D-Alanylation in the Assembly of Ansatrienin Side Chain Is Catalyzed by a Modular NRPS

Article abstract of DOI:10.1021/acschembio.6b00004

Ansatrienins are a group of ansamycins with an N-cyclohexanoyl d-alanyl side chain. Though ansatrienins have been identified for decades, the mechanism for the addition of this unique side chain was not established. Here, we report the biochemical characterization of a tridomain nonribosomal peptide synthetase (NRPS), AstC, and an N-acyltransferase, AstF1, encoded in the biosynthetic pathway of ansatrienins. We demonstrate that AstC can efficiently catalyze the transfer of d-alanine to the C-11 hydroxyl group of ansatrienins, and AstF1 is able to attach the cyclohexanoyl group to the amino group of d-alanine. Remarkably, AstC presents the first example that a modular NRPS can catalyze intermolecular d-alanylation of the hydroxyl group to form an ester bond, though alanyl natural products have been known for decades. In addition, both AstC and AstF1 have broad substrate specificity toward acyl donors, which can be utilized to create novel ansatrienins.

Full text of DOI:10.1021/acschembio.6b00004

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Letter
D-alanylation in the assembly of ansatrienin
side chain is catalyzed by a modular NRPS
Guoyin Shi, Ning Shi, Yaoyao Li, Wang Chen, Jingjing
Deng, Chao Liu, Jing Zhu, Haoxin Wang, and Yuemao Shen
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b00004 • Publication Date (Web): 25 Jan 2016
Downloaded from http://pubs.acs.org on January 26, 2016
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D-alanylation in the assembly of ansatrienin side chain is catalyzed by a
modular NRPS
†
,§
†,§
‡
‡
‡
†
Guoyin Shi, Ning Shi, Yaoyao Li, Wang Chen, Jingjing Deng, Chao Liu, Jing
†
,†
,†, ‡
Zhu, Haoxin Wang,* Yuemao Shen*
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†
State Key Laboratory of Microbial Technology, School of Life Sciences, Shandong
University, Jinan, Shandong 250100, P. R. China
‡
Key Laboratory of Chemical Biology (Ministry of Education), School of
Pharmaceutical Sciences, Shandong University, Jinan, Shandong 250012, P. R.
China
§
These authors contributed equally to this work
*
Corresponding authors. Tel.: +86 531 8838 2108; Fax: +86 531 8836 2350.
Eꢀmail: wanghaoxin@sdu.edu.cn (H. Wang), yshen@sdu.edu.cn (Y. Shen).
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Abstract
Ansatrienins are a group of ansamycins with Nꢀcyclohexanoyl Dꢀalanyl side
chain. Though ansatrienins have been identified for decades, the mechanism
for the addition of this unique side chain were not established. Here, we report
the biochemical characterization of a tridomain nonribosomal peptide
synthetase (NRPS), AstC, and an Nꢀacyltransferase, AstF1, encoded in the
biosynthetic pathway of ansatrienins. We demonstrate that AstC can
efficiently catalyze the transfer of Dꢀalanine to the Cꢀ11 hydroxyl group of
ansatrienins, and AstF1 is able to attach the cyclohexanoyl group to the
amino group of Dꢀalanine. Remarkably, AstC presents the first example that a
modular NRPS can catalyze intermolecular Dꢀalanylation of hydroxyl group to
form an ester bond, though alanyl natural products have been known for
decades. In addition, both of AstC and AstF1 have broad substrate specificity
towards acyl donors, which can be utilized to create novel ansatrienins.
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Dꢀalanylation is of broad biological significance as exemplified by that of
teichoic acids (TAs), major components of the lowꢀG+C Gramꢀpositive cell
wall. Dꢀalanylation of TAs is involved in diverse physiological process,
including antibiotic resistance, biofilm formation, virulence, and acid
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tolerance. The modification of TAs with Dꢀalanine involves enzymes encoded
by the dltABCD operon, and the whole pathway displays remarkable
parallelism to the division of labor employed by nonribosomal peptide
2
synthetases (NRPSs).
Similar pathways for the glycylation of
3
lipopolysaccharide in Gramꢀnegative bacteria had also been identified.
However, although Dꢀalanylated smallꢀmolecule natural products have been
identified for decades, the biosynthetic mechanism of this modification has not
been established.
