Biosynthesis of l-4-Chlorokynurenine, an Antidepressant Prodrug and a Non-Proteinogenic Amino Acid Found in Lipopeptide Antibiotics

Article abstract of DOI:10.1002/anie.201901571

l-4-Chlorokynurenine (l-4-Cl-Kyn) is a neuropharmaceutical drug candidate that is in development for the treatment of major depressive disorder. Recently, this amino acid was naturally found as a residue in the lipopeptide antibiotic taromycin. Herein, we report the unprecedented conversion of l-tryptophan into l-4-Cl-Kyn catalyzed by four enzymes in the taromycin biosynthetic pathway from the marine bacterium Saccharomonospora sp. CNQ-490. We used genetic, biochemical, structural, and analytical techniques to establish l-4-Cl-Kyn biosynthesis, which is initiated by the flavin-dependent tryptophan chlorinase Tar14 and its flavin reductase partner Tar15. This work revealed the first tryptophan 2,3-dioxygenase (Tar13) and kynurenine formamidase (Tar16) enzymes that are selective for chlorinated substrates. The substrate scope of Tar13, Tar14, and Tar16 was examined and revealed intriguing promiscuity, thereby opening doors for the targeted engineering of these enzymes as useful biocatalysts.

Full text of DOI:10.1002/anie.201901571

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Accepted Article
Title: Biosynthesis of L-4-Chlorokynurenine, a Lipopeptide Antibiotic
Non-Proteinogenic Amino Acid and Antidepressant Prodrug
Authors: Bradley S. Moore, Hanna Luhavaya, Renata Sigrist, Jonathan
R. Chekan, and Shaun M. K. McKinnie
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201901571
Angew. Chem. 10.1002/ange.201901571
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Biosynthetic enzymes
Biosynthesis of L-4-Chlorokynurenine, a Lipopeptide Antibiotic
Non-Proteinogenic Amino Acid and Antidepressant Prodrug
Hanna Luhavaya,[a] Renata Sigrist,[a,b] Jonathan R. Chekan,[a] Shaun M. K. McKinnie,[a] and Bradley S.
Moore*[a,c]
Abstract: L-4-Chlorokynurenine (L-4-Cl-Kyn) is a neuropharma-
ceutical drug candidate in development for the treatment of major
depressive disorder. Recently, this amino acid was naturally found as
a residue in the lipopeptide antibiotic taromycin. Herein we report the
unprecedented conversion of L-tryptophan to L-4-Cl-Kyn catalyzed by
four enzymes in the taromycin biosynthetic pathway from the marine
bacterium Saccharomonospora sp. CNQ-490. We used genetic,
biochemical, structural, and analytical techniques to establish L-4-Cl-
Kyn biosynthesis, which is initiated by the Tar14 flavin-dependent
tryptophan chlorinase and its flavin reductase partner Tar15. This
work revealed the first tryptophan 2,3-dioxygenase (Tar13) and
kynurenine formamidase (Tar16) enzymes that are selective for
chlorinated substrates. The substrate scope of Tar13, 14, and 16 was
examined revealing intriguing promiscuity, thereby opening doors for
the targeted engineering of these enzymes as useful biocatalysts.
catabolic pathway in eukaryotes, which leads to vital biochemicals
such as the neurotransmitter serotonin and the cofactor NADH.[8]
The initial and rate-limiting step of the pathway is catalyzed by the
Fe2+/heme-dependent
enzyme
tryptophan-2,3-dioxygenase
(TDO) leading to formation of N-formyl-L-Kyn (6), which is
hydrolyzed by kynurenine formamidase (KF) to give L-Kyn.
Considering the functional importance of the products of this
pathway, TDOs and KFs show specificity for L-Trp and N-formyl-
L-Kyn, respectively. Homologous enzymes have been identified
in some prokaryotes,[9] however, they are not essential for
bacterial survival, as bacteria catabolize Trp mainly through non-
oxidative degradation.[10] Recently, BGCs with co-clustered TDO-
encoding genes have been discovered, but in vitro studies of
these enzymes remain limited. Cluster-specific TDOs have been
shown to have broader substrate specificities (e.g., the
actinomycin TDO AcmG accepts L-Trp, D/L--CH3-Trp, D/L-5-
CH3-Trp, and D/L-5-F-Trp).[11-14] Only one representative of a
BGC-associated KF, from actinomycin biosynthesis, has been
tested in vitro.[15] This enzyme predominantly worked on N-formyl-
L-Kyn, but was also able to deformylate at reduced rates N‘,N--
diformyl-L-Kyn and O-formamino acetophenone. Bioinformatic
analysis of the taromycin BGC (tar) identified an unprecedented
quartet of enzymes – Tar13, 14, 15, and 16 – that show close
homology to TDO, flavin-dependent halogenase (FDH), flavin
reductase, and KF, respectively, and are encoded by adjacent
genes (Figure 1C).
