Processing 2-Methyl- l -Tryptophan through Tandem Transamination and Selective Oxygenation Initiates Indole Ring Expansion in the Biosynthesis of Thiostrepton

Article abstract of DOI:10.1021/jacs.7b05337

Thiostrepton (TSR), an archetypal member of the family of ribosomally synthesized and post-translationally modified thiopeptide antibiotics, possesses a biologically important quinaldic acid (QA) moiety within the side-ring system of its characteristic thiopeptide framework. QA is derived from an independent l-Trp residue; however, its associated transformation process remains poorly understood. We here report that during the formation of QA, the key expansion of an indole to a quinoline relies on the activities of the pyridoxal-5′-phosphate-dependent protein TsrA and the flavoprotein TsrE. These proteins act in tandem to process the precursor 2-methyl- l-Trp through reversible transamination and selective oxygenation, thereby initiating a highly reactive rearrangement in which selective C2-N1 bond cleavage via hydrolysis for indole ring-opening is closely coupled with C2′-N1 bond formation via condensation for recyclization and ring expansion in the production of a quinoline ketone intermediate. This indole ring-expansion mechanism is unusual, and represents a new strategy found in nature for l-Trp-based functionalization.

Full text of DOI:10.1021/jacs.7b05337

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Communication
Processing 2-Methyl-L-Tryptophan through Tandem
Transamination and Selective Oxygenation Initiates Indole
Ring Expansion in the Biosynthesis of Thiostrepton
zhi lin, Jia Ji, Shuaixiang Zhou, Fang Zhang, Jiequn Wu, Yinlong Guo, and Wen Liu
J. Am. Chem. Soc., Just Accepted Manuscript • DOI: 10.1021/jacs.7b05337 • Publication Date (Web): 18 Aug 2017
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Processing 2-Methyl- -Tryptophan through Tandem Transamina-
tion and Selective Oxygenation Initiates Indole Ring Expansion in
the Biosynthesis of Thiostrepton
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Zhi Lin1, Jia Ji1, Shuaixiang Zhou1, Fang Zhang2, Jiequn Wu1,4, Yinlong Guo2, and Wen Liu1,3,4,*
1 State Key Laboratory of Bioorganic and Natural Products Chemistry, Shanghai Institute of Organic Chemistry, University of Chinese
Academy of Sciences, 345 Lingling Road, Shanghai 200032, China.
2 State Key Laboratory of Organometallic Chemistry, Shanghai Institute of Organic Chemistry, Chinese Academy of Sciences, 345 Lingꢀ
ling Road, Shanghai 200032, China.
3 State Key Laboratory of Microbial Metabolism, School of Life Science & Biotechnology, Shanghai Jiao Tong University, 800
Dongchuan Road, Shanghai 200240, China.
4 Huzhou Center of BioꢀSynthetic Innovation, 1366 Hongfeng Road, Huzhou 313000, China.
Supporting Information Placeholder
ABSTRACT: Thiostrepton (TSR), an archetypal member of the
family of ribosomally synthesized and postꢀtranslationally modiꢀ
fied thiopeptide antibiotics, possesses a biologically important
quinaldic acid (QA) moiety within the sideꢀring system of its
characteristic thiopeptide framework. QA is derived from an inꢀ
dependent LꢀTrp residue; however, its associated transformation
process remains poorly understood. We here report that during the
formation of QA, the key expansion of an indole to a quinoline
relies on the activities of the pyridoxalꢀ5’ꢀphosphateꢀdependent
protein TsrA and the flavoprotein TsrE. These proteins act in
tandem to process the precursor 2ꢀmethylꢀ LꢀTrp through reversiꢀ
ble transamination and selective oxygenation, thereby initiating a
highly reactive rearrangement in which selective C2ꢀN1 bond
cleavage via hydrolysis for indole ringꢀopening is closely coupled
with C2’ꢀN1 bond formation via condensation for reꢀcyclization
and ring expansion in the production of a quinoline ketone interꢀ
mediate. This indole ringꢀexpansion mechanism is unusual, and
represents a new strategy found in nature for
tionalization.
LꢀTrpꢀbased funcꢀ
Figure 1. Bicyclic thiopeptide TSR, its ribosomally synthesized precursor
peptide and related biosynthetic pathway. (A) Structures of TSR, in which the
QA (ochre) moiety is indicated in a dashed rectangle. (B) Specific PTMs that
involve related enzymatic activities for processing the precursor
achieve QA (via quinoline ketone intermediate 1) within the sideꢀring system.
