Stereospecific biosynthesis of β-methyltryptophan from L-tryptophan features a stereochemical switch

Article abstract of DOI:10.1002/anie.201306255

Make the switch: The three-enzyme cassette MarG/H/I is responsible for stereospecific biosynthesis of β-methyltryptophan from L-tryptophan (1). MarG/I convert 1 into (2S,3R)-β-methyltryptophan, while MarG/I combined with MarH convert 1 into (2S,3S)-β-methyltryptophan. MarH serves as a stereochemical switch by catalyzing the stereoinversion of the β-stereocenter. Copyright

Full text of DOI:10.1002/anie.201306255

Angewandte
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DOI: 10.1002/anie.201306255
Amino Acid Synthesis
Stereospecific Biosynthesis of b-Methyltryptophan from l-Tryptophan
Features a Stereochemical Switch**
Yi Zou, Qi Fang, Haixing Yin, Zutao Liang, Dekun Kong, Linquan Bai, Zixin Deng, and
Shuangjun Lin*
Nonproteinogenic amino acids, especially b-methyl
amino acids, are important building blocks in the
assembly of bioactive natural products.[1] For exam-
ple, b-methyl aspartic acid is a biosynthetic precursor
of friulimicin,[2] nikkomycin,[3] streptolydigin,[4] and
vicenistatin,[5] (2S,3S)-b-methylphenylalanine is
a precursor of mannopeptimycin,[6] and the lipopep-
tide antibiotics daptomycin, calcium-dependent anti-
biotic (CDA), and A54145 contain a (2S,3R)-b-
methyl glutamate unit.[7] Intriguingly, two different
diastereoisomers of b-methyl tryptophan (b-MeTrp)
have been found to contribute to nonribosomally
synthesized peptides and tryptophan-derived alka-
loids: maremycin A and
B
(MARs)[8] and
FR900452[9] contain the (2S,3S)-b-MeTrp (5) unit,
while chaetoglobisin K,[10] indolmycin,[11] and telo-
mycin[12] contain the (2S,3R)-b-MeTrp (4) moiety
(Figure 1). This moiety is also proposed to be the
biosynthetic precursor of both streptonigrin[13] and
lavendamycin.[14] Stereospecific alkyl functionaliza-
tion of amino acids at the relatively unreactive b-
position is a synthetic challenge which has attracted
considerable interest in understanding the enzymol-
ogy of stereospecific biosynthesis of b-MeTrp from l-
tryptophan.
To investigate the biosynthesis of 5 in MARs, the
biosynthetic gene cluster of MARs has been cloned and
identified by genome sequencing of Streptomyces sp.
B9173,[8a] the MARs-producing strain, which consists of
about a 21 kb DNA sequence and 17 open reading frames
(see Table S3 in the Supporting Information). Within this
Figure 1. Chemical structures of maremycins, FR900452, indolmycin, chaetoglo-
bosin K, streptonigrin, lavendamycin, and compounds 1–6.
cluster,
a three-gene cassette, marG-marH-marI, was
revealed to encode a pyridoxal 5’-phosphate (PLP)-depen-
dent aminotransferase, a cupin-fold protein, and an S-
adenosylmethionine (SAM)-dependent C-methyltransferase
(Figure 2). Previous studies have demonstrated a two-step
conversion of a-keto acids into b-methyl amino acids
catalyzed by a methyltransferase and an aminotransferase.[15]
Therefore, we hypothesized that MarG and MarI might be
involved in furnishing b-MeTrp for the biosynthesis of MARs.