Non-proteinogenic amino acid L-4-chlorokynurenine (L-4-Cl-Kyn,
1) is a next-generation, fast-acting oral prodrug for the treatment
of major depressive disorder.[1,2] Additional studies report that this
drug candidate is effective in animal models for the treatment of
neuropathic pain, epilepsy, and Huntington’s disease.[2] After
active transport across the blood-brain barrier, 1 is enzymatically
converted into the active agent 7-chlorokynurenic acid, which is a
highly selective competitive antagonist of the N-methyl-D-aspartic
acid (NMDA) receptor.[3] To date, only synthetic routes to 1 have
been described.[3,4]
Biologically, 1 was recently identified as an amino acid
building block in the lipopeptide antibiotics taromycin A (2) and B
(3) (Figure 1A)[5,6] and in the putative glycopeptide antibiotic
complex INA-5812.[7] With the concurrent discovery of the
taromycin biosynthetic gene cluster (BGC) from the marine
actinomycete Saccharomonospora sp. CNQ-490, we sought to
establish the biosynthetic logic for the bacterial synthesis of L-4-
Cl-Kyn. Herein we report the concise three-step L-4-Cl-Kyn
pathway originating from L-tryptophan (L-Trp, 4) and present it as
an orthogonal approach to produce this promising drug candidate.
Enzymatic conversion of L-Trp to L-kynurenine (L-Kyn, 5) is
part of the kynurenine pathway (Figure 1B), the major Trp
Considering that taromycin contains a second chlorinated
amino acid residue, L-6-Cl-Trp (7), and that its BGC encodes only
one halogenase, we hypothesized that the chlorination reaction
takes place directly on L-Trp versus on a carrier protein bound
substrate[16] or post-assembly on a released peptide substrate.[17]
Phylogenetic analysis of Tar14 showed that it clades with FDHs
acting on free L-Trp (Figure S1). To validate this, we performed
an in-frame gene deletion of tar14. The availability of the 62.4 kb
tar BGC cloned in pCAP01-tarM1[6] allowed rapid genetic
interrogations of the pathway. The tar cluster showed instability
upon use of recombineering techniques, therefore, we chose an
in vitro CRISPR/Cas9 approach (Figure S9).[18] The deletion
construct, pCAP01-tarM1∆tar14, was integrated into the genome
of Streptomyces coelicolor M1146 for heterologous expression.[19]
Liquid chromatography mass spectrometry (LCMS) analysis of
the mutant culture extracts revealed abolished production of 2 and
3 (Figure 2A). Chemical complementation of the mutant strain
with 6-Cl-Trp restored taromycin production. These results
validated that free L-Trp is the substrate for Tar14, and strongly
suggested that L-4-Cl-Kyn is formed by conversion of L-6-Cl-Trp
rather than direct halogenation of L-Kyn.
[a]
Dr. H. Luhavaya, R. Sigrist, Dr. J. R. Chekan, Dr. S. M. K. McKinnie,
Prof. Dr. B. S. Moore
Center for Marine Biotechnology and Biomedicine
Scripps Institution of Oceanography
University of California, San Diego, La Jolla, CA 92093 (USA)
E-mail: bsmoore@ucsd.edu
[b]
[c]
R. Sigrist
Department of Organic Chemistry, University of Campinas
UNICAMP, Cidade Universitária Zeferino Vaz s/n
P.O. Box 6154, 13083-970, Campinas-SP (Brazil)
Prof. Dr. B. S. Moore
Next, we proceeded with the in vitro reconstitution of L-Trp
to L-4-Cl-Kyn. Genes encoding Tar13, 15, and 16 were
individually cloned and expressed in Escherichia coli (Figure S12),
while Tar14 was expressed in S. coelicolor CH999 (Figure S3).[20]
However, no yellow color associated with flavin binding was
Skaggs School of Pharmacy and Pharmaceutical Sciences
University of California, San Diego, La Jolla, CA 92093 (USA)
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201901571
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observed for Tar14, suggestive of either enzyme misfolding or a
weak binding affinity.[17,21] Upon incubation of Tar14 with flavin
and a flavin reductase system (Figure 2B, Supplementary
Information),[22] we observed conversion of L-Trp to L-6-Cl-Trp as
confirmed by high resolution mass spectrometry (HRMS) and
NMR characterization of the purified product (Supplementary
Information). Kinetic parameters of Tar14 with L-Trp were
determined: kcat=0.4 min-1, KM=12 M, kcat/KM= 0.03 min-1M-1,
and are consistent with other characterized FDHs (Figures S6-S8,
Table S6).[23]
We next probed the substrate specificity of Tar14 towards
different halide partners (Figure 2B, Table 1). Tar14 readily
brominated L-Trp, however, it exhibited an erosion of
regiospecificity, producing a 1:5 mixture of L-5-Br-Trp (8) and L-6-
Br-Trp (9) as determined by HRMS and retention time comparison
with commercial standards (Figure 2B, Figure S11). No iodinated
tryptophan products were detected when incubated with iodide.