LꢀTrp to
Thiopeptide antibiotics are a class of sulfurꢀrich, ribosomally
synthesized and postꢀtranslationally modified peptides (RiPPs)
that possess a wide variety of biological properties.1 These antibiꢀ
otics share an unusual macrocyclic core that contains a sixꢀ
membered heterocycle central to multiple azoles and dehydroamꢀ
ino acids (Figure 1). As with many RiPPs,2 the biosynthesis of
thiopeptides starts with the conversion of a peptide precursor
composed of an Nꢀterminal leader sequence and a Cꢀterminal core
sequence. A myriad of postꢀtranslational modifications (PTMs)
occur solely on the latter, exemplifying how nature develops
structural complexity from a Ser/Thr and Cysꢀrich sequence.3
ment,5 as exemplified by the biosynthesis of the bicyclic members
thiostrepton (TSR, Figure 1A) and nosiheptide (NOS). Specificalꢀ
ly, TSR bears a quinaldic acid (QA) moiety within a 27ꢀ
membered large sideꢀring system, whereas in NOS, an indolic
acid (IA) moiety is directly attached to the macrocyclic core,
forming a 19ꢀmembered compact sideꢀring system. Benefiting
from these moieties, which expand the chemical spaces available
for biological functions, bicyclic thiopeptides appear to be unique
regarding their antiꢀinfectious actions.6
Intriguingly, both the QA moiety of TSR and the IA moiety of
NOS originate from a common substrate, L
ꢀTrp (Figure 1B).7 The
The establishment of a characteristic thiopeptide framework reꢀ
quires a series of common PTMs in the formation of azoles, deꢀ
hydroamino acids and a central heterocyclic domain.4 In addition
to sequence permutation of precursor peptides, the constitution of
the thiopeptide family, which includes over 100 different entities,
depends on a number of specific PTMs for individualized treatꢀ
formation of IA relies primarily on the activity of NosL, an Sꢀ
adenosylmethionine (SAM)ꢀdependent radical protein.8 This proꢀ
tein catalyzes a complex arrangement of the carbon side chain of
LꢀTrp, i.e., the removal of the C2’ꢀN unit and a shift of the carꢀ
boxylate group onto the indole ring, and produces 3ꢀmethylꢀ2ꢀ
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The recombinant TsrA protein appeared colorless and exhibited
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activity only in the presence of exogenous pyridoxalꢀ5’ꢀphosphate
(PLP) (Figure 2). Consistent with previous findings,11 this protein
catalyzed the transformation of 2ꢀmethylꢀLꢀTrp (2) using indolꢀ
ylpyruvate (IPA) as an ammonia acceptor, yielding 2ꢀmethylꢀ
indolylpyruvate (3, [M ˗ H]ꢀ m/z: calcd. 216.0661 for C12H10NO3,
found 216.0662) and the coꢀproduct LꢀTrp (Figure 2).Therefore,
TsrA is a characteristic PLPꢀdependent aminotransferase. Intriꢀ
guingly, the ammonia acceptor is changeable, as the replacement
of IPA with other αꢀketo acids varying in αꢀsubstitution, e.g.,
phenylpyruvate (aromatic) or αꢀketoglutarate (linear), still proꢀ
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duced 3 with a different coꢀproduct, e.g.,
tively (Figure 2), indicating that TsrA is flexible with respect to
its substrate. Indeed, TsrA tolerates ꢀTrp and ꢀPhe and catalyzed
reversible conversions between ꢀTrp and indolylpyruvate and
between ꢀPhe and phenylpyruvate when each of these αꢀketo
LꢀPhe or LꢀGlu, respecꢀ
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L
L
L
L
acids served as the ammonia acceptor for the other transamination
reaction (Figure S3). TsrA functions in a stereoꢀselective manner,
because none of the tested
Dꢀamino acids, including DꢀTrp and Dꢀ
Phe, can be transformed into the corresponding αꢀketo acids.
Bioinformatics suggests that TsrE is a flavinꢀdependent protein
and is homologous to various acylꢀCoA dehydrogenases that cataꢀ
lyze acyl chain desaturation accompanied by the reduction of the
flavin cofactor from its oxidized form (e.g., flavin adenine dinuꢀ
cleotide, FAD) to reduced form (e.g., FADH2).12a Accordingly, it
was previously proposed that TsrE follows TsrA activity to dehyꢀ
drogenate indole enamine 3 in a similar manner, producing an
indole imine, which is reactive and may readily undergo hydrolyꢀ
sis to break the N1ꢀC2 bond and initiate the ring expansion proꢀ
cess (Figure 3, Route 1).9 However, in the presence of FAD, the
addition of purified TsrE protein to the TsrAꢀcontaining reaction
mixture did not result in further transformation of 3 (Figure 4A).
