Directed Evolution of a Tryptophan 2,3-Dioxygenase for the Diastereoselective Monooxygenation of Tryptophans

Article abstract of DOI:10.1002/anie.201911825

Herein, we report an engineered enzyme that can monooxygenate unprotected tryptophan into the corresponding 3a-hydroxyhexahydropyrrolo[2,3-b]indole-2-carboxylic acid (HPIC) in a single, scalable step with excellent turnover number and diastereoselectivity. Taking advantage of directed evolution, we analyzed the stepwise oxygen-insertion mechanism of tryptophan 2,3-dioxygenases, and transformed tryptophan 2,3-dioxygenase from Xanthomonas campestris into a monooxygenase for oxidative cyclization of tryptophans. It was revealed that residue F51 is vital in determining the product ratio of HPIC to N′-formylkynurenine. Our reactions and purification procedures use no organic solvents, resulting in an eco-friendly method to prepare HPICs for further applications.

Full text of DOI:10.1002/anie.201911825

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Accepted Article
Title: Directed Evolution of a Tryptophan 2,3-Dioxygenase for
Diastereoselective Monooxygenation of Tryptophans
Authors: Yanxin Wei, Chen Lu, Shengsheng Jiang, Yanyan Zhang,
Qiuchun Li, Wen-Ju Bai, and Xiqing Wang
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To be cited as: Angew. Chem. Int. Ed. 10.1002/anie.201911825
Angew. Chem. 10.1002/ange.201911825
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10.1002/anie.201911825
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Directed Evolution of a Tryptophan 2,3-Dioxygenase for
Diastereoselective Monooxygenation of Tryptophans
Yanxin Wei,† Chen Lu,† Shengsheng Jiang, Yanyan Zhang, Qiuchun Li, Wen-Ju Bai,* and Xiqing
Wang*
Abstract: We report herein the first engineered enzyme that can
monooxygenate unprotected tryptophans into their corresponding
HPICs in a single, scalable step with excellent turnover number and
diastereoselectivity. Taking advantage of directed evolution, we
analyzed the stepwise oxygen insertion mechanism of tryptophan
2,3-dioxygenases, and transformed tryptophan 2,3-dioxygenase
from Xanthomonas campestris into a monooxygenase for oxidative
cyclization of tryptophans. It was revealed that residue F51 is vital in
determining the product ratio of HPIC to N’-formylkynurenine. Our
reactions and purification procedures use no organic solvents,
representing an eco-friendly method to prepare HPICs for further
applications.
employ electrophiles like N-phenylselenophthalimide (N-PSP) or
N-bromosuccinimide (NBS) to activate the indole ring; the
activating heteroatom is then replaced with oxygen using
oxidative deselenation or solvolysis (Figure 1, 1a).[11] Unlike the
aforementioned stepwise methods, DMDO oxidation can convert
Trp derivatives to HPICs directly, but the diastereoselectivity
depends on the carboxylic acid and amine protecting groups
(Figure 1, 1b).[12] Without bulky blocking groups like
triphenylmethyl (Tr), 1:1 mixtures of HPIC diastereomers are
obtained. Recently, radical-triggered cyclizations have also been
used for stepwise preparation of HPICs from Trp derivatives.[13]
Despite these notable advances, the step-economy[14] of existing
HPIC preparations is negatively impacted by the need for
multiple protecting group manipulations. Efforts to address this
challenge have been tried via photosensitized oxygenation of
unprotected Trp in a two-step/one-pot sequence (Figure 1, 2),[15]
involving tricyclic peroxide formation and hydroperoxide
cleavage. While straightforward, this method suffers from poor
diastereoselectivity. In addition, the labile peroxide intermediate
may rapidly decompose to N’-formylkynurenine (NFK).
The tricyclic 3a-hydroxyhexahydropyrrolo[2,3-b]indole-2-
carboxylic acid (HPIC) scaffold is a common motif in alkaloids
and peptides with marvelous structural diversity and biological
2]
activity (Figure 1, left).[1,
For example, kapakahine C is
cytotoxic to P388 murine leukemia cells,[3] and most family
members of the NW-G natural products,[4] such as NW-G01,
exhibit potent and selective antibacterial activity against Gram-
positive bacteria. Besides their antibacterial properties,
himastatin[5] and chloptosin[6] also display anticancer activity.
