A Highly Promiscuous ?-Ketoacyl-ACP Synthase (KAS) III-like Protein Is Involved in Pactamycin Biosynthesis

Article abstract of DOI:10.1021/acschembio.6b01043

β-Ketoacyl-acyl carrier protein (β-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein (PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous, recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS III-like proteins may be used as versatile tools for modifications of complex natural products.

Full text of DOI:10.1021/acschembio.6b01043

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Letter
A Highly Promiscuous #-Ketoacyl-ACP Synthase (KAS)
III-like Protein is Involved in Pactamycin Biosynthesis
Mostafa E. Abugrain, Corey J. Brumsted, Andrew R. Osborn, Benjamin Philmus, and Taifo Mahmud
ACS Chem. Biol., Just Accepted Manuscript • DOI: 10.1021/acschembio.6b01043 • Publication Date (Web): 06 Jan 2017
Downloaded from http://pubs.acs.org on January 9, 2017
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A Highly Promiscuous ß-Ketoacyl-ACP Synthase (KAS) III-like Protein is
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Involved in Pactamycin Biosynthesis
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Mostafa E. Abugrain,†,¶ Corey J. Brumsted,‡,¶ Andrew R. Osborn,† Benjamin Philmus,† and
Taifo Mahmud†,‡,*
†Department of Pharmaceutical Sciences, Oregon State University, Corvallis, Oregon 97333,
United States
‡Department of Chemistry, Oregon State University, Corvallis, Oregon 97333, United States
*To whom correspondence should be addressed
¶These authors contributed equally
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ABSTRACT
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β-Ketoacyl-acyl carrier protein (β-Ketoacyl-ACP) synthase (KAS) III catalyzes the first step in
fatty acid biosynthesis, involving a Claisen condensation of the acetyl-CoA starter unit with the
first extender unit, malonyl-ACP, to form acetoacetyl-ACP. KAS III-like proteins have also been
reported to catalyze acyltransferase reactions using coenzyme A esters or discrete ACP-bound
substrates. Here, we report the in vivo and in vitro characterizations of a KAS III-like protein
(PtmR), which directly transfers a 6-methylsalicylyl moiety from an iterative type I polyketide
synthase to an aminocyclopentitol unit in pactamycin biosynthesis. PtmR is highly promiscuous,
recognizing a wide array of S-acyl-N-acetylcysteamines as substrates to produce a suite of
pactamycin derivatives with diverse alkyl and aromatic features. The results suggest that KAS
III-like proteins may be used as versatile tools for modifications of complex natural products.
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β-Ketoacyl-acyl carrier protein synthase (KAS) enzymes play a central role in type I
(modular) and type II (dissociable) fatty acid synthases (FASs) and polyketide synthases (PKSs),
as well as in chalcone synthases.1 In the type II dissociable FASs, three types of KAS enzymes
are known: KAS I (FabB) and KAS II (FabF) are responsible for the elongation steps, whereas
KAS III (FabH) catalyzes the initiation step, involving a Claisen condensation of the acetyl-CoA
starter unit with the first extender unit, malonyl-ACP, to form acetoacetyl-ACP (Figure S1a).1 In
Streptomyces, KAS III can also recruit alternative starter units, such as isobutyryl-CoA and
methylbutyryl-CoA, to form branched chain fatty acids. Less efficiently, it can also catalyze
acyl-CoA:ACP transacylase (ACAT) reactions.2
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Over the past decade, a number of KAS III-like enzymes have been reported to have unusual
catalytic functions. The cloN2 gene of the clorobiocin cluster in Streptomyces roseochromogenes
var. oscitans has been found through a gene inactivation study to be responsible for the transfer
of the pyrrolylcarbonyl unit to the sugar moiety of clorobiocin.3 The KAS III-like protein CerJ
uses malonyl-CoA analogs to form sugar esters in cervimycin biosynthesis (Figure S1b),4
whereas ChlB6 from the chlorothricin pathway transfers a 3-chloro-6-methoxy-2-methylbenzoyl
moiety from a discrete ACP, ChlB2, to a sugar moiety of chlorothricin (Figure S1c).5 The 3-
chloro-6-methoxy-2-methylbenzoyl unit is derived from a 6-methylsalicylyl (6MSA) unit, a
product of an iterative type I PKS (ChlB1). Incorporation of this PKS product to the sugar
moiety of chlorothricin requires multiple intermediary acyl transfer reactions involving two
discrete KAS III-like proteins (ChlB3 and ChlB6) and a discrete ACP (ChlB2) (Figure S1c).5
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Genes encoding proteins homologous to ChlB6 have been found in a number of natural
products biosynthetic gene clusters, such as aviN of the avilamycin A cluster in Streptomyces
viridochromogenes Tü57,6 evrI of the evernimicin cluster in Micromonospora carbonacea var.