Figure 2. In vitro transamination of 2 to 3 (top) in the absence (lower left) and
presence (lower right) of TsrA, using IPA (i), phenylpyruvate (ii) or αꢀ
ketoglutarate (iii) as an ammonia acceptor.
We thus reconsidered the nature of TsrE catalysis. In addition
to FADꢀbased dehydrogenation, O2 and FADH2ꢀdependent oxyꢀ
genation or halogenation is a direct alternative for achieving inꢀ
dole ring activation and expansion (Figure 3, Routes 2, 3 and 4).12
Consequently, Fre,13 a flavin reductase, was purified from E. coli
(Figure S2) and complemented to the above reaction mixture to
recycle FADH2 from oxidized FAD with dihydronicotinamide
adenine dinucleotide phosphate (NADPH) in situ. In the presence
of Fre, FAD and saturated NADPH, TsrE drove TsrAꢀmediated
reversible transamination forward by effectively converting 3,
indolic acid for further incorporation. In contrast, the process
through which QA is formed remains poorly understood. This
process results in more complex changes in the structure of LꢀTrp,
including ring expansion by cleavage of the C2ꢀN1 bond for inꢀ
dole ring opening and the connection of C2’ of the carbon side
chain to N1 for reꢀcyclization.7b Focusing on this key transforꢀ
mation, we here dissect an unusual mechanism for indole ring
expansion and demonstrate that processing the precursor 2ꢀ
methylꢀLꢀTrp through tandem transamination and selective oxyꢀ
genation triggers an intramolecular rearrangement during the forꢀ
mation of a quinoline ketone intermediate.
completed the consumption of the precursor 2ꢀmethylꢀLꢀTrp (2) in
the racemate and produced the quinoline ketone intermediate 1 in
a timeꢀdependent manner (Figure 4A). This transformation failed
to occur under strictly anaerobic conditions, supporting the notion
that O2 is indispensable. TsrE could not transform indolylpyruvate
or 2, indicating that indole ring expansion follows the activities of
2ꢀmethylation and transamination. In addition, omitting halide
ions from the reaction buffer had little effect on the production of
1. More likely, this protein is a flavinꢀdependent oxygenase essenꢀ
tial for an intramolecular rearrangement.
We previously demonstrated that, in the TSRꢀproducing Strep-
tomyces laurentii strain, the formation of the QA moiety involves
a ringꢀexpanded intermediate, quinoline ketone 1 (Figure 1B).9
The genes tsrT, tsrA and tsrE are related to the biogenesis of 1
because the inactivation of each gene completely abolished the
production of TSR, which was then restored by feeding 1 into the
corresponding mutant strain. Among these genes, the only one
that has been functionally characterized thus far is tsrT, which
encodes a SAMꢀdependent radical protein for the 2ꢀmethylation
Careful analysis of the TsrA and TsrEꢀinvolving transformation
that proceeded at 30°C revealed a minor compound ([M + H]+
m/z: calcd. 206.0812 for C11H12NO3, found 206.0817) (Figure 4A),
which displays an ultraviolet absorption spectrum different from
those associated with 2, 3 and 1 (Figure S4). According to the
established molecular formula and the hypothesis that TsrE catalꢀ
ysis is essentially an oxygenation process, a shunt product was
proposed to arise from the decarboxylation of an uncharacterized
oxygenated intermediate during the formation of 1. Two related 2ꢀ
methylꢀindole derivatives, αꢀhydroxyl carboxylate 4 from Route 2
and lactone 5 from Route 4 (Figure 3), were then synthesized and
served as standards for structural determination (Supplementary
Results). Highꢀperformance liquid chromatography with mass
of LꢀTrp to produce 2ꢀmethylꢀL
ꢀTrp (2) (Figure 1B).10 To deterꢀ
mine whether the remaining genes code for the transformation of
2, tsrA and tsrE were coꢀexpressed heterologously in Escherichia
coli, where an excess of 2ꢀmethylꢀDLꢀTrp ([M + H]+ m/z: calcd.
219.1134 for C12H15N2O2, found 219.1134), a racemate syntheꢀ
sized in this study (Supplementary Results), was supplemented.
The production of quinoline ketone 1 was observed; however, it
was completely abolished by omitting either tsrA or tsrE (Figure
S1). The necessity of tsrA and tsrE was further confirmed using
the recombinant E. coli cell homogenate to transform 2 because 1
was produced (Figure S1). Both TsrA and TsrE were then purified
from E. coli to assay their specific activities in vitro (Figure S2).
