in
KtzN adenylation assays,15 CoA-S-Glu as PCP mimic as well
as a noncognate PCP domain from the CDA biosynthetic
cluster10 were considered and the results are summarized in
Table S2.
The method introduced herein, inspired by work of the Bruner
group where phosphopantetheinyl thioester mimics, including
amino-coenzyme A (NH2-CoA), were used to manipulate the
geometry of carrier domains in multidomain NRPS assemblies18
involves replacement of the labile thioester linkage with a
hydrolytically stable amide bond. Amino-coenzyme A was used
as a synthetic precursor to prepare a nonhydrolyzable amino-
The free amino acid were not accepted as substrates by
KtzO or KtzP and therefore a precursor synthesis pathway
can be excluded. KtzO and KtzP were found to be highly
(27) Begley, T. P.; Kinsland, C.; Strauss, E. Vitam. Horm. 2001, 61, 157–
171.
(26) Kopp, F.; Linne, U.; Oberthur, M.; Marahiel, M. A. J. Am. Chem.
Soc. 2008, 130, 2656–2666.
(28) Clarke, K. M.; Mercer, A. C.; La Clair, J. J.; Burkart, M. D. J. Am.
Chem. Soc. 2005, 127, 11234–11235.
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threo- and erythro-Hydroxyglutamate Biosynthesis
A R T I C L E S
Figure 5. QTOF-MS analysis of CoA-HN-glutamic acid loading on A*PCP3 and hydroxylation assays. (A) A*PCP3 incubated with Sfp and CoA-HN-Glu.
The mass shift of 453 Da compared to apo-A*PCP3 (Figure 4A, 22343 Da) is in agreement with the calculated ppant-HN-Glu addition. (B, C) Incubation
of ppant-HN-Glu loaded A*PCP3 with KtzO (B) or KtzP (C) yielded in a mass shift of 16 Da, suggesting hydroxylation.
Figure 6. Proposed reaction mechanism for glutamic acid activation, incorporation and hydroxylation during kutznerides assembly.
specific for hydroxylation of PCP-bound L-glutamic acid;
PCP-S-D-Glu was not a substrate. The D-hGlu moieties as
observed in mature kutznerides are thus likely to be generated
exclusively by the action of the epimerization domain, which
is present in the third module of KtzH.
From this biochemical characterization of KtzO and KtzP,
in addition to the studies of KtzN-mediated Glu transfer onto
the third PCP domain of KtzH, a reaction mechanism for
the origin of the threo- and erythro-D-3-hydroxyglutamic acid
moieties found in mature kutznerides can be proposed (Figure
6). The stand-alone adenylation domain KtzN first activates
L-glutamic acid as amino acyl adenylate, which then can be
transferred site-specifically to the third PCP domain of KtzH.
The specificity of this interaction is mediated by the presence
of the truncated A domain (A*) as well as one hydroxylase,
either KtzO or KtzP. Both A* and a hydroxylase are required
for Glu-AMP transfer. Subsequently, the PCP-bound glutamic
acid is hydroxylated by KtzO to yield to L-threo-3-hydroxy-
glutamic acid or by KtzP to afford L-erythro-3-hydroxy-
glutamic acid. Epimerization of the two PCP-S-L-hGlu to
D-amino acids, catalyzed by the E domain, will also convert
the relative positioning of the hydroxyl group such that that
the KtzO product becomes erythro-D-hGlu and the KtzP
product threo-D-hGlu. Subsequently, condensation and pep-
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A R T I C L E S
Strieker et al.
Table 1. Kinetic Parameters for the Hydroxylation Reactions Catalyzed by KtzO and KtzP and Comparison with Free Amino Acid
Hydroxylating Non-Heme Iron Oxygenases AsnO10 and VioC12
substrate
enzyme
KM [µmol]
kcat [min-1
]
kcat/KM [min-1µmol-1
]
A*PCP3-HN-ppant-Glu
A*PCP3-HN-ppant-Glu
CDA-T9-HN-ppant-Glu
CDA-T9-HN-ppant-Glu
asparagine
KtzO
KtzP
KtzO
KtzP
AsnO
VioC
121.5 ( 28.1
168.9 ( 44.0
123.3 ( 10.9
148.5 ( 21.1
479 ( 6.7
0.34 ( 0.03
0.44 ( 0.05
0.33 ( 0.01
0.42 ( 0.02
298.8 ( 19.2
2611 ( 196
2.8 · 10-3
2.6 · 10-3
2.7 · 10-3
2.8 · 10-3
0.62
arginine
3400 ( 450
0.76
tide elongation occur, and release of the mature peptide is
achieved by cyclization by the thioesterase domain.