Ansatrienins belong to a unique group of ansamycin antibiotics produced by
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Streptomyces. These small molecules contain a 21ꢀmembered macrocyclic
lactam ring and a cyclohexanoylꢀalanyl side chain attached to the Cꢀ11
hydroxyl group of ansa ring. The biosynthesis of ansatrienins is presumed to
follow the same manner as suggested for other ansamycins, which are
produced by multimodular type I polyketide synthases (PKSs) using 3ꢀaminoꢀ
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ꢀhydroxybenzoic acid (AHBA) as the starter unit, and released by amide
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synthases followed by postꢀPKS modifications. Although the biosynthetic
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gene clusters for ansatrienins have been reported,
cyclohexanoylꢀalanyl side chain has not been revealed.
the attachment of the
Previously, feeding experiments with Streptomyces collinus Tϋ 1892 have
demonstrated that the Dꢀalanine moiety of ansatrienins is derived directly from
free Dꢀalanine rather than Lꢀalanine, and cyclohexanoylꢀalanyl side chain is
8
not incorporated as an intact moiety but added one component at a time. On
the basis of bioinformatic analysis of the myc gene cluster in Streptomyces
flaveolus DSM 9954, Qu et al. proposed that the NRPS MycC is involved in
adenylating alanine residue, which is then transferred from MycC to the Cꢀ11
hydroxyl group of ansa ring by the esterase MycF4, followed by Nꢀcyclohexyl
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formylation catalyzed by the Nꢀacetyltransferase MycF1. Recently, as part of
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our efforts to exploit novel ansamycins from AHBA synthase gene positive
9ꢀ11
strains,
an ansatrienin gene cluster (ast, GenBank accession number:
KF813023.1 and KP284551) was activated by constitutive expression of the
pathwayꢀspecific positive regulator gene astG1 in Streptomyces sp. XZQH13,
resulting in the isolation of two known ansatrienins, hydroxymycotrienin A (1)
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and thiazinotrienomycin G (2) (Figure 1a). However, sequence analysis
revealed that the homolog of mycF4 gene was absent in the ast gene cluster,
suggesting that MycF4 or its homologs may be not required for the transfer of
the alanine residue. Accordingly, an isolate tridomain (adenylationꢀthiolationꢀ
thioesterase, AꢀTꢀTE) NRPS, namely AstC, a homolog of MycC, was
speculated to be responsible for the attachment of Dꢀalanyl group to the ansa
ring. This type of AꢀTꢀTEꢀtridomain NRPSꢀmediated intermolecular amino acid
esterification is unprecedented among known esterases or NRPSs. Here we
demonstrate that astC and astF1 are essential genes for the attachment of
the cyclohexanoylꢀalanyl side chain onto ansatrienin ansa ring, AstC can
catalyze the attachment of Dꢀalanine into the biosynthetic intermediate (3)
(Figure 1a), and AstF1 is responsible for the attachment of the cyclohexanoyl
moiety.
a
b
XZQH13
XZQH13/pJTU824ꢀastG1(OE)
●
1
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●
●
2●
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●
XZQH13OE∆astD1
XZQH13OE∆astC
★
★
★
★
★
3
★
★
XZQH13OE∆astC/pPM927ꢀastC
XZQH13OE∆astF1
XZQH13OE∆astF1/pPM927ꢀastF1
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Time (min)
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Figure 1. The astC and astF1 genes are required for the attachment of the
cyclohexanoylꢀalanyl side chain onto ansatrienin ansa ring in Streptomyces sp.
XZQH13/pJTU824ꢀastG1(OE). a) Structures of the representative ansatrienins
isolated from the strain XZQH13. b) HPLC analysis (275 nm) of metabolites
produced by the strain XZQH13 and the corresponding mutant strains.
Uncharacterized ansatrienin analogs were identified by diode array UV comparison
with that compounds 1–3 in the strains XZQH13/pJTU824ꢀastG1(OE) (●) and
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XZQH13OEꢁastC (★), respectively. It is worth noting that the intermediates
accumulated by XZQH13OEꢁastF1 are refractory to isolations thus that we have not
obtained enough amount for full structure elucidation.