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Figure 3. Potential routes of indole ring expansion toward the formation of quinoline ketone 1 (ochre, shown in the solid rectangle). The TsrEꢀinitiated ringꢀ
expansion process is indicated in the dashed rectangle, where the C3 stereochemistry of related chemicals is proposed based on the assignment of the stereochemꢀ
istry of 7. The atom 18O is indicated in red when using 18O2.
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spectrometric detection (HPLCꢀMS) indicated that this product
differs from 4. Instead, it is identical to 5, leading to a hypothesis
that TsrEꢀcatalyzed oxygenation directly targets the pyrrole part
of the indole ring. Furthermore, TsrE was found to hydroxylate 2ꢀ
methylꢀ3ꢀpropylꢀindole (6, a synthetic mimic of 3, [M + H]+ m/z:
calcd. 174.1277 for C12H16N, found 174.1278) at C3 and trigger a
double bond shift within the pyrrole ring, yielding an imine prodꢀ
uct, 7 ([M + H]+ m/z: calcd. 190.1226 for C12H16NO, found
190.1226) (Figure 4B). Chiral separation of 7 on HPLC and subꢀ
sequent electronic circular dichroism (ECD) calculation validated
the stereoꢀselectivity of TsrE (Figures S6 and S7), which proꢀ
duced a (3S)ꢀisomer with an enantiomeric excess (ee) value of up
to 96%. Using 18O2 in the TsrEꢀcatalyzed hydroxylation of 6 led
to the production of single 18Oꢀlabeled 7 ([M + H]+ m/z: calcd.
192.1269 for C12H16N18O, found 192.1265). Consequently, these
observations provide strong evidence that TsrE can catalyze the
selective oxygenation of 3 and generate (S)ꢀ3ꢀ(3ꢀhydroxyꢀ2ꢀ
methylꢀ3Hꢀindolꢀ3ꢀyl)ꢀpyruvic acid (8) (Figure 3), which exists in
an imine form and appears to be too unstable to be detected at
30°C.
stable 2,3ꢀdihydroxylated pyrroline intermediate. Breaking the
N1ꢀC2 bond of 9 would result in intermediate 10 ([M ˗ H]ꢀ m/z:
calcd. 250.0721 for C12H12NO5, found m/z 250.0720; and calcd.
252.0763 for C12H12NO418O, found 252.0766). Ring expansion
relies on the condensation occurring between the released amino
group and the αꢀketo group of 10 to produce a reꢀcyclized interꢀ
mediate, which would undergo dehydration/aromatization to form
quinoline ketone 1. Alternatively, intermediate 8 could undergo
the oxidative decarboxylation of its carbon side chain (particularly
in the presence of O2 and FADH2, which react to form the oxidizꢀ
ing adduct FADꢀ4aꢀOOH), and the subsequent nucleophilic attack
of the newly generated carboxylate group onto C2 would furnish
an indole lactone to produce shunt product 5 (Figure S9). The
production of double 18O–labeled 5 ([M ˗ H]ꢀ m/z: calcd. 208.0751
for C11H12NO18O2, found 208.0749) in the presence of 18O2 supꢀ
ported this conversion. The oxidative decarboxylation of the carꢀ
bon side chain appears to compete with the process of indole ring
expansion, and the yield of 5 evidently increased with the deꢀ
crease in incubation temperature (Figure S5).
In conclusion, we demonstrate that the formation of quinoline
ketone intermediate 1 in TSR biosynthesis involves the tandem
activities of TsrA and TsrE for processing the precursor 2ꢀmethylꢀ
By lowering the incubation temperature, we slowed the transꢀ
formation of the precursor 2 that involves both TsrA and TsrE
activities. Remarkably, the reaction proceeding at 4°C did accuꢀ
mulate 8 ([M ꢀ H]ꢀ m/z: calcd. 232.0615 for C12H10NO4, found
232.0612), which results from 3ꢀhydroxylation (or 8’ from 2,3ꢀ
epoxidation, an oxygenation alternative that cannot be completely
excluded at this time because the facile tautomerization between 8
and 8’ could occur in solution), as judged by careful HPLCꢀhigh
resolution (HR)ꢀMS and MS/MS analyses (Figure S8). At this
temperature, using 18O2 in the reaction mixture produced single
18Oꢀlabeled 8 (or 8’, [M ꢀ H]ꢀ m/z: calcd. 234.0658 for
C12H10NO318O, found 234.0659), consistent with the notion that
TsrEꢀmediated indole ring expansion is initiated by a cryptic seꢀ
lective oxygenation of 3 rather than dehydrogenation (Route 1) or
halogenation (Route 3). These analyses revealed a set of related
derivatives, further supporting the following conversion process
toward the formation of 1 through Route 4 (Figures 3 and S8).
