A fungal nonribosomal peptide synthetase module that can synthesize thiopyrazines

Article abstract of DOI:10.1021/ol200288w

A nonribosomal peptide synthetase-like enzyme (NRPS325) from Aspergillus terreus was reconstituted in vitro and was shown to synthesize thiopyrazines using an unprecedented mechanism. Substrate promiscuity of NRPS325 toward different amino acids and free

Full text of DOI:10.1021/ol200288w

ORGANIC
LETTERS
2011
Vol. 13, No. 7
1758–1761
A Fungal Nonribosomal Peptide
Synthetase Module that can Synthesize
Thiopyrazines
Kangjian Qiao,† Hui Zhou,† Wei Xu,† Wenjun Zhang,† Neil Garg,‡ and Yi Tang*,†,‡
Department of Chemical and Biomolecular Engineering and Department of Chemistry
and Biochemistry, University of California Los Angeles, Los Angeles, California 90095,
United States
yitang@ucla.edu
Received January 29, 2011
ABSTRACT
A nonribosomal peptide synthetase-like enzyme (NRPS325) from Aspergillus terreus was reconstituted in vitro and was shown to synthesize
thiopyrazines using an unprecedented mechanism. Substrate promiscuity of NRPS325 toward different amino acids and free thiols was explored
to produce >60 different thiopyrazine compounds.
Filamentous fungi are important microorganisms that
biosynthesize structurally diverse and pharmaceutically
important natural products.1 Among them, polyketides
(such as lovastatin) and nonribosomal peptides (NRPs,
such as penicillin) are of particular interest because of their
important biological activities.2 The biosynthetic enzymes
from fungi that assemble these molecules, such as polyke-
tide synthases (PKSs) and NRP synthetases (NRPSs) are
giant megasynthases with a multidomain architecture.
Fungal PKSs iteratively utilize a single set of domains to
assemble complex metabolites,3 while a typical module of
fungal NRPS only catalyzes one round of (i) adenylation
of an amino acid by the Adenylation (A) domain and
transfer to the phosphopantetheinyl (pPant) arm of Thio-
lation (T) domain and (ii) condensation between the
nucleophilic amino group and an electrophilic carbonyl
by the Condensation (C) domain.4 Using this biosynthetic
logic, fungal NRPS modules can synthesize tetramic acids
when fused to a PKS,5 diketopiperizines when paired in
tandem,6 and other oligopeptides7 and indole alkaloids8
when multiple modules are connected in an assembly line
like fashion.
We previously reconstituted the activities of ApdA, an
Aspergillus nidulans PKS-NRPS megasynthetase that
synthesizes the tetramic acid preaspyridone (Figure 1).9
We demonstrated that the ApdA PKS and NRPS modules
of the megasynthetase can be dissected and can interact
functionally in trans. When ApdA PKS module and
† Department of Chemical and Biomolecular Engineering.
‡ Department of Chemistry and Biochemistry.
(1) Hoffmeister, D.; Keller, N. P. Nat. Prod. Rep. 2007, 24, 393.
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Kleinkauf, H.; Keller, U. Biochemistry 1986, 25, 550. (b) Daniel, J. F.;
Filho, E. R. Nat. Prod. Rep. 2007, 24, 1128.
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3351. (c) Gao, X.; Chooi, Y.; Ames, B. D.; Wang, P.; Walsh, C. T.; Tang,
Y. J. Am. Chem. Soc. 2011, 133 (8), 2729–2741.
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J. W.; Schmidt, E. W. J. Am. Chem. Soc. 2008, 130, 11149. (c) Halo, L. M.;
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r
10.1021/ol200288w
Published on Web 03/08/2011
2011 American Chemical Society