To further investigate its biocatalytic halogenation potential, Tar14
was interrogated with a library of Trp derivatives (Table 1, Figures
S16-S21, Table S7). Tar14 was capable of generating a wide
variety of dihalogenated Trp species by accepting
monohalogenated substrates. Previously, only the kutzneride
FDH KtzR was shown to have L-7-Cl-Trp as its preferred
substrate yielding L-6,7-diCl-Trp,[24] while FDH KrmI from a
sponge metagenome was able to chlorinate L-7-F-Trp,[25] albeit at
trace level.
We solved the X-ray crystal structure of Tar14 with bound
flavin cofactor to a resolution of 1.74 Å (PDB: 6NSD) using the
structure of C6 Trp halogenase Th-Hal (PDB: 5LV9, 73%
identity)[23] as a molecular replacement model (Supplementary
Information, Table S5). Two protomers were observed in the
asymmetric unit, consistent with the homodimeric form of Tar14
in solution (Figure S3). Each monomer adopts the classical FDH
fold comprised of pyramid-like and box-shaped domains (Figure
3A).[21] Catalytic lysine and glutamate residues identified for all
characterized FDHs, remain conserved in Tar14. Bioinformatic
analysis of Tar14 and other characterized Trp FDHs clearly shows
existance of two sub-types of C6 halogenases: ThaI[26] and
BorH[27] are closely related to C7 Trp halogenases, while Tar14,
together with SttH,[28] Th-Hal,[23]and KtzR,[24] forms a separate
clade on the phylogenetic tree, and shows more sequence and
structural similarity to C5 Trp halogenase PyrH[29] (Figures S1, S2,
S4, Table S4). Superimposition of Tar14 with related Th-Hal and
SttH structures shows conservation of proposed active site
residues (Figure 3B). However, structural superimposition with
the phylogenetically distinct ThaI reveals that active site of these
two enzymes are formed by different protein regions (Figure 3):
residues of L-Trp interacting region of ThaI do not have
equivalents in Tar14, while Tar14 residues of regions  and ,
positioned in the vicinity of the proposed active site, are missing
in ThaI. In light of the observed substrate flexibility of Tar14, we
looked for distinct structural and sequence features of Tar14 in
comparison to SttH, Th-Hal, and KtzR. Sequence alignment
superimposed onto the Tar14 structure showed that the putative
substrate binding region remains conserved among these
enzymes, and amino acid variations in Tar14 are distantly located
from the putative active site (Figure S5).
a new early eluting compound (Figure 2C). HRMS and NMR data
confirmed that the new peak corresponded to N-formyl-L-4-Cl-
Kyn (10) (Supplementary Information). Kinetic parameters of
Tar13 with L-6-Cl-Trp were determined: kcat=0.03 s-1, KM=112.3
M, kcat/KM= 0.27 min-1mM-1 (Figure S14). Upon mildly acidic
purification conditions, 10 showed partial deformylation to L-4-Cl-
Kyn (1). Therefore, to test the activity of KF Tar16, we performed
a coupled Tar13/Tar16 assay in which the substrate for Tar16 was
generated in situ (Supplementary Information). Addition of Tar16
to the reaction resulted in full conversion of 7 to 1, thus confirming
the role of Tar16 in the biosynthesis of 1. We additionally explored
the possibility of directly converting L-Trp to L-4-Cl-Kyn in a one-
pot reaction. The expected product was detected, however, the
production of L-4-Cl-Kyn remained below 1% largely due to the
incompability of optimal assay conditions for each individual
enzyme in vitro (Figure 2C, Supplementary Information).