africana ATCC 39149,7 calO4 of the calicheamicin cluster in Micromonospora echinospora
subsp. calichensis,8 pokM2 of the polyketomycin cluster in Streptomyces diastatochromogenes
Tü6028,9 tiaF of the tiacumicin cluster in Dactylosporangium aurantiacum subsp. hamdenensis
NRRL 18085,10 and esmD1 of the esmeraldin pathway in Streptomyces antibiotics Tü 2706.11
Except esmeraldin, all of these natural products contain one or more sugar moieties decorated by
6MSA or orsellinic acid derivatives. However, in contrast to the chlorothricin pathway, they lack
the genes that code for the second KAS III and the discrete ACP in their clusters.
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A gene encoding a protein homologous to KAS III was also found in the pactamycin cluster
in Streptomyces pactum (Figure S2).12 This gene (ptmR) may be involved in the attachment of
6MSA, which is synthesized by the iterative type I PKS PtmQ,12 to the aminocyclopentitol core
unit in pactamycin biosynthesis. Also present in the cluster are genes that code for a KAS I
protein (PtmK), a discrete ACP (PtmI), and a putative hydrolase/acyltransferase (PtmO). The
roles of these genes in pactamycin biosynthesis are currently unknown, but their possible
involvement in the attachment of 6MSA cannot be ruled out.
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O
H3C
H3C
O
H3C
H3C
O
N
N
NH2
NH2
NH2
O
O
NH
O
NH
H
NH
H
N
H
N
O
7
N
H3C
CH3
CH3
H3C
H3C
CH3
7
R1
H3C
9
9
9
H3C
H3C
HO OH
HO OH
OR
HO OH
OR2
OR
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O
O
O
Pactamycate (4)
Pactamycin (1)
2
1
H3C
OH
R= H3C
OH
H3C
OH
R =OH, R =
TM-026 (6) R=
De-6MSA-pactamycin (2)
R1=OH, R2=H
De-6MSA-pactamycate (5) R=H
TM-025 (7) R=H
De-6MSA-7-deoxypactamycin (3)
R1=R2=H
Figure 1. Chemical structures of pactamycin analogs.
RESULTS AND DISCUSSION
To investigate the involvement of ptmI, ptmK, ptmO, and ptmR in the transfer of the 6MSA
moiety, we generated mutant strains of S. pactum by either in-frame deletion or gene disruption
with aac(3)IV (Figures S3 and S4), and characterized their products by ESI-MS and HPLC.
Inactivation of ptmI, ptmK, or ptmO completely abrogated the production of pactamycin and its
analogs (Figure S5),12 suggesting that these genes are involved in the early steps of the pathway,
not in the attachment of 6MSA, which occurs later in the pathway.12, 13 On the other hand, the
ΔptmR mutant produced de-6MSA-pactamycin (2) and its degradation product, de-6MSA-
pactamycate (5) (Figures 1, S6a and S7), consistent with the absence of 6MSA-transferase
activity in this mutant. Furthermore, we inactivated ptmR in a mutant strain of S. pactum,
ΔptmH, which produces 7-deoxy-7-demethylpactamycin (TM-026, 6).14 The double gene
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knockout mutant, ΔptmH/ΔptmR, produces de-6MSA-7-deoxy-7-demethylpactamycin (TM-025,
7) (Figures S6g and S8), confirming the function of PtmR as a 6MSA-transferase. These results
were further corroborated by a gene complementation experiment, in which an integrative
plasmid harboring intact ptmR was introduced into ΔptmH/ΔptmR. As expected, the resulting
conjugants were able to produce TM-026 (Figure S6f).
