Fatty acyl incorporation in the biosynthesis of WAP-8294A, a group of potent anti-MRSA cyclic lipodepsipeptides

Article abstract of DOI:10.1039/c5ra20784c

WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2. Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of functionally overlapping ACLs.

Full text of DOI:10.1039/c5ra20784c

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Fatty acyl incorporation in the biosynthesis of
WAP-8294A, a group of potent anti-MRSA cyclic
lipodepsipeptides†
Cite this: RSC Adv., 2015, 5, 105753
*
*
Haotong Chen,‡ Andrew S. Olson,‡ Wei Su, Patrick H. Dussault and Liangcheng Du
WAP-8294A is a family of at least 20 cyclic lipodepsipeptides exhibiting potent anti-MRSA activity. These
compounds differ mainly in the hydroxylated fatty acyl chain; WAP-8294A2, the most potent member of
the family that reached clinical trials, is based on (R)-3-hydroxy-7-methyloctanoic acid. It is unclear how
the acyl group is incorporated because no acyl-CoA ligase (ACL) gene is present in the WAP-8294A
gene cluster in Lysobacter enzymogenes OH11. Here, we identified seven putative ACL genes in the
OH11 genome and showed that the yield of WAP-8294A2 was impacted by multiple ACL genes with the
ACL6 gene having the most significant effect. We then investigated several (R)-3-hydroxy fatty acids and
their acyl SNAC (N-acetylcysteamine) thioesters as substrates for the ACLs. Feeding (R)-3-hydroxy-7-
methyloctanoate-SNAC to the ACL6 gene deletion mutant restored the production of WAP-8294A2.
Finally, we heterologously expressed the seven ACL genes in E. coli and purified six of the proteins. While
these enzymes exhibit a varied level of activity in vitro, ACL6 showed the highest catalytic efficiency in
converting (R)-3-hydroxy-7-methyloctanoic acid to its CoA thioester when incubated with coenzyme A
and ATP. These results provided both in vivo and in vitro evidence to support the fact that ACL6 is the
main player for fatty acyl activation and incorporation in WAP-8294A2 biosynthesis. The results also
suggest that the molecular basis for the acyl chain diversity in the WAP-8294A family is the presence of
functionally overlapping ACLs.
Received 7th October 2015
Accepted 7th December 2015
DOI: 10.1039/c5ra20784c
www.rsc.org/advances
family, exhibit 14-fold greater anti-MRSA activity compared
with vancomycin; an IV formulation was in phase-I clinical
studies in 2010.12,17,18
Although WAP-8294A compounds were rst isolated nearly
two decades ago, the study of their biosynthetic mechanism was
reported only in 2011.11,12 Improved understanding of the
molecular mechanism of biosynthesis could open rational
approaches to generate new analogs with improved clinical
properties. We have identied the WAP biosynthetic gene
Introduction
The discovery of new anti-infective drugs is a pressing and
continual need for human health due to the constant emer-
gence of drug resistant pathogens.1,2 Nosocomial infections
caused by methicillin-resistant Staphylococcus aureus (MRSA)
have become an especially serious clinical problem. It is
imperative to discover and develop new antibiotics. Natural
products (NPs) are the most signicant source of anti-infectives3
and we have been using Lysobacter species as a new source for
bioactive NP discovery.4–10 WAP-8294A (Fig. 1), a family of
compounds displaying anti-MRSA activity, are cyclic lip-
odepsipeptides originally isolated from Lysobacter staph-
ylocidin.11–13 Cyclic depsipeptides belong to a large and diverse
family of nonribosomally synthesized peptides that have
a wide variety of important biological activities.14–16 WAP-
8294A2 (lotilibicin), the most potent antibacterial of the
Department of Chemistry, University of Nebraska-Lincoln, Lincoln, NE, 68588-0304,
USA. E-mail: ldu3@unl.edu; pdussault1@unl.edu
† Electronic supplementary information (ESI) available: Descriptions of primer
design and sequences, plasmid construction, protein expression and
purication, kinetic data, organic synthesis and detailed protocols for
experimental procedures. See DOI: 10.1039/c5ra20784c
‡ HC and ASO contribute equally.
Fig. 1 Structure of the two main members of the WAP-8294A family.