To solidify the link between the marG-marH-marI
cassette and the biosynthesis of 5, these enzymes were
characterized in vitro. MarI was overexpressed and purified
from E. coli BL21(DE3)pLysS as an N-terminal His6-tagged
fusion protein (His6-MarI; see Figure S1a). The MarI-cata-
lyzed b-methylation was measured by using either indolepyr-
[*] Y. Zou, Q. Fang, H. Yin, Z. Liang, D. Kong, Prof. Dr. L. Bai,
Prof. Dr. Z. Deng, Prof. Dr. S. Lin
State Key Laboratory of Microbial Metabolism
School of Life Sciences & Biotechnology
Shanghai Jiao Tong University
800 Dongchuan Road, Shanghai 200240 (P.R. China)
E-mail: linsj@sjtu.edu.cn
[**] We thank HKI, Jena (Germany) and Prof. Osamu Tamura from
Showa Pharmaceutical University, Tokyo (Japan) for providing the
Streptomyces sp. B9173 strain and maremycin A standard, respec-
tively. We thank Prof. Peter F. Leadlay from University of Cambridge
for insightful discussions and critical proofreading. This work was
financially supported by the 973 program (2010CB833805 for S.L.
and 2009CB118901 for Z.D.) from the MOST, the key project
(311018) from MOE and NSFC (31070057 and 21372154 for S.L.;
31121064 for Z.D.).
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306255.
Figure 2. Putative gene cassettes for the conversion of l-tryptophan
into b-MeTrp.
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uvate (InPy; 1) or l-Trp as the substrate. When 1 was
incubated with MarI and SAM, a new peak was observed and
identified as b-methyl indolepyruvate (b-MeInPy; 2; m/z
218.1 [M + H]+) by LC-MS analysis (Figure 3a, trace 2), while
MarI was unable to directly install a methyl group onto the b-
carbon atom of l-Trp under the same assay conditions
(Figure S1b).
covert l-Trp into b-MeTrp. When MarG and MarI were
incubated with l-Trp, aKG, and SAM, the expected b-MeTrp
(m/z 219.1 [M + H]+) was observed upon LC-MS analysis
(Figure 3b, trace 1). Assays without the exogenous aKG or
SAM established that SAM, but not aKG, is an essential
cofactor for the MarG/I-coupled reaction (Figure 3b, traces 2
and 3). Therefore, the aminotransferase MarG and methyl-
transferase MarI are necessary and sufficient for the biosyn-
thesis of b-MeTrp from l-Trp.
To determine whether the enzymatic product is the
desired 5, the configuration of the a-carbon atom of the b-
MeTrp was first identified as 2S based on the l(s)-specificity
of MarG. No formation of b-MeTrp was observed from d-Trp
in the MarG/I-catalyzed coupled reaction (see Figure S3a).
The stereospecificity of MarI was determined by identifying
the configuration of the b-carbon atom of the b-MeTrp. b-
MeTrp was prepared by biotransformation of l-Trp in
recombinant E. coli BL21(DE3) overexpressing both marG
and marI (see the Supporting Information), and proved to be
identical to the product of the MarG/I-catalyzed in vitro
reaction as judged by HPLC analysis (Figure S3b). The
identity of the b-MeTrp was confirmed by NMR spectroscopy
(Figure S3c). Unexpectedly, its optical rotation value ([a]D27
À33.98, c 1.70, 0.1m HCl) is close to that reported for 4.[17] This
value indicates that the b-MeTrp formed by the MarG/I-
catalyzed reaction is 4 rather than the desired 5. To
corroborate the assignment of the 3R configuration,
a DmarI mutant that abolished the production of MARs
(see Figures S4a,b, and c, trace iii) was constructed for
carrying out feeding experiments. The compound 4 was
unable to restore the production of MARs when it was fed to
the DmarI mutant strain (Figure S4c, trace iv). This result
confirmed the 3R configuration, and suggested that 4 must be
epimerized prior to incorporation into the nonribosomal
peptide synthetase assembly line proposed for MARs.