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To characterize the catalytic function of PtmR in vitro, we cloned the gene in the expression
vector pET-20b(+) and heterologously expressed it in Escherichia coli BL21(DE3) pLysS to give
a 39-kDa C-terminal His6-tagged protein (Figure S9). Initial enzymatic experiments were carried
out using cell-free extracts of E. coli containing PtmR, with de-6MSA-pactamycin (2), de-
6MSA-7-deoxypactamycin (3), de-6MSA-pactamycate (5), or TM-025 (7) as substrates. The
synthetically prepared N-acetylcysteamine (NAC) thioester of 6MSA, which mimics an ACP-
bound substrate, was used as a model acyl donor substrate. ESI-MS and HPLC analyses of the
reaction products revealed the conversion of the de-6MSA analogs to their corresponding 6MSA
esters (Figures S10 and S11). Similar experiments using purified PtmR protein and de-6MSA-
pactamycin also gave pactamycin, and its degradation product, pactamycate (Figure 2), which
unambiguously confirm the 6MSA-transferase activity of PtmR. Incubations of the enzyme and
TM-025 with 6MSA free acid as substrate did not give any products, indicating PtmR only
recognizes an activated substrate (data not shown). Moreover, addition of 6MSA to ΔptmQ and
ΔptmQ/ΔptmH cultures did not give pactamycin or TM-026 (Figure S12), suggesting that free
6MSA is not involved in the pathway.
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a)
b)
c)
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boiled PtmR + 2 + 6MSA-SNAC
*
1
*
PtmR + 2 + 6MSA-SNAC
Pactamycin (1)
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d)
e)
Pactamycate (4)
6MSA-SNAC
2
f)
de-6MSA-pactamycin (2)
5
g)
de-6MSA-pactamycate (5)
7.5
10.0 12.5 15.0 17.5 20.0 22.5 25.0
27.5 30.0 32.5 35.0 37.5
Minutes
Figure 2. HPLC analyses of PtmR reactions using de-6MSA-pactamycin (2) and 6MSA-
SNAC as substrates. Stars indicate the products of PtmR. Compounds 1 and 2 can non-
enzymatically convert to 4 and 5, respectively.
However, in saphenamycin biosynthesis, it is proposed that 6MSA is first activated by a
6MSA adenylase (EsmD2) to its AMP derivative and loaded onto a carrier protein (EsmD3)
before being transferred to saphenic acid by EsmD1, a ChlB6 homologue.11 In fact, a gene
encoding a putative AMP-forming acyl-CoA synthetase is present in the pactamycin cluster. The
gene product (PtmS) has been proposed to catalyze the activation of 3-aminobenzoic acid
(3ABA), the precursor of the 3-aminoacetophenone moiety of pactamycin.12, 15 However, its role
in the pactamycin pathway has yet to be experimentally established.
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To investigate the possible involvement of PtmS in the 6MSA transfer, we inactivated the
gene in S. pactum by in frame-deletion and analyzed the products by ESI-MS. Similar to the
ΔptmI, ΔptmK, and ΔptmO mutants, ΔptmS was not able to produce pactamycin or its analogs
(Figure S13), suggesting that it is involved in an early step of the pathway. However, the results
cannot rule out the possibility of PtmS playing a dual role in the pathway – that is activating both
3ABA and 6MSA. This may also be the case with the discrete ACP PtmI. To test this possibility,
we carried out co-culture experiments using ΔptmQ/ΔptmH + ΔptmI and ΔptmQ/ΔptmH +
ΔptmS. Co-cultures of ΔptmQ/ΔptmH + ΔptmJ (which lacks the glycosyltransferase activity)12
were used as a positive control. It is expected that the ΔptmQ/ΔptmH mutant product (TM-025)
will be taken up by ΔptmJ and converted to TM-026. A similar phenomenon should occur in the
ΔptmQ/ΔptmH + ΔptmI and ΔptmQ/ΔptmH + ΔptmS cultures. However, if PtmI and/or PtmS are
involved in the 6MSA attachment, no conversion from TM-025 to TM-026 should be observed.