Therefore, HPIC-containing natural products are popular targets
for synthetic and pharmacological investigations. Moreover,
HPICs may be useful intermediates for synthesis of
pyrrolobenzoxazine alkaloids, such as paeciloxazine, CJ-12662,
and CJ-12663,[7] via oxidation.[8] Additionally, monocyclic
peptides bearing an HPIC skeleton can be transformed into
bicyclic peptides with Savige-Fontana tryptathionylation for
potency improvement.[9] Thus, in addition to being present in a
variety of bioactive molecules, HPICs are also important
synthetic intermediates.
An ideal but unrealized strategy is the direct
monooxygenation
of
Trps.
Besides
reactivity
and
chemoselectivity, controlling the diastereoselectivity of such a
process represents a key challenge. Since enzymatic oxidation
of an indole moiety is believed to be fundamental in biosynthesis
of HPIC-containing natural products, we believed that an
appropriate enzyme would overcome these obstacles. Recent
studies on the biosynthesis of some HPIC-containing natural
products reveal that certain monooxygenases can promote
oxidative cyclizations on the indole cores of complicated
diketopiperazines and cyclodepsipeptides,[16] lending credence
to our initial proposal. However, due to the substrate-specific
nature of these enzymes, applying them to the
monooxygenation of simple Trp is problematic and not
surprisingly, oxygenases that are suitable for this transformation
have not been reported. As a result, we examined several Trp
oxygenases and were intrigued by two heme-containing Trp
dioxygenase enzymes: tryptophan 2,3-dioxygenase (TDO) and
indoleamine 2,3-dioxygenase (IDO). Mechanistic studies on
these enzymes reveal that Trp is converted to NFK via stepwise
oxygen insertions (Figure 2).[17] The first oxygen insertion from
Construction of HPIC motifs from tryptophan (Trp)
derivatives has been enthusiastically explored by synthetic
chemists (Figure 1, right). Pioneering work by Danishefsky
enabled diastereoselective preparation of HPICs from Trp in 3-4
steps, which represents a powerful strategy for synthesizing
complex natural pruducts.[10] These cation-triggered cyclizations
[a]
Y. Wei, C. Lu, S. Jiang, Prof. Dr. Q. Li, Prof. Dr. X. Wang
College of Bioscience and Biotechnology, Yangzhou University
Yangzhou, Jiangsu 225009, China
E-mail: xiqingwang@yzu.edu.cn
the heme iron-bound dioxygen generates
a 2,3-epoxide
intermediate, which is opened by the incipient iron-oxo species.
This intermediate then undergoes oxidative cleavage of the C2-
C3 bond to yield NFK (Path A). We envisioned that these two
oxidations could be decoupled by impairing the coordination of
the 2,3-epoxide to the metal center, possibly by using directed
evolution.[18] Interrupting the second oxygen insertion would
enable HPIC production by allowing for intramolecular, nitrogen-
assisted epoxide opening followed by cyclization onto the
resulting imine (Path B). Herein, we report our findings.
[b]
[c]
Dr. Y. Zhang
Testing Center, Yangzhou University
Yangzhou, Jiangsu 225009, China
Dr. W.-J. Bai
Department of Chemistry, Stanford University
Stanford, California 94305, USA
E-mail: bai.stanford@gmail.com
Present address: Amgen Inc., 1 Amgen Center Drive, Thousand
Oaks, CA 91320.
The use of wild-type TDOs and IDOs with Trp afforded NFK
as the major product and delivered only trace amounts of
[†]
These authors contributed equally to this work.
HPIC.[17h]
Among
examined
IDOs
and
TDOs,
Supporting information for this article is given via a link at the end of
the document.
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10.1002/anie.201911825
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Figure 1. Left: Examples of HPIC-containing natural products & CJ-12662. Right: Synthetic approaches towards HPIC skeletons.