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cluster from L. enzymogenes OH11, which includes two large
nonribosomal peptide synthetase (NRPS) genes. The NRPSs
comprise a total of 12 modules that make 45 functional domains,
which are responsible for the assembly of the 12-amino acid
core structure of WAP-8294A compounds.11
The WAP-8294A family all contains a 3-acyloxy amide linkage
derived from a (R)-3-hydroxy fatty acyl chain; individual family
members differ in the length of the chain and the presence or
absence of a branching methyl group (Fig. 1). The fatty acids
must be activated before incorporation into the peptide. The
mechanism is likely to involve activation of fatty acids to their
corresponding acyl coenzyme A (CoA) thioesters by a family of
acyl CoA ligases (ACL). This catalytic process occurs in two steps
and involves requisite formation of an acyladenylate (acyl-AMP)
intermediate.19 Acyl CoA ligases are usually located within the
biosynthetic cluster associated with formation of the remainder
of the natural product. For example, the gene clusters for
amphi-enterobactin,20 daptomycin16 and calcium-dependent
antibiotic (CDA)21 all host a dedicated ACL and an ACP (acyl
carrier protein) for the activation and incorporation of the fatty
acyl chain. It is therefore interesting that there is no ACL gene
present in or near the WAP gene cluster,11 a phenomenon
previously observed with the gene cluster for surfactin.22 In
addition, no dedicated ACP is present within the WAP cluster.
These features suggest that the WAP-8492A biosynthetic
pathway likely recruits an ACL in trans and that the fatty acids
are directly activated as acyl-S-CoA thioesters, instead of acyl-S-
ACP, for the incorporation into the peptide core.
The aim of this work is to determine the molecular basis for
activation and incorporation of fatty acyl chains in the biosyn-
thesis of members of the WAP-8294A family, particularly for the
most eminent member WAP-8294A2. Here we describe both the
in vivo and in vitro results obtained from the study of seven
putative ACLs found in the genome of L. enzymogenes OH11.
Our study shows that multiple ACLs contribute to WAP-8294A
biosynthesis and that ACL6 is the most important enzyme in
the activation of (R)-3-hydroxy-7-methyloctanoic acid and
incorporation into WAP-8294A2.
Fig. 2 HPLC analysis of metabolites from various strains of Lysobacter
enzymogenes OH11. (A) OH11 wild type; (B–H) disruption mutants of
ACL1 through ACL7, respectively; (I) WAP-8294A2 standard. The
detection wavelength was 280 nm.
ACL6 in subsequent experiments. The results suggest that
multiple ACLs contribute to the fatty acyl chain activation and
incorporation in WAP-8294A2 biosynthesis, and the ACLs are
functionally redundant.
Results and discussion
Multiple acyl-CoA ligases contribute to WAP-8294A
Feeding fatty acyl precursor restored WAP-8294A2 production
in ACL6 in-frame deletion mutants
biosynthesis
To eliminate potential polar effects caused by insertion of
We identied seven putative ACL genes in the genome of the conjugal vector in the gene disruption mutants, we also
L. enzymogenes OH11 that are associated with secondary generated in-frame deletion mutants for ACL6 gene (Fig. S3†).
metabolism (Fig. S1A†). The ACL genes are either associated WAP-8294A2 production in this mutant strain was monitored
with NRPS-PKS gene clusters, or adjacent to genes related to by HPLC and MS (Fig. 3). Similar to that in the ACL6 disruption
biosynthesis and regulation of natural products, such as genes mutant, WAP-8294A2 production in the in-frame deletion
encoding lantibiotic-modifying enzyme, enoyl-CoA hydratase, mutant was nearly completely eliminated. We chemically
or two-component regulatory system. To test if any of the ACLs synthesized the N-acetyl cysteamine thioester (SNAC) of (R)-3-
is involved in WAP-8294A biosynthesis, we disrupted each of hydroxy-7-methyloctanoic acid (Fig. 4); details are provided in
the 7 ACL genes (Fig. S2 and S3†). The HPLC and MS analysis ESI.† This compound was used as the acyl-CoA surrogate to
showed that the disruption of ACL1, ACL5, and ACL7 had little be fed to the ACL6 deletion mutant. HPLC analysis clearly
impact on the yield of WAP-8294A2, whereas the disruption of showed that WAP-8294A2 production was restored in the ACL6
ACL2, ACL3, ACL4, and ACL6 signicantly reduced the yield mutant (Fig. 3). This result demonstrated that ACL6 is the
(Fig. 2). In particular, the ACL6 mutant produced a barely predominant enzyme responsible for fatty acyl incorporation
detectable amount of WAP-8294A2. Thus we mainly focused on into WAP-8294A2.
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Fig. 3 HPLC analysis of metabolites in strains of Lysobacter enzy-
mogenes OH11. (A) ACL6 in-frame deletion mutant; (B) the ACL6
mutant supplemented with (R)-3-hydroxy-7-methyloctanoic acyl-
SNAC; (C) WAP-8294A2 standard.
Fig. 5 HPLC and MS analysis of in vitro reaction products of ACL6
incubated with 3-hydroxy-7-methyloctanoic acid, ATP, and CoA. The
ESI-MS signals were measured in positive single ion mode. (A) HPLC of
control reaction (without enzyme); (B) HPLC of ACL6 reaction; (C) ESI-
MS analysis of the ACL6 reaction product, the distinct peak in HPLC as
indicated by a star. The calculated mass of 3-hydroxy-7-methyl-
octanoyl-CoA is 923.23 (see Fig. S5†).