When we tested L-Trp as a substrate for Tar13, we only
measured trace activity. This stark difference in the substrate
preference of Tar13 was illuminated in a comparative in vitro
substrate consumption assay of L-Trp versus L-6-Cl-Trp (Figure
S15). Tar13’s preference for the halogenated substrate was
clearly evident by the slow rate of L-Trp consumption and failure
to achieve its complete conversion. Bioinformatic analysis of the
draft genome sequence of S. sp. CNQ-490 revealed a second pair
of TDO/KF enzymes with less than 30% identity to Tar13/Tar16
and that are likely associated with L-Trp metabolism (Figures S32,
S33). We hypothesize that Tar13 and Tar16 diverged from
catabolic TDOs and KFs, respectively, and coevolved to serve a
specialized role in L-4-Cl-Kyn biosynthesis after gene duplication.
Collectively, our results indicate that Tar13 and Tar16 are the first
representatives of their enzyme families to prefer chlorinated
substrates.
We further probed Tar13 and Tar16 activity with a variety of
substrate analogues (Table 1) and identified a preference of these
enzymes towards C6 halogenated Trp derivatives (Figures S22-
S25, Table S7). Phylogenetic analysis revealed that Tar13 and
Tar16 are evolutionarily distinct from catabolic TDO and KFs,
respectively (Figures S30, S31). Interestingly, they also branch
out from other BGC-associated enzymes and form a separate
clade with uncharacterized homologs. Inspection of gene
neighborhoods of these homologs revealed nonribosomal peptide
synthetase (NRPS) BGCs that also contain putative Trp FDH, KF,
and adenylation (A) domain with predicted specificity towards Kyn
(Table S9). This observation suggests halogenated kynurenine
residues may be more widespread than previously thought in
peptide natural products, and that Tar13/Tar16 sequences may
be convenient “search hooks” for their discovery.
The in vitro substrate flexibilities of Tar13 and Tar16
encouraged us to probe whether analogous promiscuity is
retained in vivo. The incorporation of Trp and Kyn derivatives
additionally relies on the relaxed specificity of the corresponding
NRPS A domains (A1 and A13). We fed a variety of Trp derivatives
(Table 1) to S. coelicolor M1146-tarM1∆tar14. Analysis of LC-
HRMSn data showed that all analogues, apart from 12, were
incorporated into residue-1 by A1 (Table 1, Figures S26-S29,
Table S8). However, we observed limited incorporation of non-
native Kyn derivatives at residue-13 by A13. This mirrors the in
vitro activities of Tar13/Tar16, suggesting that the inability to
generate corresponding Kyn derivatives in vivo likely precludes
We next examined the activity of TDO Tar13. Incubation of
Tar13 with L-6-Cl-Trp (7) resulted in its complete conversion into
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In summary, we genetically and biochemically validated the
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Acknowledgements
This work was supported by NIH grant R01-GM085770 to B.S.M.,
the Simons Foundation Fellowship of the Life Science Research
Foundation to J.R.C., an NSERC postdoctoral fellowship to
S.M.K.M., São Paulo Research Foundation FAPESP Proc.
2016/25735-1 to R.S. We thank Dr. P. Jensen and Dr. W. Fenical
for S. sp. CNQ-490, Dr. M. Bibb for S. coelicolor M1146, Dr. S.
Nair for PtdH and SsuE expression plasmids, Dr. P. Leadlay for
pCJW93 and S. coelicolor CH999. We thank Dr. J. Noel and Dr.
G. Louie for providing beamtime and assistance with beamline
operation.
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Conflict of Interest
[30]
[31]
[32]
The authors declare no conflict of interest.
Keywords: biocatalysis • biosynthesis • chlorokynurenine •
halogenation • natural product
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Figure 1. A. Chemical structures of taromycins A (2) and B (3). B. Proposed enzymatic route from L-Trp
(4) to L-4-Cl-Kyn (1). C. Gene organization of the taromycin BGC; tar13,14,15,16 gene numbers are
labeled red.