Thus, we turned our attention to the gene marH which
encodes a small protein (129 aa). Although sequence analysis
shows MarH of unknown function, the secondary structure
prediction indicates that MarH possesses a cupin fold[18] (see
Figure 2 and Figure S5a). The cupin superfamily is a group of
proteins with diverse functions including epimerases and
isomerases catalyzing isomerizations of sugars involved in the
biosynthesis of cell-wall carbohydrates in bacteria.[18] There-
fore, MarH was proposed to be involved in the biosynthesis of
5 by catalyzing a stereochemical inversion of the b-methyl
group. To test this hypothesis, purified His6-MarH (Fig-
ure S5b) was added to the MarG/I-catalyzed coupled reaction
with SAM and l-Trp as substrates. A new peak was eluted
with a retention time that was different from that of 4, but had
the same ion peak at m/z 219.1 for [M + H]+ (Figure 3b, trace
4). To elucidate its structure, it was prepared by the
biotransformation of l-Trp in the recombinant E. coli BL21-
(DE3) overexpressing marG/H/I (Figure S5c). The NMR
spectra confirmed this new peak to be b-MeTrp (Figure S5d).
It is exciting that its optical rotation value ([a]2D9 + 46.58, c
1.70, 0.1m HCl) is almost identical to that reported for 5.[17]
When the DmarI mutant was fed with 5, the production of
MARs was perfectly restored (see Figure S4c, trace v), thus
confirming that the product from the MarG/H/I-catalyzed
Figure 3. Characterization of MarG, MarI, and MarH in vitro. LC-MS
profiles (UV 280 nm) of biochemical assays of MarG, MarI, and MarH
(flow rate is 0.6 mLminÀ1
.
l-Trp, 1, & 2, 4, 5). a: 1) 1
~
&
*
^
(standard); 2) MarI incubated with 1 and SAM; 3) MarG incubated
with l-Trp and aKG; 4) l-Trp (standard). b: 1) MarG/I incubated with
l-Trp, SAM and aKG; 2) MarG/I incubated with l-Trp and SAM;
3) MarG/I incubated with l-Trp and aKG; 4) MarG/H/I incubated with
l-Trp and SAM.
Next, His6-MarG was overexpressed and purified for
testing its activity in vitro (see Figure S2a). UV/Vis spectros-
copy shows that MarG was purified with the cofactor PLP
(Figure S2b),[16] so no exogenous PLP was added to the assays
of MarG. When MarG was incubated with l-Trp and a-
ketoglutarate (aKG), a new peak was observed and con-
firmed to be 1 by comparison with a standard of 1 using HPLC
analysis (Figure 3a, trace 3). This result confirmed that MarG
functions as a l-Trp aminotransferase, which is crucial for the
supply of 1 (the substrate of MarI) during the biosynthesis of
b-MeTrp.
Furthermore, a transamination is required to convert the
b-MeInPy into b-MeTrp. Within the mar cluster, marG is the
only identified aminotransferase gene (see Table S3), so
MarG would be the candidate to catalyze the second round
transamination. MarG was added to the previous MarI-
catalyzed methylation reaction of 1 with a commonly used
+
amino donor (l-Glu or NH4 ). However, the expected
products were not detected (see Figure S2c). Further assays
with other natural amino acids as putative amino donors
showed that l-Trp is the best amino donor for conversion of
b-MeInPy into b-MeTrp by MarG (Figure S2c). MarG was
also able to utilize l-tyrosine, l-phenylalanine, l-histidine,
and l-methionine as the amino donor, albeit in low efficiency
(Figure S2c).
Subsequently, a coupled deamination/methylation/trans-
amination enzymatic assay was developed using MarG/I to
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coupled reaction is the biosynthetic precursor (5) of MARs.
Taken together, these results suggest that MarI is R specific
and introduces the methyl group from the Re face onto the b-
carbon atom of 1 to form (R)-b-MeInPy (2) (Scheme 1).
Scheme 1. Pathways for the MarG/I- and MarG/H/I-catalyzed biosyn-
thesis of 4 and 5 from l-Trp via the intermediates 1, 2, and 3.