The production of ansatrienins is low in the strain XZQH13 but increased
tremendously by overexpressing the regulator gene astG1 thus that many
uncharacterized
extra
peaks
were
presented
in
the
mutant
1
2
XZQH13/pJTU824ꢀastG1(OE) (Figure 1b). All these extra peaks were
identified to be ansatrienins by HPLC comparison of the fermentation
products of the PKS gene (astD1)ꢀdisrupted mutant XZQH13OE∆astD1, and
XZQH13/pJTU824ꢀastG1(OE) with that of ansatrienins (Figure 1b ，
Supplementary Figure S4), supporting that the ast gene cluster is responsible
for the biosynthesis of ansatrienins. To confirm the roles of astC and astF1 in
the incorporation of the cyclohexanoylꢀalanyl side chain, the individual geneꢀ
disrupted mutants were generated and complemented in trans of a fullꢀlength
copy of the disrupted gene under the control of the promoter of the
erythromycin resistance gene (ermE*) (Supplementary Figure S5). Both of the
resultant mutants XZQH13OEꢁastC and XZQH13OEꢁastF1 abolished the
production of compounds 1 and 2 along with the uncharacterized analogs,
and recovered the production of ansatrienins after complementing the astC
and astF1 genes (Figure 1b), respectively, indicating that both genes are
required for the attachment of the cyclohexanoylꢀalanyl side chain onto
ansatrienin ansa ring.
To investigate the function of AstC, we identified the metabolic
intermediates accumulated in XZQH13OEꢁastC. Compound 3 was obtained
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from the fermentation products of XZQH13OEꢁastC (Figure 1a). Its highꢀ
resolution mass spectrometry (HRMS) showed a quasi molecular ion at m/z
ꢀ
527.2152 for 3 [M(C28
H
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N
2
O
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S) – H] (Supplementary Figure S11). Its
structure was established by the NMR comparison with that of
thiazinotrienomycin E (Supplementary Table S3, Supplementary Figures S7 –
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0). The NMR spectroscopic data of both compounds were similar, except
for the absence of the cyclohexanoylꢀalanyl side chain at Cꢀ11 in 3, which
indicated that the astC gene may be involved in the incorporation of the Dꢀ
alanine residue in ansatrienins.
The astC gene encodes a singleꢀmodule AꢀTꢀTEꢀtridomain NRPSꢀlike
protein. The closest homologs in NCBI are AnsC and MycC (protein
accession numbers AHW80292.1 and AFG19416.1) of S. seoulensis and S.
flaveolus DSM 9954, respectively, both shared 88% identity of amino acid
sequences with AstC. However, none of these two proteins have been
functionally characterized. To obtain direct evidence for the role of AstC, we
amplified the 2541ꢀbp fragment of astC gene using PCR and cloned it into
pET28a(+) for expression in E. coli BL21(DE3). To produce the holoꢀenzyme,
heterologously expressed AstC was treated in vitro with the
phosphopantetheinyl transferase Sfp. The activity of holoꢀAstC was assayed
using compound 3 as acyl acceptor and D/Lꢀalanine as acyl donors. LCꢀMS
analysis showed the presence of an extra peak (4) with a quasi molecular ion
ꢀ
at m/z 598.2515 [M – H] (Figure 2a, Supplementary Figure S12), in
accordance with the attachment of an alanyl residue to 3, in the reaction with
Dꢀalanine. In contrast, reactions either with boiled AstC or Lꢀalanine did not
afford any evident products (Figure 2a). These results show that AstC
catalyzes the alanylation of compound 3 with Dꢀalanine but not Lꢀalanine as
the acyl donor to produce compound 4 (Figure 2c), which is consistent with
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the selective use of Dꢀalanine in feeding experiments.
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Figure 2. Reconstitution of AstC activity in vitro. a) HPLC traces (260 nm) the
incubations of Lꢀ/Dꢀalanine and 3 with holoꢀAstC and holoꢀAstCS545A. b) AstC follows
the “pingꢀpong” mechanism. c) AstC was proposed to incorporate the Dꢀalanine
residue at Cꢀ11 of 3.