Figure 1. Selected tetramic acid natural products.
cyclopiazonic acid synthetase10 (CpaS) NRPS module
from Aspergillus flavus were combined, a tryptophan-
containing preaspyridone analog was obtained. Inspired
by this result of combinatorial biosynthesis, we set out to
funcationally identify other heterologous NRPS modules
from different sequenced fungal species. In this study, we
show that serendipitously, the NRPS module (NRPS325)
of the only PKS-NRPS megasynthetase (ATEG00325) in
Aspergillus terreus can synthesize thiol-substituted pyra-
zines. The thiopyrazine synthetase activities were indepen-
dent of any upstream PKS activities. During the prepar-
ation of this manuscript, the natural role of ATEG00325
was genetically identified to be involved in the biosynthesis
of isoflavipucine and dihydroisoflavipucine (Figure 1),11
highlighting the unexpected biosynthetic potential of this
NRPS module unlocked during our genome mining
studies.
Bioinformatic analysis predicts that the NRPS325 is
capped with a C-terminus NADPH-binding reductase
(R) domain that is homologous to those found in
equisetin,5b tenellin,5c and CpaS synthetases.10 To analyze
the activities of NRPS325 in vitro, we expressed and
purified the 158 kDa holo-NRPS module consisting of
C-A-T-R from the engineered E. coli BAP1/pKJ75 (Figure
S1, Supporting Information).12 The relative specificity of
the A domain was assessed using a pyrophosphate release
assay in the presence of different amino acids (Figure S2,
Supporting Information). NRPS325 displayed substrate
promiscuity toward nearly all the natural aliphatic and
aromatic amino acids, including L-Ile, L-Met, L-Leu, L-Val,
L-Phe, L-Tyr and L-Trp. Toward L-Leu, which is the amino
acidthatisconfirmed by isotopic-labelingexperiment tobe
incorporated intoisoflavipucine,11 the A domaindisplayed
kcat of 17.2( 2.4 min-1 and KM of 310.0( 15.5μM (Figure
S3, Supporting Information).
Figure 2. Identification and in vitro reconstitution of NRPS325.
(A) Organization of ATEG00325 gene cluster from A. terreus.
Highlighting in gray is the 12.4 kb hybrid PKS-NRPS. (B)
HPLC analysis (320 nm) of ethyl acetate extracts from the in
vitro assays. Each assay contains 10 μM NRPS325 in the
presence of different reagents partly shown on the right of the
traces. The final concentrations of the different components
when added were: 10 μM ApdA-PKS; 10 μM ApdC; 5 mM thiol;
10 mM amino acid; 2 mM NADPH; 1 mM SAM; and 20 mM
ATP. All reactions were performed at room temperature for 12 h
in phosphate buffer (pH = 7.4).
afforded preaspyridone as a single product. In sharp
contrast, the reaction extract analyzed by LC-MS revealed
an unexpected, conjugated product 1a (Figure 2B, i)
(Molecular weight (MWT) = 344) with λmax at 319 nm
(Figure S16, , Supporting Information). Removal of
ApdA-PKS had no effect on the product profile, suggest-
ing the turnover of 1a is entirely from the standalone
functions of NRPS325 (Figure 2B, ii). Excluding either
L-Leu or ATP from the above reaction mixture resulted in
no product formation. Formation of 1a was absolutely
dependent on NADPH (Figure 2B, iii), which points to a
likely role of the R domain in reductive product release.
Substitution of L-Leu with L-Val (Figure 2B, iv) in the
reaction mixture led to the formation of 1b (MWT = 316)
with identical UV absorption. The decrease of 28 mu is
consistent with the loss of two methylene groups and is
therefore indicative of the product originating from two
molecules of the provided amino acid.
Interestingly, removal of the ∼0.5 mM DTT that was
present in the buffer used for enzyme storage totally
abolished the production of 1a (Figure 2B, v). The role of
the thiol in the formation of 1a was investigated by
substituting DTT with N-acetylcysteamine (NAC). A dif-
ferent product 2a (MWT = 309) with identical UV spec-
trum to 1a was formed (Figure 2B, vi). The decrease of 35
mu between 1a and 2a is consistent with the molecular
weight difference between DTT and NAC. Taking to-
gether, these observations ledus to predictthat1a is derived
from two molecules of L-Leu and one molecule of DTT.
We then attempted to functionally characterize
NRPS325 in vitro in the presence of equal molar amounts
of ApdA-PKS, along with enoyl reductase ApdC, mal-
onyl-CoA, L-Leu, SAM, NADPH and ATP (Figure 2B).
The same mixture with the natural AdpA NRPS partner
(10) Liu, X.; Walsh, C. T. Biochemistry 2009, 48, 8746.
(11) Gressler, M.; Zaehle, C.; Scherlach, K.; Hertweck, C.; Brock, M.
Chem. Biol. 2011, 18, 198.
(12) Pfeifer, B. A.; Admiraal, S. J.; Gramajo, H.; Cane, D. E.;
Khosla, C. Science 2001, 291, 1790.
Org. Lett., Vol. 13, No. 7, 2011
1759