MarH is necessary for formation of 5 by catalyzing the
stereochemical inversion. A time course study shows that the
biosynthesis of both 4 and 5 is time dependent (see Figure S6).
MarG and MarI catalyze the formation of 4 from l-Trp
with 1 and 2 as intermediates. However, the addition of MarH
to the MarG/I-catalyzed coupled reaction led to the forma-
tion of 5, thus raising the question of when and how MarH
inverts the configuration of the b-methyl group. It is
reasonable to propose that MarH-catalyzed stereochemical
inversion is most likely to occur with 2, which is epimerized to
3, with subsequent transamination by MarG to eventually
form 5 (Scheme 1).
Figure 4. Timing and mechanism of MarH-catalyzed stereochemical
inversion. a) HPLC profiles (UV 280 nm and the flow rate of
0.5 mLminÀ1
.
4, 5) of 2 incubated with MarG alone (1) and with
~
^
MarG and MarH (2). b) Extracted ion chromatograms of m/z [M+H]+
219.1128 and 220.1191 corresponding to unlabeled and monodeute-
rium-labeled 5. 1) 6 (standard); 2) unlabeled 5; 3) monodeuterium-
labeled 5; 4) 4 (standard); 5) 5 (standard).
suggest that two possible mechanisms may be involved in the
epimerization of 2 into 3 (Scheme 2). In the one-base
mechanism, the deuterium on the b-carbon atom should be
retained, while in the two-base mechanism, the hydrogen in
To examine this hypothesis, we synthesized [3,3-2H2]InPy
(6; Figure 4b, trace 1; see also the Supporting Information),
and used MarI to generate the monodeuterated 2 as
a substrate for MarH. The monodeuterated 2 (synthesized
in situ; see the Supporting Information for details) was
incubated with MarG and with or without MarH for 1 hour.
In contrast to the MarH-free reaction that produced 4, the
reaction with MarH generated 5 (Figure 4a). Surprisingly,
both loss (m/z 219.1128 [M + H]+) and retainment (m/z
220.1191 [M + H]+) of deuterium were observed in 5 at a ratio
of 4.40:1 (Figure 4b, traces 2 and 3). These observations
À
the newly formed C H bond is expected to be solvent
derived.[19]
To differentiate whether the MarH-catalyzed epimeriza-
tion follows the one-base or two-base mechanism, a series of
site-directed mutageneses of MarH were performed. Mutiple
sequence alignments reveal that MarH possesses two con-
served motifs (HxHxxxE and PxGxxH) which are character-
istic of the cupin superfamily of proteins (see Figure S5a).[18]
Scheme 2. Two possible mechanisms of MarH-catalyzed epimerization of 2 to 3. In the two-base mechanism (a), a catalytic base deprotonates
the b proton of 2 to form an enolate intermediate which is then reprotonated by the acid form of a second base. In the one-base mechanism (b),
a single base is used in both deprotonation and reprotonation.
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Given that His and Glu or Asp usually act as a general-base
dyad for formation of an enolate intermediate in most
epimerases,[19] the mutants H62A, H64A, E68A, and H107A
were constructed (see Figure S8a). The H62A and H64A
mutants have no effect on the activity of MarH (Figure 5a,
Information for details). Complementing the result obtained
with 6, the deuterated (m/z 220.1191 [M + H]+) and non-
deuterated (m/z 219.1128 [M + H]+) forms of 5 were observed
at a ratio of 4.42:1 (see Figure S9).
Notably, three homologues of MarH were identified and
annotated as hypothetical proteins [StnK3 from the biosyn-
thetic pathway of streptonigrin[25] (similarity/identity, 86%/
82%), ACPL_6197 from the genome of Actinoplanes sp.
SE50/110 (82%/75%), and Cwoe_4835 (57%/41%) from the
genome of Conexibacter woesei DSM 14684; see Figures 2
and S5a]. stnK3 and ACPL_6197 were cloned, and
Cwoe_4835 was synthesized (see the Supporting Information
for details). These three homologues were also overexpressed
and purified from E. coli BL21(DE3)pLysS (see Figure S8b).