KtzO and KtzP are both able to hydroxylate glutamic acid
bound to a noncognate PCP, in this case the ninth PCP domain
of the CDA NRPS assembly line (CDA-T9)10 (Supporting
Information, Figure S4). The kinetic parameters for this reaction
were determined as well for both hydroxylases (Supporting
Information, Figure S5) and it was found that KtzO and KtzP
were similarly efficient in hydroxylating CDA-T9-HN-Glu
compared with A*PCP3-HN-Glu in Vitro (Table 1). These
findings make ktzO and ktzP valuable targets for gene transfer
into NRPS gene clusters, which produce natural products with
a glutamic acid moiety. A strong overproduction of KtzO and
KtzP within these clusters could yield in NRPS products
containing hGlu moieties, which might improve the bioactivity
of these natural products.
which simultaneously restores the ability of an NRPS
assembly line to produce a secondary metabolite.
To determine the kinetic parameters of KtzO and KtzP
catalyzed hydroxylations, the problem of the labile thioester
bond of A*PCP3-S-Glu was circumvented by using synthetic
coenzyme A analogs coupled to glutamic acid where the
thioester was replaced by a hydrolytically stable amide bond.
This method, adapted from previous work,18,21,28 is a simple
and robust way to investigate carrier proteins, which have
substrates bound via a prosthetic ppant arm. The applications
of this methodology are broad and it may be used in
investigations of related peptidyl, fatty acid or acyl carrier
proteins that involve substrate trapping, NMR and X-ray
crystallography studies or whenever a labile thioester is a
problem.
Finally, KtzO and KtzP exclusively hydroxylate PCP-
bound glutamic acid, but do not require the cognate PCP of
module three of KtzH for in Vitro activity. If the NRPS online
modification by noncluster enzymes can be transferred to in
ViVo applications, the insights from this study might assist
in future reengineering approaches of NRPS gene clusters
in order to generate new secondary metabolites.
Conclusions
KtzO and KtzP are nonheme iron(II)/RKG-dependent
hydroxylases. Notably, they both work on PCP tethered
glutamic acid. One could expect from the kutzneride struc-
tures, where the threo and erythro 3-hydroxyglutamic acids
are found in similar amounts, that it would be more
economical for the kutzneride NRPS to have only one enzyme
for Glu hydroxylation. This enzyme could catalyze ꢀ-hy-
droxylation of PCP-S-L-Glu, providing racemic mixture in a
kind of epimerization mechanism. Instead, evaluation of KtzO
and KtzP shows there are two online hydroxylating enzymes
in kutzneride biosynthesis, which stereospecifically catalyze
the formation of PCP-S-L-threo-hGlu (KtzO) and PCP-S-L-
erythro-hGlu (KtzP).
Acknowledgment. We thank Antje Scha¨fer and Volker
Gatterdam for excellent technical assistance, and Dr. Uwe Linne
for help with MS measurements. We also thank Dr. Eric R.
Strieter for providing the expression plasmids containing genes
for the coenzyme A biosynthesis enzymes and for sharing
NH2-CoA synthesis experience. Financial support was provided
by the Deutsche Forschungsgemeinschaft and Fonds der Che-
mischen Industrie (M.S. and M.A.M.) as well as NIH grant
GM43998 (C.T.W.) and a NIH postdoctoral fellowship (E.M.N.).
The transfer of the glutamic acid onto the third PCP domain
of KtzH was investigated. The truncated adenylation domain
of the third module of KtzH is nonfunctional, and the stand-
alone A domain KtzN, which was shown to activate glutamic
acid as aminoacyl adenylate,15 acts as in trans to restore the
NRPS assembly line. Assembly line recovery and transfer
of glutamic acid onto the PCP is only achieved when the A*
domain and at least one of the hydroxylases are present in
Vitro. To the best of our knowledge, this is the first
characterization of an in trans acting adenylation domain,
Supporting Information Available: Sequence alignments,
experimental details of amino CoA synthesis and coupling
of CoA/NH2-CoA to amino acids, SDS-PAGE analysis of
the studied enzymes, hydroxylation assay analysis of KtzO
and KtzP with PCP3-S-Glu, CDA-T9-HN-Glu and kinetic
data. This material is available free of charge via the Internet
at http://pubs.acs.org.
JA9054417
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13530 J. AM. CHEM. SOC. VOL. 131, NO. 37, 2009