To characterize the enzymatic properties, the holoꢀAstCꢀcatalyzed reaction
was quantified by HPLC. The purified holoꢀAstC showed optimal activity at pH
8.0, and exhibited more than 75% of its maximum activity between pH 7.0 and
1
0.0 (Supplementary Figure S3a). The optimal reaction temperature was
0 °C at pH 8.0, with > 75% activity between 25 and 35 °C (Supplementary
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Figure S3b). The kinetic parameters of holoꢀAstC were measured under the
optimal conditions, showing the conversion with an apparent K of 40.21 ±
M
ꢀ1
2
M
0.60 ꢂM and kcat of 0.70 ± 0.20 min for compound 3, and an apparent K of
ꢀ
1
5.57 ± 1.56 ꢀM and kcat of 0.27 ± 0.03 s for Dꢀalanine (Supplementary
Figures S3d, e). To determine the mechanism of reaction catalyzed by AstC,
the concentrations of Dꢀalanine were varied with different constant
concentrations of compound 3. When plotting the reciprocal of V
reciprocal of the Dꢀalanine concentrations, parallel lines were obtained (Figure
b), showing that AstC transfers the Dꢀalanyl to the Cꢀ11 hydroxyl group of 3
via the “pingꢀpong” mechanism.
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To address the substrate specificity of AstC, sodium propionate, βꢀalanine,
glycine and 12 Dꢀamino acids were selected as acyl donors. The strongest
conversion was detected with Dꢀalanine, followed by Dꢀserine, βꢀalanine,
glycine, Dꢀthreonine, and Dꢀvaline (4–9; Supplementary Figure S6). The other
tested Dꢀamino acids were not accepted at the detected levels. This relaxed
substrate specificity toward acyl donors suggests a rather nonꢀspecific
activation of amino acids by the Aꢀdomain of AstC, which will allow for
chemoenzymatic synthesis of new ansatrienin analogs. We also assayed the
tolerance of AstC toward different acyl acceptors by performing the acylation
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assay
with
various
ansamycins,
including
Nꢀdesmethylꢀ4,5ꢀ
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desepoxymaytansinol, geldanamycin and hygrocins. However, no positive
results were obtained (data not shown), suggesting a strict substrate
specificity of AstC for the acyl acceptors.
The notable feature of the AstC is the presence of only three individual
domains (AꢀTꢀTE) responsible for adenylation, thiolation and thioester
cleavage, and the absence of the traditional condensation (C) domain.
Previously, only a few such AꢀTꢀTEꢀtridomain, one module NRPS proteins
have been characterized, all are involved in the homodimerizations of
16ꢀ18
aromatic αꢀketo acids by catalyzing carbonꢀcarbon bond formation.
AstC
represents a new member of AꢀTꢀTEꢀtridomain NRPS enzymes but with quite
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different function.
The Dꢀalanylation catalyzed by AstC presents a unique
21,
acylation strategy, which differs markedly from any other acyltransferases.
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In the latter biosynthetic manifolds, the enzymeꢀmediated acylations usually
require coenzyme A (CoA) thioesters or Nꢀacetylcysteamine (NAC) thioesters
23
as substrates to produce the acylated products. By contrast, AstC utilizes
free amino acids as acyl donors that are activated as aminoacyl adenylates by
the Aꢀdomain, and then transferred to the Tꢀdomain, transiently forming
aminoacyl thioesters. Indeed, when the active site Ser545 within the
conserved GGXSXLA motif of the Tꢀdomain was mutated to alanine, the
mutant AstC was no longer capable of generating 4 from 3 (Figure 2a).
Transfer of the activated acyl moiety from the Tꢀdomain to the TEꢀdomain
would form the acylꢀOꢀTE enzyme intermediate (Figure 2c). At this point, we
proposed that the acyl carbonyl carbon of this intermediate was
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nucleophilically attacked by the Cꢀ11 hydroxyl oxygen of 3 to complete the
acylation (Figure 2c). However, it is not yet clear how AstC recognizes and
interacts with the acyl acceptor, 3. Moreover, so far, only a PCPꢀCꢀdidomain
(Fum14) and a freestanding Cꢀdomain (SgcC5) NRPSꢀlike proteins have been
biochemically characterized to use free alcohols as the acyl acceptors to
couple with acyl carrier proteinꢀtethered acyl donors for ester bond
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formations.
To our knowledge, AstC is the first example of an AꢀTꢀTEꢀ
tridomain NRPS protein that is capable of catalyzing intermolecular ester
bond formation, particularly, using free amino acids as acyl donors.