Fig. 4 Chemical synthesis of (R)-3-hydroxy fatty acids and 3-hydroxy
fatty acyl-SNAC (acyl thioester of N-acetyl cysteamine).
hydroxyoctanoic acid, CoA and ATP (data not shown). The result
indicates that ACL5 is able to catalyze the acyl-CoA formation
from (R)-3-hydroxyoctanoic acid and CoA.
Heterologous expression of ACL genes and in vitro assay of the
enzyme activity
To further conrm the specicity of the enzymes, we per-
formed kinetic analysis, using EnzChek Pyrophosphate Assay
Kit. The inorganic pyrophosphatase in this kit catalyzes
conversion of PPi into two equivalents of Pi. The Pi is then
consumed by the 2-amino-6-mercapto-7-methyl-purine ribo-
nucleoside (MESG) by purine ribonucleoside phosphorylase
(PNP) and the product, 2-amino-6-mercapto-7-methyl purine, is
detected by an increase in absorbance at 360 nm. The EnzChek
Pyrophosphate Assay Kit had been used for the quantitation
of PPi in solution or for the continuous determination of PPi
released in enzymatic reactions.23–25 To measure the activity,
ACL was incubated with free fatty acid, ATP, CoA and the
coupled reaction system provided by the kit as described above.
The kinetic parameters were determined by initial velocity
We cloned the seven putative ACL genes in pET28a and heter-
ologously expressed the genes in E. coli. The expressed proteins
were puried, except ACL2 which was produced mostly as an
insoluble protein (Fig. S4†). As judged by SDS-PAGE, the
observed molecular masses of ACL1 through ACL7, including
the His6 tag, were consistent with the predicted masses (58.7,
58.8, 60.4, 59.8, 61.0, 47.2, 58.6 kDa) calculated from the amino
acid sequences (Fig. S4†).
The enzyme activity of ACLs 1–7 was measured by incubating
the enzymes with (R)-3-hydroxy-7-methyloctanoic acid, ATP, and
CoA. A distinct peak in HPLC was detected around 9.2 min in
the reaction containing ACL6. This peak was collected and
analyzed by MS, which gave a [M + H]+ of m/z 924.93, consistent
with the mass of 3-hydroxy-7-methyloctanoyl-CoA (calculated
mass 923.23) (Fig. 5 and S5†). Tandem mass spectrometry
was used to fragment the analytes into product ions. Both the
fatty acyl-CoA precursor ion (m/z 924.30) and its product ions
(m/z 428.10, 417.20) were detected, while the control reaction
(w/o enzyme) only gave the product ions of CoA (m/z 480.10 and
m/z 261.20). The results support the hypothesis that ACL6
catalyzes acyl-CoA formation from the fatty acid and CoA. Other
ACLs also showed some activity, but the product was formed
in such minute amounts that the peak could only be detected
by MS. In addition, we tested (R)-3-hydroxyoctanoic acid, which
corresponds to the fatty acyl portion of WAP-8294A1 (Fig. 1).
HPLC and MS data showed a peak around 9 min that gave
a [M + H]+ of m/z 911.00 when ACL5 was incubated with (R)-3-
Fig. 6 Proposed mechanism for fatty acid incorporation in WAP-
8294A2 biosynthesis. The fatty acid, (R)-3-hydroxy-7-methyloctanoic
acid, is activated by ACL6, and the resultant acyl-CoA is then recog-
nized by the condensation (C) domain of the NRPS, which catalyzes
the amide bond formation between the PCP-bound L-serine and the
fatty acyl-CoA.
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experiments using the coupled assay (Tables S2A and B†). These was demonstrated by the conversion of the chemically synthe-
data were globally tted to Michaelis–Menten equation, sized (R)-3-hydroxy fatty acids to the corresponding CoA thio-
yielding Km values for (R)-3-hydroxy-7-methyloctanoic acid and esters, when the puried ACLs were incubated with the
(R)-3-hydroxyoctanoic acid. The data conrmed that ACL6 substrate, CoA and ATP. The kinetic data revealed that (R)-3-
favored (R)-3-hydroxy-7-methyloctanoic acid as substrate, while hydroxy-7-methyloctanoic acid is the preferred substrate for
ACL5 had a faster rate when (R)-3-hydroxyoctanoic was the ACL6. These observations are consistent with the results ob-
substrate. The relatively slow reaction rates might explain the tained from ACL mutants and in vivo feeding experiment.
very low yield of WAP-8294A in L. enzymogenes.11 No steady Together, the results support that ACL6 is the most important
signal was detected in the enzyme-free or ATP-free control enzyme for fatty acyl incorporation into WAP-8294A2, and ACL5
reactions, showing that the detected signals resulted from the may be an important contributor for the fatty acyl incorporation
enzymatic activity of ACLs.
into WAP-8294A1. Based on the results presented in this study,
we propose a mechanism for the initiation of the biosynthesis
of WAPs, as shown in Fig. 6.