As expected, the ΔptmQ/ΔptmH + ΔptmJ cultures produced TM-026 as a major metabolite
(Figure S14). The same result was also observed in the cultures of ΔptmQ/ΔptmH + ΔptmI and
ΔptmQ/ΔptmH + ΔptmS, indicating that both PtmI and PtmS do not play a role in 6MSA
attachment. Altogether, the results show that PtmR is responsible for a direct transfer of the
6MSA moiety from the iterative type I PKS PtmQ to the aminocyclopentitol unit in pactamycin
biosynthesis (Figure 3).
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O
H3C
H3C
PtmQ
N
NH2
O
NH
AT
H
N
O
H3C
H3C
DH
H3C
HO
N
NH2
CH3
KS
KR
O
NH
ACP
H
N
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KAS III-like
(PtmR)
H3C
HO
H3C
HO OH
O
CH3
+
O
S
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H3C
O
HO OH
OH
OH
De-6MSA-pactamycin (2)
OH
Pactamycin (1)
Figure 3. Proposed catalytic activity of PtmR.
The fact that PtmR does not only process de-6MSA-pactamycin but also de-6MSA-7-
deoxypactamycin, de-6MSA-pactamycate, and TM-025 indicates that it has relaxed substrate
specificity. To explore if PtmR can also transfer acyl groups other than 6MSA, we synthesized an
additional 13 NAC thioesters with a variety of alkyl and aromatic features ranging from acetyl-
SNAC to 3-aminobenzoyl-SNAC to cycloheptanecarbonyl-SNAC (Table 1), and incubated them
with TM-025 and a cell-free extract of E. coli containing PtmR. ESI-MS analysis of the
enzymatic reactions revealed the ability of PtmR to use all 13 NAC thioesters, resulting in a suite
of new pactamycin analogs with diverse functionalities (Table 1 and Figures S10, S15–S17).
Parallel experiments with benzoyl-CoA, acetyl-CoA, propionyl-CoA, and butyryl-CoA did not
give any products (Figure S18), indicating that PtmR does not recognize CoA esters as
substrates. Scaled-up enzymatic reactions using 4-chlorobutyryl-SNAC furnished TM-107,
whose complete chemical structure was determined by 1D and 2D NMR spectroscopic data
(Figures S48–S52).
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Table 1. Conversion of TM-025 to various pactamycin analogs by PtmR.
7
8
9
O
O
H3C
H3C
H3C
H3C
N
N
NH2
NH2
O
O
NH
NH
H
N
H
N
PtmR
7
7
CH3
CH3
H3C
H3C
9
H
N
9
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H3C
H3C
HO OH
HO OH
S
R
OH
OR
O
TM-025
HR ESI-MS (m/z)
Relative
Mol.
Formula
Conversiona
R
Product
Calcd
Observed
O
529.2657
529.2664
TM-026
TM-104
TM-105
TM-106
TM-107
C27H36N4O7
C21H32N4O6
C22H34N4O6
C23H36N4O6
100%
71%
[M+H]+
[M+H]+
OH
O
437.2395
437.2396
[M+Na]+
M+Na]+
O
473.2371
473.2383
87%
[M+Na]+
[M+Na]+
O
465.2708
465.2718
91%
[M+H]+
[M+H]+
O
499.2318
499.2327
Cl
C23H35ClN4O6
C23H36N4O6
100%
[M+H]+
[M+H]+
O
465.2708
465.2718
TM-108
83%
[M+H]+
[M+H]+
479.2864
479.2874
O
TM-109
TM-110
TM-111
TM-112
C24H38N4O6
C26H40N4O6
C24H38N4O6
C26H34N4O6
61%
44%
20%
43%
[M+H]+
[M+H]+
O
505.3021
505.3025
[M+H]+
[M+H]+
O
479.2864
479.2872
[M+H]+
[M+H]+
O
499.2551
499.2558
[M+H]+
[M+H]+
O
514.2660
514.2690
H2N
TM-113
C26H35N5O6
17%
[M+H]+
[M+H]+
O
513.2708
513.2738
TM-114
TM-115
TM-116
C27H36N4O6
C26H40N4O6
C27H42N4O6
95%
57%
87%
[M+H]+
[M+H]+
O
505.3021
505.3022
[M+H]+
[M+H]+
O
519.3177
519.3182
[M+H]+
[M+H]+
aBased on relative intensity of (+)-ESI-MS substrate (m/z 395) and observed product peak
The ability of PtmR to utilize a broad range of substrates is somewhat surprising, as so far
KAS III-like proteins, e.g., CloN2, ChlB6, CalO4, AviN, and EvrI, have only been associated
with the transfer of an aryl or pyrrolylcarbonyl group to a sugar moiety. A phylogenetic analysis
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of PtmR and homologous proteins showed that PtmR falls within a clade of KAS III-like
enzymes, but it forms a separate sub-clade together with a number of hypothetical proteins from
Streptomyces niveiscabiei NRRL B-24457, Streptomyces acidiscabies 84-104, and Streptomyces
sp. DSM 15324 (Figure S19). Genome mining studies revealed that these genes are part of gene
clusters that resemble the pactamycin cluster of S. pactum. Similar to ptmR, they are also located
next to a gene encoding an iterative type I PKS (6MSA synthase). Therefore, we predict that the
encoding proteins are also responsible for the attachment of 6MSA or its analogs to the
corresponding natural products.