AstF1 identified as an acyltransferase was suggested to catalyze the
transfer of cyclohexanoyl moiety to the alanyl nitrogen at Cꢀ11 of
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ansatrienins. To establish the function of AstF1, the astF1 gene (927 bp) was
amplified from fosmid 17ꢀ10F by PCR, and inserted into pET22b for
expression in E. coli BL21(DE3). Due to lack of the intermediates
accumulated by the astF1ꢀdeleted mutant (XZQH13OEꢁastF1), we directly
examined whether compound 4 can be used as the acyl acceptor in the
transacyl reaction. Incubation of the recombinant AstF1 protein with the
reaction mixture of AstC assay containing compound 4 and the chemically
synthesized cyclohexanoylꢀSNAC (I) (Figure 3a) led to the formation of
thiazinotrienomycin E (10) accompanied by the decrease of 4 (Figure 3b). To
confirm this result, an identical incubation without AstF1 was conducted; no
conversion of 4 was detected. These results clearly demonstrated the
predicted acyltransferase activity of AstF1 (Figure 3a).
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4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
O
O
a
O
NH
OH
NHAc
NH
OH
R
S
S
I ꢀ VI
S
NH
AstF1
NH
HO
O
HO
O
SNAC
O
O
OMe
O
O
OMe
O
H2N
4
R
N
10 - 15
H
R =
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
I, 10
4
II, 11
III, 12
IV, 13
V, 14
VI, 15
b
3
I
×10
.0
5
710.3479
692.3373
i)
2
ii)
1.0
0
1
0
732.3296
iii)
II
×
10
5
708.3314
i)
2
.0
ii)
690.3210
730.3134
1
.0
1
1
iii)
i)
0
III
×
10
5
698.3478
80.3373
6
1.0
ii)
12
0.5
0
7
20.3297
iii)
IV
i) ×10
5
666.3212
2
.0
6
84.3315
ii)
1
.0
0
706.3129
1
3
iii)
V
×
10
5
664.3053
682.3156
i)
3
.0
ii)
iii)
2.0
14
1.0
0
704.2975
VI
i) ×10
5
684.3315
66.3211
6
2
.0
ii)
1
.0
706.3135
15
iii)
0
6
00 640 680 720 760 800
6
8
10 12 14 16 18 20
Time (min)
m/z
Figure 3. Reconstitution of AstF1 activity in vitro. a) The reactions catalyzed by
AstF1. b) LCꢀMS analysis of the reaction products 10–15 of AstF1 incubated with Nꢀ
acetylcysteamine (SNAC) thioesters I–VI and 4 (produced by the incubation of 3 and
Dꢀalanine with holoꢀAstC), respectively. i), SNAC thioesters; ii), SNAC thioesters and
4
without AstF1; iii), SNAC thioesters and 4 with AstF1.
Broad substrate specificity with regard to the acyl donors of
acyltransferases has been commonly observed and utilized to generate
27
analogs of natural products with varied acyl substituents. We examined
whether cyclohexꢀ1ꢀenecarbonylꢀSNAC (II), 4ꢀmethylpentanoylꢀSNAC (III), 2ꢀ
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methylbutanoylꢀSNAC (IV), 2ꢀmethylbutꢀ2ꢀenoylꢀSNAC (V), and 3ꢀ
methylbutanoylꢀSNAC (VI) could replace I as the acyl donors in the acylations
of 4. As expected, all these SNAC thioesters can serve as substrates for
AstF1, but with different turnover rates, to produce 11 – 15 (Figure 3b). The
demonstrated ability of the recombinant AstF1 to use various acylꢀSNAC
substrates is consistent with the substitution profile of identified natural
ansatrienins. The mutasynthesis study by Song et al. has also shown that a
diversity of small molecule organic acids can be attached to the Dꢀalanyl
nitrogen of ansatrienins, which is supposed to be catalyzed by a homolog of
10
11
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AstF1 in S. seoulensis IFBꢀA01. Therefore, the remarkable substrate
tolerance of AstF1 to acyl donors provides an efficient chemoenzymatic route
to new ansatrienin analogs.
In conclusion, we have confirmed that the AꢀTꢀTEꢀtridomain NRPS protein
AstC catalyzes the attachment of Dꢀalanyl to the Cꢀ11 hydroxyl group of
ansatrienin backbone, which represents an unprecedented acylation
mechanism, and AstF1 catalyzes the sequential attachment of the
cyclohexanoyl group to the Dꢀalanyl nitrogen during ansatrienins biosynthesis.