Xanthomonas campestris TDO (xcTDO) was chosen for further
evaluation due to its highest activity (Table S1), no substrate
inhibition,[19] and extensive mechanistic studies. Going forward,
the amino acid residues surrounding the substrate and heme
binding pocket of xcTDO were subjected to three rounds of site-
saturation mutagenesis (Figure S1). Enzymatic reactions were
carried out with cell-free lysates of Escherichia coli expressing
xcTDO variants, then high-performance liquid chromatography
(HPLC) was used to screen the libraries for HPIC yield.
Authentic cis and trans HPIC diastereomers prepared via
photooxygenation of Trp were used as the standards to assign
and quantify the HPIC peak in the HPLC traces at 295 nm.[15]
The first-round of site-saturation mutagenesis targeted
xcTDO residues located in the αB (F51 and H55) and αD (Y113,
R117, and L120) helices and the amino-terminal residues from
the other monomer (Y24, Y27, and L28). They are all situated
on one side of the active site and within 7 Å from the substrate
(Figure S1). During screening, most lysates in the F51X
library exhibited significantly more HPIC production than that of
wild-type xcTDO, whereas little or no HPIC was detected with
lysates from the other libraries. Moreover, only cis-HPIC was
observed, demonstrating superb diastereoselectivity of this
enzymatic reaction. These results indicated that the replacement
of F51 could change the reaction pathway to favor HPIC
formation. Accordingly, we focused on the F51 site and purified
a complete set of mutant xcTDO proteins with amino acid
substitutions at this residue. At optimized pH 6.0 (Table S2),
HPIC yields and total conversions of all xcTDO-F51X variants
were evaluated (see Figures S3-S5) and summarized in Figure
3. Of the mutations that retained good activities (i.e. total
conversion> 50%), xcTDO variants with smaller amino acid side
chains (e.g. A, L, V, and I) at residue 51 exhibited higher HPIC
productivity than those with sterically hindered side chains (e.g.
F, W, and H). This suggests that the van der Waals forces
imposed by bulky amino acid side chains at residue 51
preferentially orient the tryptophan-2,3-epoxide intermediate for
the second oxygen insertion (Figure 4). The high conservation in
all wild-type TDOs and IDOs and the same spatial conformation
in the structures of xcTDO, human TDO, and human IDO
(Figure S2) implies that F51 is important in selecting for NFK
production by inhibiting the intramolecular cyclization pathway
involved in HPIC formation. These findings further support the
stepwise oxygen insertion mechanism proposed for tryptophan
dioxygenases and validate our strategy of tuning a dioxygenase
for HPIC production.
The F51X mutant plasmids that expressed xcTDO proteins
with TONs > 250 were used as templates to further improve
HPIC production. Residues 121-125, 127, and 253-255 in the
αD-αE and αJ-αK loops, all of which reside on the other side of
the active site and within 7 Å from the substrate (Figure S1),
were targeted in the second-round of site-saturation
mutagenesis. Again, only cis-HPIC was observed. xcTDO-
F51M/Q127Y exhibited increased overall reactivity (>99%
conversion), excellent TON for HPIC formation (3,312), and
moderately enhanced HPIC/NFK ratio (2:1) (Table 1, entry 1;
Figure S6).
Figure 2. The stepwise oxygen insertion mechanism of xcTDO. Both radical
and electrophilic addition mechanisms have been proposed for the first oxygen
insertion (Step 1).
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10.1002/anie.201911825
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Figure 3. The effects of amino acid substitution at residue 51 on HPIC production and overall activity of xcTDO. [xcTDO]=1 μM; [Trp]=5 mM; total
conversion=([HPIC]+[NFK])/(5 mM). Note that F51P mutant was not tested due to poor stability.
.
Q127, which is also a highly conserved residue in the tryptophan
dioxygenase family (Figure S2A), resides on the opposite side of
F51 in all of the xcTDO, human TDO, and human IDO structures
(Figures 4 and S2B). Replacing it with a Tyr residue might
indirectly affect the active site via interactions with the amino-
terminus from the other monomer and the αD-αE loop. Even
though the F51M substitution was not prominent (Figure 3), the
xcTDO-F51M/Q127Y mutant showed higher HPIC productivity
The third-round screening employed xcTDO-F51M/Q127Y
and xcTDO-F51S/T254S as templates and targeted residues
involved with the heme-iron binding (W102, L105, F126, Y131,
R132, W236, H240, V244, I248, G256, F262, and L263) (Figure
S1). No improvement was observed, suggesting that residues
coordinated with the heme-iron are trivial for HPIC/NFK product
distribution.