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In conclusion, the present work showed that a highly promiscuous KAS III-like enzyme is
responsible for the attachment of 6MSA directly from an iterative type I PKS to an
aminocyclopentitol unit in pactamycin biosynthesis. The results also serve as a starting point for
the development of KAS III-like proteins as versatile tools for creation of new libraries of
complex natural products.
METHODS
Construction of ΔptmR and ΔptmH/ΔptmR mutant strains. The target genes were
inactivated using a gene in-frame deletion strategy (Figure S3). Two ~1 kb PCR fragments
upstream (HindIII/EcoRI) and downstream (EcoRI/XbaI) of the ptmR gene were fused and
cloned into the HindIII/XbaI site of pBluescript II SK(–) vector to generate pTMM056 (Table
S2). The fused PCR fragment was excised and cloned into the HindIII/XbaI site of pTMN002 to
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generate pTMM057. The plasmid was then introduced into the wild-type and the ΔptmH mutant
strains of S. pactum ATCC 27456 by conjugation using E. coli ET12567/pUZ8002 as a donor
strain. Apramycin resistant strains representing single crossover mutants were obtained and
grown on BTT [glucose (1%), yeast extract (0.1%), beef extract (0.1%), casein hydrolysate
(0.2%), agar (1.5%), pH 7.4] agar plates containing apramycin (50 µg mL-1). Subsequently,
apramycin sensitive colonies were counter-selected by replica plating on BTT agar with and
without apramycin (50 µg mL-1). The resulting double-crossover candidate strains were
confirmed by PCR amplification with ptmR-F1 and ptmR-R2 primers (Table S3) flanking the
respective targeted gene (Figure S3).
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Complementation of ΔptmH/ΔptmR mutant. For complementation of the
ΔptmH/ΔptmR mutant, the ptmR gene was amplified by PCR using the primers PtmR-pET-F
and PtmR-C-R (Table S3). The PCR products were digested with BglII and EcoRI and ligated
into the BamH/EcoRI sites of the cloning vector pBluescript II SK(–) to generate pTMM058.
DNA sequencing confirmed the correct sequence of the construct. The resulting plasmid was
digested with Ndel/EcoRI and the DNA fragment was ligated into the integration vector
pTMW50, predigested with the same restriction enzymes, to generate pTMM059. The
pTMM059 plasmid was then transferred into E. coli ET12567/pUZ8002, which was
subsequently used to transform ΔptmH strain by conjugation.16 Selection of the exconjugants
(ΔptmH/ΔptmR mutants) was performed on BTT agar containing apramycin (50 µg mL-1) and
ampicillin (100 µg mL-1).
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Analysis of ΔptmR, ΔptmH/ΔptmR, and ΔptmH/ΔptmR+pTMM059 metabolic profiles.