Moreover, kinetic studies of the Dꢀalanyl transfer reaction catalyzed by AstC
demonstrated that the reaction occurred by the “pingꢀpong” mechanism. In
addition, both of AstC and AstF1 display broad substrate specificity toward the
acyl donors. The ability to decorate the biosynthetic intermediates of
ansatrienins with combinations of various amino acids and small organic acids
via the actions of AstC and AstF1 should provide routes to new variants in the
ansamycin class.
Methods
Inactivation of astD1, astF1 and astC in XZQH13/pJTU824-astG1
The genomic library of Streptomyces sp. XZQH13 was constructed and
screened by PCR using AHBA synthaseꢀspecific degenerate primers to
identify fosmid 17ꢀ10F. The target genes in fosmid 17ꢀ10F were replaced by
R
the apramycin resistance cassettes (acc(3)IV(Apr ) + oriT) by λꢀRED
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1
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2
2
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2
2
2
2
2
2
3
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5
6
mediated PCRꢀtargeting method. The mutant fosmids were introduced into E.
coli ET12567/pUZ8002 for conjugation with XZQH13/pJTU824ꢀastG1(OE).
Apramycin resistant exconjugants were selected and verified by PCR to
generate XZQH13OEꢁastD1, XZQH13OEꢁastC and XZQH13OEꢁastF1,
respectively (Supplementary Figure S4).
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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7
8
9
0
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8
9
0
In vitro assays for AstC and AstCS545A
The holoꢀAstC and holoꢀAstCS545A were obtained by incubating apoꢀAstC or
apoꢀAstCS545A (2.5 ꢀM) and CoA (100 ꢀM) with Sfp (1 ꢀM) in a 50 ꢀL reaction
2
containing TrisꢀHCl (50 mM, pH 8.0), MgCl (10 mM), and DTT (1 mM). After 1
h incubation at 30 °C to prime the Tꢀdomain, the reaction was initiated by the
addition of ATP (2 mM), amino acids (1 mM), and compound 3 (26 ꢀM). After
incubation for 2 h at 30 °C, a 100 ꢀL reaction was quenched by the addition of
100 ꢀL methanol and centrifuged for 10 min at 13,000 rpm. The supernatant
was analyzed by HPLC (Supporting Information). The details of
characterization of AstC see Supporting Information.
In vitro assay for AstF1
Six acylꢀSNACs were synthesized as the substrates for AstF1 (Supporting
Information). The reaction mixture of formation of compound 4 contained Trisꢀ
2
HCl (50 mM, pH 8.0), MgCl (10 mM), holoꢀAstC (2.5 ꢀM), compound 3 (26
ꢂM), Dꢀalanine (1 mM), ATP (2 mM), and DTT (1 mM). After incubation for 2 h
at 30 °C to synthesize 4, the reaction was initiated by the addition of acylꢀ
SNAC (2 mM), glycerol (5%), AstF1 (5 ꢀM) and DTT (1 mM). Reactions
without AstF1 were served as the control. After incubation for 5 h at 30 °C, a
75 ꢀL reaction was quenched by the addition of 75ꢀL methanol and
centrifuged for 10 min at 13,000 rpm. The supernatant was analyzed by LCꢀ
MS similar as that for AstC, but in positive mode (Supporting Information).
Associated Content
Supporting Information
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The isolation of compound 3, the synthesis of acylꢀSNACs, Tables S1–3, and
Figures S1–30 are available free of charge via the Internet from
http://pubs.acs.org.
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Author Information
Corresponding Author
*
Eꢀmail: wanghaoxin@sdu.edu.cn (H. Wang), yshen@sdu.edu.cn (Y. Shen).
Notes
The authors declare no competing financial interests.
Acknowledgements
This work was jointly supported by the 973 programs (2012CB721005,
2
010CB833802), the NSFC projects (81530091, 31570039), and the Program
for Changjiang Scholars and Innovative Research Team in University
IRT13028).
(
References
(
1) Santa Maria, J. P., Jr., Sadaka, A., Moussa, S. H., Brown, S., Zhang, Y. J., Rubin,
E. J., Gilmore, M. S., and Walker, S. (2014) Compound-gene interaction
mapping reveals distinct roles for Staphylococcus aureus teichoic acids, Proc.
Natl. Acad. Sci. U S A 111, 12510–12515.
(2) Roy, H. (2009) Tuning the properties of the bacterial membrane with
aminoacylated phosphatidylglycerol, IUBMB Life 61, 940–953.