Scheme 1. Proposed Biosynthetic Pathway for 2a
To obtain a sufficient amount of 1a for structural
elucidation, E. coli BAP1/pKJ75 induced with IPTG was
supplemented with 5 mM DTT and 10 mM L-Leu. How-
ever, the presence of DTT at millimolar concentrations
inhibited growth of E. coli. Instead, NAC was supplemen-
ted to BAP1/pKJ75 to give 2a at a final titer of 30 mg/L
three days after induction. The molecular formula of 2a
was determinedtobeC16H27N3OSby highresolutionmass
spectrometry ([M þ H]þ m/z: observed 310.1951; calcu-
lated: 310.1948) (Figure S11, Supporting Information).
The structure of 2a as shown in Scheme 1 was established
by extensive 1D and 2D NMR analysis (Table S3 and
Figures S12-15, Supporting Information). A comparison
of the 1H and 13C NMR spectra of 2a with those of pure
NAC and L-Leu revealed that 2a contains an intact NAC
molecule and two isobutyl chains. The remaining 1D
signals (H-5 at δH 8.25; C-2 at δC 153.9; C-3 at δC 153.6;
C-5 at δC 139.0; C-6 at δC 151.7) suggested the presence of
a 6-member aromatic heterocycle containing two nitrogen
atoms. The structure of 2a was finalized through key
1H-13C and 1H-15N HMBCcorrelations(showninTable
S3, Supporting Information) to be 2-(S-NAC)-3,6-diiso-
butylpyrazine. NRPS325 was thus confirmed to synthesize
a thiopyrazine instead of a diketopiperazine from two
molecules of L-Leu and one molecule of NAC, resulting
in the NAC being attached to C-2 of the pyrazine via an
aryl sulfide linkage.
The proposed mechanism of thiopyrazine synthesis is
shown in Scheme 1. Since two molecules of L-Leu must be
activated sequentially by a single A domain, we propose
NRPS325 must transfer the first leucyl moiety from the T
domain to the free thiol of NAC to form leucyl-S-NAC 3
bytransthioesterification. Inthemixture containingL-Leu,
ATP, NAC and NRPS325, a compound with [M þ H]þ m/
z = 233 can be identified using selected ion monitoring,
and its retention time matched precisely to a chemically
synthesized 3 (Figure S5, Supporting Information). This
transthioesterification reaction frees up the T domain to be
loaded with the second leucyl group, and is consistent with
the thiol-dependent formation of 2a. 3 as an intermediate
in the reaction can be further supported through the
synthesis of 2a by NRPS325 in the presence of only 3
and NADPH (Figure S6, Supporting Information). In this
case, the T domain can be loaded with leucyl group
through transthioesterification between the pPant arm of
holo-NRPS325 and 3, hence no ATP or free L-Leu is
required for activation. Attack of the R-amino group of
the second leucyl moiety on the carbonyl of 3 then yields a
tetrahedral intermediate, which is dehydrated to afford the
ethanimidothioate 4. Notably, the formation of the pro-
posed intermediate 4 requires an unusual dehydration step
occurred on the tetrahedral intermediate instead of the
expected thiol elimination to form the amide bond. This
step may be reminiscent of the cyclodehydration reactions
catalyzed by the cyclization (Cy) domain in some bacterial
NRPSs to afford oxazole and thiazole rings.13 Subse-
quently, reductive release of aldehyde 5 by the R domain
(Scheme 1), followed by imine formation and air oxidation
result in the formation of 2a.
To gain further insights into the unusual mechanism of
thiopyrazine synthesis as shown in Scheme 1, the roles of
the individual domains were probed. First, apo-NRPS325
lost the ability to produce 2a, confirming the dependence
onthe pPantarm of the loadedT domain. The C domain of
NRPSs catalyzes the canonical C-N bond formation, and
therefore should play a role in the formation of the
tetrahedral intermediate in the proposed pathway.4 The
two histidine residues located within the signature motif of
C domain HHxxxDG were mutated to Ala separately.14
Both H193A and H194A mutants of NRPS325 were
impaired in the synthesis of thiopyrazine compounds, as
the apparent rate of 2a formation was <10% of the wild
type NRPS325 (Figure S8, Supporting Information). A
comparable ∼13-fold reduction in the rate of 2a synthesis
was also observed when we truncated the C domain and
used the ATR tridomain (Figure S1, Supporting
Information) for the synthesis of 2a. Therefore, the pro-
posed nucleophilic attack of the free amine leucyl-S-T on
the carbonyl of 3 can take place spontaenously, but its rate
can be significantly enhanced in the presence of the C
domain. The C domain may achieve rate enhancement
through favored binding of the two substrates. Similar
observations in reduction in C-N bond formation rates
upon C domain inactivation were also observed in the
vibriobactin biosynthetic enzymes VibF (C2) and the free-
standing VibH C domain.15
(13) (a) Duerfahrt, T.; Eppelmann, K.; Muller, R.; Marahiel, M. A.
Chem. Biol. 2004, 11, 261. (b) Kelly, W. L.; Hillson, N. J.; Walsh, C. T.
Biochemistry 2005, 44, 13385.
(14) (a) Samel, S. A.; Schoenafinger, G.; Knappe, T. A.; Marahiel,
M. A.; Essen, L. O. Structure 2007, 15, 781. (b) Bergendahl, V.; Linne, U.;
Marahiel, M. A. Eur. J. Biochem. 2002, 269, 620.
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Org. Lett., Vol. 13, No. 7, 2011