The ΔptmR, ΔptmH/ΔptmR, and ΔptmH/ΔptmR+pTMM059 strains were grown on BTT agar at
30 °C for 3 days. Single colonies were used to inoculate the BTT seed cultures [medium for
ΔptmH/ΔptmR+pTMM059 was supplemented with apramycin (10 µg mL-1) and ampicillin (20
µg mL-1)] and incubated at 30 °C for 2 days. Production cultures were prepared in modified
Bennett’s medium (50 mL)12 and inoculated with seed cultures [10% (v/v)]. The production
cultures were incubated at 30 °C for 5 days under vigorous shaking (200 rpm). The mycelia were
centrifuged and the supernatants were extracted twice with equal volumes of EtOAc followed by
extraction with n-BuOH. The organic solvent from each extraction was evaporated in vacuo and
the residues dissolved in MeOH and analyzed by reversed-phase HPLC and/or ESI-MS. Analysis
of the metabolites of the mutants and those complemented with ptmR was carried out by
reversed-phase HPLC with a C-18 column (Supelcosil LC-18-DB 15 cm x 4.6 cm, 5 µm) using
H2O [95% (v/v)] and CH3CN [5% (v/v)] containing TFA [0.1% (v/v)] as mobile phase at 1 mL
min-1 flow rate.
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Construction of ΔptmI, ΔptmO, and ΔptmS mutant strains. The target genes were
inactivated using a gene in-frame deletion strategy (Figure S3a). For details, see the Supporting
Information.
Construction of ptmK::aac(3)IV mutant. The ptmK gene (1.7 kb) was inactivated using a gene
disruption strategy (Figure S3b). For details, see the Supporting Information.
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Feeding experiments with 6-MSA to ΔptmQ and ΔptmH/ΔptmQ mutants. The ΔptmQ and
ΔptmH/ΔptmQ mutants were streaked on BTT agar [glucose (1% (w/v)), yeast extract (0.1%
(w/v)), beef extract (0.1% (w/v)), casein hydrolysate (0.2% (w/v)), agar (1.5% (w/v)), pH 7.3]
and incubated at 30 °C for 3 days. Spores of the ΔptmQ and ΔptmH/ΔptmQ mutants were
individually grown in two Erlenmeyer flasks (125 mL) containing seed medium [glucose (1%
(w/v)), yeast extract (0.1% (w/v)), beef extract (0.1% (w/v)), casein hydrolysate (0.2% (w/v)),
pH 7.3] (50 mL) for 3 days at 30°C and 200 rpm. Each of these seed cultures (10 mL) was used
to inoculate 4 Erlenmeyer flasks (250 mL) containing modified Bennett’s medium (100 mL).
After incubation for 18 h under the same conditions, the cultures were grouped into two groups;
the first group was supplemented with 6-MSA (5 mM, 250 µL) and the second group was used
as control. The feeding was repeated every 12 h for 2 days. All experiments were done in
triplicate. After five days of incubation, the cultures were centrifuged. The metabolites of each
group were extracted with ethyl acetate (2 x 100 mL). The organic solvent was evaporated using
rotary evaporator and the products were analyzed by MS.
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Co-culture experiments. For details on co-culture experiments with ΔptmJ, ΔptmI, ΔptmS, and
ΔptmH/ΔptmQ mutants, see the Supporting Information.
Production of PtmR. The ptmR gene was amplified using primer pairs PtmR-pET-F and PtmR-
pET-R (Table S3). High Fidelity Taq DNA Polymerase (Invitrogen) was used for PCR, and the
resulting 1.0-kb PCR product was cloned into the pET-20b(+) vector (Novagen) to generate
expression vector pTMM060, which was introduced into E. coli BL21(DE3) pLysS (Invitrogen).
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For protein production, the bacteria were grown in LB medium supplemented with ampicillin
(100 µg mL-1) and chloramphenicol (25 µg mL-1) at 30 °C with shaking at 250 rpm until an
OD600 of 0.8–1 was reached. The culture was shaken at 16 °C for 1 h. Protein expression was
induced by addition of IPTG (0.5 mM) with further cultivation for 36 h. The cells were harvested
by centrifugation and resuspended in sodium phosphate buffer (40 mM, pH 7.5) containing NaCl
(300 mM) and imidazole (10 mM), and then disrupted by sonication. After centrifugation of the
sample, the supernatant was directly loaded onto a Ni-NTA spin column (Qiagen). The
recombinant PtmR was eluted using sodium phosphate buffer (40 mM, pH 7.5) containing NaCl
(300 mM) and imidazole (250 mM). The PtmR-containing fractions were dialyzed (3 times, 5 h
each) in 1 L solution containing sodium phosphate buffer (20 mM, pH 7), MgCl2 (10 mM),
glycerol (10% (w/v)) and dithiothreitol (0.5 mM) at 4 °C.