(
3) Henderson, J. C., Fage, C. D., Cannon, J. R., Brodbelt, J. S., Keatinge-Clay, A. T.,
and Trent, M. S. (2014) Antimicrobial peptide resistance of Vibrio cholerae
results from an LPS modification pathway related to nonribosomal peptide
synthetases, ACS Chem. Biol. 9, 2382–2392.
(
4) Sugita, M., Sasaki, T., Furihata, K., Seto, H., and Otake, N. (1982) Studies on
mycotrienin antibiotics, a novel class of ansamycins. II. structure elucidation
and biosynthesis of mycotrienins I and II, J. Antibiot. 35, 1467–1473.
5) Kang, Q., Shen, Y., and Bai, L. (2012) Biosynthesis of 3,5-AHBA-derived natural
products, Nat. Prod. Rep. 29, 243–263.
6) Chen, S., von Bamberg, D., Hale, V., Breuer, M., Hardt, B., Muller, R., Floss, H.
G., Reynolds, K. A., and Leistner, E. (1999) Biosynthesis of ansatrienin
(mycotrienin) and naphthomycin. Identification and analysis of two separate
(
(
13
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ACS Chemical Biology
Page 14 of 16
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
biosynthetic gene clusters in Streptomyces collinus Tu 1892, Eur. J. Biochem.
261, 98–107.
7) Qu, X., Lei, C., and Liu, W. (2011) Transcriptome mining of active biosynthetic
(
(
(
pathways and their associated products in Streptomyces flaveolus
, Angew.
Chem. Int. Ed. 50, 9651–9654.
8) Wu, T. S., Duncan, J., Tsao, S. W., Chang, C. J., Keller, P. J., and Floss, H. G.
1987) Biosynthesis of the ansamycin antibiotic ansatrienin (mycotrienin) by
Streptomyces collinus J. Nat. Prod. 50, 108–118.
(
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
,
9) Wang, H., Chen, Y., Ge, L., Fang, T., Meng, J., Liu, Z., Fang, X., Ni, S., Lin, C.,
Wu, Y., Wang, M., Shi, N., He, H., Hong, K., and Shen, Y. (2013) PCR
screening reveals considerable unexploited biosynthetic potential of
ansamycins and a mysterious family of AHBA-containing natural products in
actinomycetes, J. Appl. Microbiol. 115, 77–85.
(10) Li, S., Li, Y., Lu, C., Zhang, J., Zhu, J., Wang, H., and Shen, Y. (2015) Activating
a cryptic ansamycin biosynthetic gene cluster to produce three new
naphthalenic octaketide ansamycins with
n-pentyl and n-butyl side chains,
Org. Lett. 17, 3706–3709.
(
(
(
(
11) Zhang, J., Qian, Z., Wu, X., Ding, Y., Li, J., Lu, C., and Shen, Y. (2014)
Juanlimycins A and B, ansamycin macrodilactams from Streptomyces sp., Org.
Lett. 16, 2752–2755.
12) Xie, C., Deng, J., and Wang, H. (2015) Identification of AstG1, a LAL family
regulator that positively controls ansatrienins production in Streptomyces sp.
XZQH13, Curr. Microbiol. 70, 859–864.
13) Smith, A. B., Barbosa, J., Hosokawa, N., Naganawa, H., and Takeuchi, T. (1998)
(
+)-Thiazinotrienomycin E: relative and absolute stereochemistry,
Tetrahedron Lett. 39, 2891–2894.
14) Li, S., Lu, C., Chang, X., and Shen, Y. (2015) Constitutive overexpression of
asm18 increases the production and diversity of maytansinoids in
Actinosynnema pretiosum
, Appl. Microbiol. Biotechnol., Epub ahead of print
(DOI: 10.1007/s00253-00015-07127-00257).
(15) Lu, C., Li, Y., Deng, J., Li, S., Shen, Y., Wang, H., and Shen, Y. (2013)
Hygrocins C-G, cytotoxic naphthoquinone ansamycins from gdmAI-disrupted
Streptomyces sp. LZ35, J. Nat. Prod. 76, 2175–2179.
16) Wackler, B., Schneider, P., Jacobs, J. M., Pauly, J., Allen, C., Nett, M., and
Hoffmeister, D. (2011) Ralfuranone biosynthesis in Ralstonia solanacearum
suggests functional divergence in the quinone synthetase family of enzymes,
Chem. Biol. 18, 354–360.