Table 1. Subset of Selected Products that are Synthesized by NRPS325
R1
R2 = CH2NHCOCH3
R2 = CH(OH)CH(OH)CH2SH
R2 = CH2OH
R2 = CH2CH2CH2OH
CH2CH(CH3)2
CH(CH3)2
2a, 100%a
2b, 18%
2c, 51%
2e, 71%
2g, 19%
2h, 25%
1a, 52%
1b, 14%
1c, 50%
1e, 29%
1g, 23%
1h, 36%
6a, 60%
6b, 9%
7a, 36%
7b, 8%
CH2CH2SCH3
CH2Ph
6c, 39%
6e, 29%
6g, 29%
6h, 17%
7c, 25%
7e, 19%
7g, 30%
7h, 26%
CH2CH(CH3)CF3
CH2CH2N3
a The percentages in the table indicate the relative yields of thiopyrazines normalized to the yield of 2a.
Clearly, the proposed reductive release of aldehyde 5 by
the R domain is a critical requirement for thiopyrazine
formation. The R domain contains the intact catalytic
triad Ser-Tyr-Lys and the well-conserved NADPH bind-
ing site GxxGxxG found in short chain dehydrogenase/
reductase.5b,10,11 Mutation of the GxxGxxG motif to
GxxAxxA completely abolished the production of 2a
(Figure S7, Supporting Information). The requirement of
NADPH by NRPS325 was also monitored spectrometri-
cally at 340 nm (Figure S9, Supporting Information).
Consumption of NADPH was only observed in the pre-
sence of all the required building blocks, including L-Leu,
ATP and NAC. Only background change in absorbance at
340 nm was observed in the absence of the free thiol,
thereby excluding the possibility of direct reduction of
aminoacyl-S-T by the R domain. The reductive release of
5 observed here is consistent with the proposed role of the
R domain in the synthesis of isoflavipucine.11 A number of
R domains in fungal PKS-NRPSs were previously identi-
fied to lack reductive function and instead catalyze Dieck-
mann condensation to form tetramic acids.5a-c,10 As
expected, no trace of thiopyrazines were detected when
the NRPS modules from ApdA and CpaS were assayed as
standalone enzymes. Lastly, formation of 2a by the trun-
cated TR didomain in the presence of 3 and NADPH
(Figure S7, Supporting Information) suggests that the R
domain may also be involved in the dehydration of the
tetrahedral intermediate to form the ethanimidothioate,
instead of the thermodynamically favored peptide bond.
However, this putative new function of the R domain
remains unverified.
foming transthioesterification with different free thiols, we
tested the biocatalytic prowess of NRPS325 in the synth-
esis of a library of trisubstituted pyrazines. Using different
combinations of amino acids and free thiol substrates, we
showed that 63 different compounds can be synthesized by
this single NRPS in good yields (Table S4 and Figure
S16-46, Supporting Information). A subset of this is
shown in Table 1, in which six different amino acids and
four different thiols were combinatorially mixed to pro-
duce24compounds in vitro. Notably, theunnatural amino
acids trifluoroleucine (Tfl) and azidohomoalanine (Aha)
were each incorporated into pyrazine scaffolds efficiently.
The only previously reported microbial source of thiopyr-
azine is the marine bacterium Sulfitobacter pontiacus
(BIO-007).16 Our work here uncovers the hidden cap-
abilities of a fungal NRPS module in the synthesis of
thiopyrazines, which is vastly different from the recently
confirmed natural role of the parent enzyme. Such
unexpected findings further underscore the untapped
biocatalytic potential of megasynthetases from natural
product biosynthetic pathways.
Acknowledgment. We thank Dr. Liansuo Zu, Dr. Xin-
kai Xie and Dr. Yit-Heng Chooi (UCLA) for insightful
discussions, Prof. David Tirrell (Caltech) for a gift of Tfl
and Aha, and Prof. Christopher Walsh (Harvard Medical
School) and Prof. Mohamed Marahiel (Philipps-Univer-
sity Marburg) for advice. This research is supported by
NIH 1R01GM092217 and by the Packard Foundation
Supporting Information Available. Experimental de-
tails, NMR spectra of 2a and biochemical data. This
material is available free of charge via the Internet at
http://pubs.acs.org.
Given the broad substrate specificities of the A domain
in activating different amino acids and NRPS325 in per-
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