Figure 2. A. LCMS extracted ion chromatograms (EIC) showing recovery of taromycin production by
S. coelicolor M1146-tarM1∆tar14 mutant fed with 6-Cl-Trp; m/z [M+2H]2+ 820.8 corresponds to
taromycin A (2), m/z [M+2H]2+ 827.8 – taromycin B (3). B. Total ion chromatograms (TIC) of Tar14-
catalyzed halogenation of L-Trp. C. TIC and EIC confirming activity of Tar13 and Tar16, and one-pot
conversion of L-Trp to L-4-Cl-Kyn. * not relevant to the reaction product; m/z [M+H]+ 271.0 corresponds
to N-formyl-L-4-Cl-Kyn, 243.0 – L-4-Cl-Kyn.
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Figure 3. A. Cartoon representation of the Tar14 structure. Monomer on the left is colored by domains:
green – box-shaped domain, pink – pyramid domain; monomer on the right is colored by secondary
structure: -helices – blue, -sheets – red, flexible loops – sand. Flavin molecules and catalytic K88 and
E373 residues are labeled. B. Superimposition of putative active site residues of Tar14 (blue), Th-Hal
(green), and SttH (salmon). C. Protein sequence alignment of Tar14 and selected Trp FDHs. Residues that
form the active site in ThaI are highlighted by pink lines, sequences surrounding the putative active site in
Tar14 – blue lines. D. Superimposition of Tar14 (blue) and ThaI (pink) structures illustrating difference in
arrangement of the putative active site. Greek letters correspond to the respective sequence regions from
the alignment above.
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Table 1. Evaluation of L-4-Cl-Kyn biosynthesis enzymes against a panel of non-native substrate
analogues. nt – not tested; «–» – no activity; # – substrates generated in situ by Tar13; * – judged by
comparison to taromycin yields by S. coelicolor M1146-tarM1.
Enzyme assays, % conversion
Feeding, % incorporation*
Substrate
Tar14, Cl- Tar14, Br- Tar13 Tar16# A1 domain
A1 +A13 domain
1, L-4-Cl-Kyn
4, L-Trp
5, L-Kyn
7, L-6-Cl-Trp
8, L-5-Br-Trp
9, L-6-Br-Trp
11, D-Trp
12, L-4-Br-Trp
13, L-7-Br- Trp
14, D/L-5-Cl-Trp
15, D/L-4-F-Trp
16, D/L-6-F-Trp
17, D/L-4-CH3-Trp
18, D/L-5-CH3-Trp
19, L-5-CH3O-Trp
20, L-5-OH-Trp
21, D/L-5-NO2-Trp
22, serotonine
23, L-tyrosine
<1
100
30
–
<1
–
15
<1
100
<1
10
25
100
50
<1
5
5
100
30
<5
–
<1
20
<5
100
<1
15
25
75
25
<5
<1
–
nt
15
nt
100
<1
100
<1
–
–
<1
–
5
–
nt
100
nt
100
100
100
40
nt
nt
100
nt
100
nt
nt
–
nt
–
50
–
nt
<5
nt
100
–
20
–
–
–
–
15
90
–
–
–
<5
30
15
<5
40
35
15
10
60
<1
<1
–
–
–
70
100
–
–
–
–
–
–
–
nt
–
–
nt
nt
nt
24, L-phenylalanine
–
–
nt
This article is protected by copyright. All rights reserved.

10.1002/anie.201901571
Angewandte Chemie International Edition
COMMUNICATION
Figure 4. LC/ESI-Q-TOF MS2 analysis of mono- (2-F, red) and di-fluorinated (2-F2, blue) analogues of
taromycin A (2) and their comparison to 2 (black).
COMMUNICATION
Two paths diverge: The unique
Hanna Luhavaya, Renata Sigrist,
Jonathan R. Chekan, Shaun M. K.
McKinnie, and Bradley S. Moore*
enzymatic route to neuroactive L-4-
chlorokynurenine has been revealed
using a blend of in vitro experiments,
X-ray crystallography, and
Page No. – Page No.
CRISPR/Cas9-mediated gene
deletion. Tar13 and Tar16 are the first
tryptophan 2,3-dioxygenase and
kynurenine formamidase homologs,
respectively, to preferentially accept
chlorinated substrates. Enzymes
characterized here are promising
candidates for chemoenzymatic
synthesis.
Biosynthesis of L-4-
Chlorokynurenine, a Lipopeptide
Antibiotic Non-Proteinogenic Amino
Acid and Antidepressant Prodrug
This article is protected by copyright. All rights reserved.