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Acyltransferase assay. The acyltransferase reaction was typically carried out in 50 µL mixtures
containing TM-25 or de-6MSA-pactamycin (1 mM), NAC thioester (2 mM), purified PtmR or a
cell free extract of E. coli containing PtmR (47 µL), and MgCl2 (10 mM) in sodium phosphate
buffer (50 mM, pH 7.5). Boiled protein or cell free extract of empty pET-20b(+) was used as
negative control. The reaction was incubated at 30 °C. After 4 h, the reaction was quenched by
the addition of one volume MeOH, centrifuged at 14,000 rpm for 15 min, and the supernatants
were analysed by reversed-phase HPLC and/or ESI-MS.
Scaled-up enzymatic reaction and isolation of TM-107. Scaled-up enzymatic reactions (100
µL x 100, 10 mL total) were carried out using the cell free extract of E. coli containing PtmR in
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sodium phosphate buffer (40 mM, pH 7.5, contains 10 mM MgCl2 and 10% (w/v) glycerol), TM-
025 (1 mM), and 4-chlorobutyryl-SNAC (2 mM). The mixtures were incubated at 30 °C for 3 h.
The reactions were then quenched by addition of one volume of MeOH and centrifuged at
14,000 rpm for 15 min. The supernatants were pooled and subjected to rotary evaporator to
remove MeOH, and the aqueous mixture was extracted twice with two volumes of EtOAc. The
EtOAc extract was dried in vacuo and the residue was dissolved in MeOH (400 µL) and
subjected to reverse-phase HPLC (YMC-Pack ODS-A, 250 x 10 mm I.D., 5 µm), solvent
gradient: CH3CN-H2O (5:95) to CH3CN (100%) containing TFA [0.1% (v/v)] over 60 min, flow
rate 4.7 mL min-1, detection at 254 nm) to give TM-107 (1.2 mg).
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TM-107: a white powder, [α]D = +37° (c 0.267, MeOH, 20 °C); UV (MeOH) λmax 240 nm (ε 1.6
x 103), 264 nm (ε 4.65 x 102), and 354 (ε 24.4); IR (KBr, MeOH) ν 3359, 2919, 2856, 1679,
1536, 1483, 1206, and 1138 cm-1. 1H NMR (700 MHz, CD3OD): Table S5; 13C NMR (175 MHz,
CD3OD): Table S5.
Chemical synthesis of NAC thioesters. For details, see the Supporting Information.
Associate Content
Supporting Information
The Supporting Information is available free of charge on the ACS Publication Website at
DOI:…
Supporting methods, figures and tables (PDF).
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Author Information
9
Corresponding Authors
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*Address: 203 Pharmacy Building, Oregon State University, Corvallis, Oregon 97331, United
States. Phone: (541) 737-9679. Fax: (541) 737-3999. E-mail: Taifo.mahmud@oregonstate.edu.
Notes
The authors declare no competing financial interest.
Acknowledgements
The authors thank P. Proteau for a critical reading of this manuscript, N. Roongsawang, E. Wan-
Knight, and K. W. Wen for technical assistance, and A. deBarber for HR-ESI-MS analysis. The
project described was supported by the Oregon State University College of Pharmacy Faculty
Development Funds.
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cyclopentane ring formation, J. Antibiot. 60, 492-503.
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Streptomyces Genetics", The John Innes Foundation Norwich, England.
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Graphic for TOC
O
H3C
H3C
N
NH2
O
NH
H
N
H3C
HO
CH3
9
PtmQ
H3C
HO OH
OH
O
H3C
DH
AT
N
CH3
NH
NH2
O
NH
H3C
KR
H
N
O
H3C
HO
ACP
KS
CH3
9
KAS III-like
S
or
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HO OH
O
O
O
S
PtmR
O
OH
R
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O
H3C
H3C
N
NH2
O
NH
H
N
H3C
HO
CH3
9
PtmQ
H3C
HO OH
OH
O
H3C
DH
AT
N
CH3
NH
NH2
O
NH
H3C
KR
H
N
O
H3C
HO
ACP
3K4 S
CH3
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KAS III-like
S
or
H3C
HO OH
O
O
O
S
PtmR
O
OH
R
R