17) Balibar, C. J., Howard-Jones, A. R., and Walsh, C. T. (2007) Terrequinone A
biosynthesis through L-tryptophan oxidation, dimerization and bisprenylation,
Nat. Chem. Biol. 3, 584–592.
18) Zhu, J., Chen, W., Li, Y. Y., Deng, J. J., Zhu, D. Y., Duan, J., Liu, Y., Shi, G. Y.,
Xie, C., Wang, H. X., and Shen, Y. M. (2014) Identification and catalytic
characterization of a nonribosomal peptide synthetase-like (NRPS-like)
enzyme involved in the biosynthesis of echosides from Streptomyces sp. LZ35,
Gene 546, 352–358.
19) Ishikawa, F., Konno, S., Suzuki, T., Dohmae, N., and Kakeya, H. (2015) Profiling
nonribosomal peptide synthetase activities using chemical proteomic probes
for adenylation domains, ACS Chem. Biol. 10, 1989–1997.
20) Ishikawa, F., Miyamoto, K., Konno, S., Kasai, S., and Kakeya, H. (2015)
Accurate detection of adenylation domain functions in nonribosomal peptide
synthetases by an enzyme-linked immunosorbent assay system using active
site-directed probes for adenylation domains, ACS Chem. Biol. 10, 2816–2826.
(
(
(
(
(
14
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Page 15 of 16
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1
2
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4
5
6
7
8
9
(21) Wang, F., Wang, Y., Ji, J., Zhou, Z., Yu, J., Zhu, H., Su, Z., Zhang, L., and
Zheng, J. (2015) Structural and functional analysis of the loading
acyltransferase from avermectin modular polyketide synthase, ACS Chem.
Biol. 10, 1017–1025.
(
22) Gay, D. C., Spear, P. J., and Keatinge-Clay, A. T. (2014) A double-hotdog with a
new trick: structure and mechanism of the trans-acyltransferase polyketide
synthase enoyl-isomerase, ACS Chem. Biol. 9, 2374–2381.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
(
23) Koryakina, I., McArthur, J., Randall, S., Draelos, M. M., Musiol, E. M., Muddiman,
D. C., Weber, T., and Williams, G. J. (2013) Poly specific trans-acyltransferase
machinery revealed via engineered acyl-CoA synthetases, ACS Chem. Biol. 8
,
200–208.
(
24) Zaleta-Rivera, K., Xu, C., Yu, F., Butchko, R. A., Proctor, R. H., Hidalgo-Lara, M.
E., Raza, A., Dussault, P. H., and Du, L. (2006) A bidomain nonribosomal
peptide synthetase encoded by FUM14 catalyzes the formation of tricarballylic
esters in the biosynthesis of fumonisins, Biochemistry 45, 2561–2569.
(25) Lin, S., Van Lanen, S. G., and Shen, B. (2009) A free-standing condensation
enzyme catalyzing ester bond formation in C-1027 biosynthesis, Proc. Natl.
Acad. Sci. U S A 106, 4183–4188.
(
26) Lin, S., Huang, T., Horsman, G. P., Huang, S.-X., Guo, X., and Shen, B. (2012)
Specificity of the ester bond forming condensation enzyme SgcC5 in C-1027
biosynthesis, Org. Lett. 14, 2300–2303.
27) Moss, S. J., Bai, L., Toelzer, S., Carroll, B. J., Mahmud, T., Yu, T. W., and Floss,
H. G. (2002) Identification of Asm19 as an acyltransferase attaching the
(
biologically essential ester side chain of ansamitocins using
desepoxymaytansinol, not maytansinol, as its substrate, J. Am. Chem. Soc.
24, 6544–6545.
N-desmethyl-4,5-
1
(
28) Song, Y. N., Jiao, R. H., Zhang, W. J., Zhao, G. Y., Dou, H., Jiang, R., Zhang, A.
H., Hou, Y. Y., Bi, S. F., Ge, H. M., and Tan, R. X. (2015) New ansamycin
derivatives generated by simultaneous mutasynthesis, Org. Lett. 17, 556–559.
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The A-T-TE-tridomain NRPS protein AstC catalyzes the D-alanylation of C-11 hydroxyl group, and the
acyltransferase AstF1 catalyzes the sequential attachment of acyl group to the D-alanyl nitrogen during the
biosynthesis of ansatrienins.
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