Table 1. Improved product distribution during the 2nd round screening.
than
xcTDO-F51I/Q127Y
(TONHPIC=457)
and
xcTDO-
HPIC
(TON)[a]
NFK
(TON)[a]
HPIC/NFK
ratio
Entry
xcTDO variant
F51L/Q127Y (TONHPIC=633) (Table S3), suggesting that the
F51M and Q127Y substitutions imposed a cooperative effect on
the enzyme activity (Figure 4). Furthermore, two other mutants,
xcTDO-F51S/T254S and xcTDO-F51Q/G255S, were found to
enhance the HPIC/NFK ratio to 6.2:1 and 3.1:1, respectively, at
the expense of catalytic activity (Table 1, entries 2 and 3).
Molecular dynamic simulations suggest that T254 modulates the
H-bond interaction between the NH3+ group and epoxide oxygen
of the 2,3-epoxide intermediate.[20] Thus, a subtle change from
Thr to a structurally similar Ser residue or a perturbation from
the mutation in the neighboring G255 might further destabilize
the transition state for the second oxygen insertion, thereby
increasing the likelihood of the desired intramolecular cyclization
(Figure 2). Further improvement efforts by combining the
mutations of F51M, Q127Y, and T254S resulted in a decrease in
the TON for HPIC production (Table 1, entry 4).
1
2
3
4
F51M/Q127Y
F51S/T254S
3312
468
809
340
1655
76
2.0:1
6.2:1
3.1:1
2.6:1
F51Q/G255S
264
133
F51M/Q127Y/T254S
[a]Determined by HPLC.
To demonstrate the scalability and practicality of this
enzymatic reaction, the xcTDO-F51M/Q127Y mutant was
selected for scale-up because of its impressive TON. At a
catalyst loading of 0.03 mol% and optimal pH 6.0 (Table S4), 1
mmol of Trp was smoothly converted to cis-HPIC[15] in 60% yield
and perfect diastereoselectivity, making this transformation
synthetically useful (eq. 1).
Given that some HPIC-containing natural products bear
chlorine substituents on the aromatic ring (e.g. NW-G01 and
chloptosin in Figure 1), monooxygenation of unprotected 5-Cl-
and 6-Cl-tryptophans was also investigated. Without further
engineering, direct application of xcTDO-F51M/Q127Y delivered
Figure 4. Schematic of the active site of xcTDO (PDB: 2NW8). The fragment
colored in pink comes from the other monomer in the crystal structure of
xcTDO.
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a process that has not been
synthetically realized before. It was found that residue F51 was
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Acknowledgements
We thank Dr. Christopher A. Kalnmals (Stanford University) for
critical reading of the manuscript. X. W. acknowledges the
financial support from National Natural Science Foundation of
China (Grant 31670792) and Talent Support Program of
Yangzhou University.
Keywords: biocatalysis • monooxygenation • protein
engineering • pyrroloindole • tryptophan 2,3-dioxygenase
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Entry for the Table of Contents (Please choose one layout)
Layout 1:
COMMUNICATION
Taking advantage of directed
evolution, we interrupted the dual
oxygen insertion process and
transformed the xcTDO
Yanxin Wei, Chen Lu, Shengsheng
Jiang, Yanyan Zhang, Qiuchun Li,
Wen-Ju Bai,* and Xiqing Wang*
tryptophan dioxygenase into a
monooxygenase. Our engineered
xcTDO exhibits excellent TON
and high diastereoselectivity, and
enables the scalable synthesis of
HPICs from Trps in one step.
Page No. – Page No.
Directed Evolution of a
Tryptophan 2,3-Dioxygenase for
Diastereoselective
Monooxygenation of
Tryptophans
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