Intermediate 8 (or 8’), which possesses a highly reactive pyrrole
imine ring, could be readily hydrated to generate 9 ([M ˗ H]ꢀ m/z:
calcd. 250.0721 for C12H12NO5, found 250.0721; and calcd.
252.0763 for C12H12NO418O, found 252.0757), an extremely unꢀ
LꢀTrp (2) through reversible transamination and selective oxygenꢀ
ation. TsrA, a flexible PLPꢀdependent aminotransferase, activates
C2’ of the carbon side chain through transamination to generate αꢀ
keto acid 3. TsrE, a flavinꢀdependent protein, selectively oxygenꢀ
ates 3 to produce a highly reactive indole imine, which then unꢀ
dergoes a rearrangement process through intermediates 9 and 10
for ring expansion. More likely, this unstable intermediate is
C3(S)ꢀhydroxylated 8 according to data presented here and a simiꢀ
lar intermediate resulting from unusual FADꢀdependent protein
activity, which initiates indoloterpenoid cyclization through a
selective C3ꢀhydroxylation of indole in xiamycin biosynthesis.14
The subsequent steps in the biosynthetic pathway of TSR include
a selective ketone reduction to generate a (12S)ꢀquinoline alcoꢀ
hol,9 which could then be activated, epoxidated and appended
onto the core peptide part of the precursor peptide prior to the
formation of the thiopeptide framework.15 The closure of the sideꢀ
ring system follows the removal of the leader peptide sequence,
and both are postꢀthiopeptide PTMs that depend on the dual activꢀ
ity of TsrI, an unusual α/βꢀhydrolase fold protein.16
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Supporting Information
Supplementary Methods, Results, Figures and Tables. This mateꢀ
rial is available free of charge via the Internet at
http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
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*wliu@mail.sioc.ac.cn
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Notes
The authors declare no competing financial interests.
ACKNOWLEDGMENT
This work was supported in part by grants from NSFC
(21520102004, 31430005 and 81302674), CAS (QYZDJꢀSSWꢀ
SLH037 and XDB20020200), STCSM (14JC1407700,
15JC1400400 and 17JC1405100), K. C. Wang Education Foundaꢀ
tion and ChangꢀJiang Scholars Program of China.
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Figure 4. Characterization of the role of TsrE in the formation of 1. (A) In vitro
assays of TsrE activity. Substrate 3 was prepared in situ using TsrAꢀcatalyzed
transamination reaction in which the ammonia acceptor is phenylpyruvate.
FADH2 was produced by reducing FAD with NADPH in the presence of Fre. i,
authentic 1; ii, transforming 3 with cofactor FAD; iii, iv and v, transforming 3
with cofactor FADH2 in the absence (negative control) and presence of TsrE
(aerobically and anaerobically), respectively; vi and vii, transforming IPA and
2; and viii, transforming 3 in the absence of halide ions. (B) TsrEꢀcatalyzed
selective hydroxylation of 6 to 7 (top, the atom 18O is indicated in blue) upon
HPLC analysis (bottom). i, authentic 7; and ii and iii, transforming 6 in the
absence (negative control) and presence of TsrE.
Indole ring expansion was previously observed during the bioꢀ
synthesis of quinoxaline and quinoloneꢀcontaining natural prodꢀ
ucts.17 In that process, the 2, 3ꢀdioxygenation of an
LꢀTrp precurꢀ
sor cleaves the C2ꢀC3 bond within the indole ring, followed by
deformylation and transamination to release both the amino and
αꢀketo groups between which condensation occurs to form an
expanded ring. In contrast, in the biosynthesis of TSR, quinoline
formation is initiated by a selective hydroxylation of a 2ꢀ
substituted LꢀTrp derivative and subsequent hydrolysis to break
the indole ring (by cleaving the N1ꢀC2 bond), and ring reconstrucꢀ
tion (by forming the N1ꢀC2’ bond) immediately couples with ring
destruction. Notably, the formation of cinchona alkaloids, such as
quinine, an antimalarial agent that has been widely used in the
clinic for nearly two centuries, also requires N1ꢀC2 bond cleavage
and transformation of a 2ꢀsubstituted LꢀTrp derivative into a quinꢀ
oline product.18 Determining whether these cinchona alkaloids
share a similar biochemical mechanism with TSRꢀtype thiopepꢀ
tides for indole ring expansion would be extremely interesting.
(18) Leete, E. Acc. Chem. Res. 1969, 2, 59.
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Indole Ring Expansion in the Biosynthesis of Thiostrepton
81x27mm (300 x 300 DPI)
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