When they were individually added to the MarG/I-catalyzed
coupled reactions, all of them entirely complement the
activity of MarH to allow the biosynthesis of 5 (Figure 5b).
A C-methyltransferase (the homologue of MarI) and an
aminotransferase (the homologue of MarG) were found to be
clustered with this cupin-fold protein as MarG/H/I except for
C. woesei DSM 14684 (Figure 2). Phylogenetic analysis
indicated that MarG/H/I and their homologues formed
a separate clade from the aminotransferases, cupin-fold
epimerases, and methyltransferases, respectively (Fig-
ures S10–S12). The recognition of such enzymatic cassettes
and biochemical characterization of MarG/H/I suggest
a
common biosynthetic strategy for the formation of
Figure 5. HPLC profiles (UV 280 nm and the flow rate of 0.6 mLminÀ1
.
(2S,3R)- and (2S,3S)-b-MeTrp in the biosynthesis of the b-
MeTrp-derived natural products.
~
*
^
l-Trp, 4, 5) of the activity assays of MarH mutants (a) and
MarH homologous proteins (b) by coupling with MarG/I.
In conclusion, a three-gene cassette, marG-marH-marI,
was identified in the biosynthetic gene cluster of MARs from
Streptomyces sp. B9173 by genome sequencing and bioinfor-
matic analyses. It was biochemically demonstrated that
MarG/I specifically catalyzes the biosynthesis of (2S,3R)-b-
MeTrp from l-Trp, while MarG/H/I catalyzes the specific
formation of (2S,3S)-b-MeTrp from l-Trp with MarH as
a stereochemical switch. By using isotope-labeled substrate
and protein mutageneses, MarH-catalyzed stereochemical
inversion has been demonstrated to occur on (3R)-b-MeInPy
putatively through the one-base mechanism with H107-E68
acting as a base dyad for deprotonation. Three analogues of
MarH were also biochemically characterized and show the
same function as MarH. To unveil the detailed mechanism of
this conversion, efforts are underway to solve the structure of
MarH by X-ray crystallography and NMR spectroscopy.
traces 2 and 3), and contrary to the typical cupin superfamily
of proteins in which two His residues of the motif 1 are
essential for activity by acting as metal ligands.[18] However,
both E68A and H107A mutants completely lost the activity
(Figure 5a, traces 4 and 5), thus confirming that E68 and
H107 are essential for MarH activity and may act as the dyad
for deprotonation.
The only difference between the one-base and two-base
models is the existence of the second base for reprotonation
for the latter (Scheme 2). Several typical two-base epimerases
such as the epimerization (E) domain in nonribosomal
peptide synthetases[20] and dTDP-4-keto sugar epimer-
ases[21–24] have been confirmed to employ Tyr for reprotona-
tion. Therefore, the only conserved and putative proton
donor, Tyr70, was mutated to Ala (see Figure S5a). The
resulting mutant remained active (Figure 5a, trace 6), thus
indicating that MarH most likely catalyzes epimerization of
the b-stereocenter by the one-base mechanism, rather than by
the two-base mechanism (Scheme 2).
However, the washout of deuterium from the basic
residue, His, of MarH may be due to the fact that the
formed conjugated anionic enolate intermediate has a longer
residence time on the protein surface so that the base can
exchange with the media (Scheme 2b).[19] To examine this
hypothesis, we carried out an antiparallel study. The com-
pound 2 was synthesized in situ by the MarI-catalyzed
methylation of 1 instead of 6, and incubated with MarH and
MarG under deuterated conditions (see the Supporting
Received: July 18, 2013
Revised: September 4, 2013
Published online: October 25, 2013
Keywords: b-methylamino acids · biosynthesis · epimerases ·
.
cupin proteins · reaction mechanism
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