Conclusions
Cyclic lipopeptides are an important group of bioactive natural
products; this is exemplied by the new antibiotic daptomycin
(Cubicin). Although many of the peptides exhibit activity, WAP-
8294A2 is exceptionally potent against MRSA (ED50 14-fold
lower than vancomycin). WAP-8294A2 reached the phase-I
clinical studies, but appears to have been discontinued from
the clinical development.18 The reason for discontinuation has
not been published, but could indicate undesirable pharmaco-
properties. The understanding of WAP-8294A biosynthetic
mechanism is an essential step toward rational engineering the
biosynthetic machinery to produce new analogs with improved
properties. In WAP-8294A biosynthesis, the mechanism by
which the 3-hydroxy fatty acyl chain is incorporated into the
peptide is the least understood step.
To get more insights into the molecular basis for the fatty
acyl incorporation and WAP-8294A structural diversity, we
searched the genome of L. enzymogenes OH11 and identied
seven putative ACL genes. We disrupted each of the seven ACL
genes and found that none of the mutants completely lost
WAP production. The result supports the notion that multiple
ACL genes are involved in the acyl incorporation, and the
function of the ACLs is likely redundant. Among the seven
mutants, ACL6 appeared to have the highest impact on the yield
Experimental procedures
Chemicals, bacterial strains, plasmids, and general
procedures for DNA manipulation
Chemicals used in this study were purchased from Fisher
Scientic, Sigma, and Acros. Oligonucleotide primers for PCR
were synthesized by Eurons MWG Operon. Plasmid prepara-
tion and DNA extraction were carried out with Qiagen kits
(Valencia, CA), and all other DNA manipulations were carried
out according to standard methods. Ni-NTA agarose was
purchased from Qiagen (Valencia, CA). Escherichia coli strain XL
blue was used as the host for general plasmid DNA propagation,
and the cloning vector was pANT841. Vector pET28a was used
for protein expression in E. coli BL21 (DE3). E. coli S17-1 was
used as the conjugal strain to transfer DNA into Lysobacter. L.
enzymogenes and other bacterial strains were grown in Luria-
Bertani (LB) broth medium, 1/10-strength tryptic soy broth
(1/10 TSB, Sigma) or NYGB/A medium. EnzChek Pyrophosphate
Assay Kit was purchased from Sigma.
Generation of gene disruption mutants
of WAP-8294A2. To prove this, we generated in-frame deletion To construct plasmids for gene disruption, an internal fragment
mutant of ACL6 to eliminate potential polar effects caused by was amplied from each of the open reading frames (ORFs)
gene disruption. We then chemically synthesized the acyl-CoA using the primer pairs described in ESI (Table S1†). Genomic
surrogate, (R)-3-hydroxy-7-methyloctanoyl SNAC. WAP-8294A2 DNA from the wild type L. enzymogenes OH11 served as the PCR
production in the ACL6 deletion mutant was restored upon template. The homologous regions of these ACLs were ampli-
feeding the synthetic acyl-CoA surrogate. The result conrms ed by PCR and introduced into the conjugation vector
ACL6's role in WAP-8294A2 biosynthesis; it also suggests that pJQ200SK to produce pJQ200SK-ACLs. Each of the pJQ200SK
ACL6 is required for substrate activation, but not NRPS priming. constructs was transformed into E. coli S17-1, which was mated
The results support that the WAP-8492A biosynthetic pathway with L. enzymogenes OH11 for conjugal transfer of the vectors.
recruits ACL in trans and that the rst condensation domain The positive colonies grown on LB plates containing gentamicin
in the WAP-8492A NRPS can accept acyl-S-CoA for the incorpo- (20 mg mLꢀ1) were picked up and inoculated into liquid cultures
ration of the fatty acyl chain into the peptide core. Interestingly, containing gentamicin. Genomic DNA was prepared from each
a phylogenetic analysis of the condensation domains of a group of the cultures, and diagnostic PCR was performed to identify
of selected NRPS showed that the rst C domain of WAP-8492A mutants that resulted from a homologous recombination. To
NRPS (WAP_WAPS1_C1) falls into the same subgroup that screen the ACL gene disruption mutants, PCR was performed
includes the rst C domain of CDA and daptomycin, whose using the diagnostic primers listed in Table S1,† which would
biosynthesis uses acyl-ACP as substrate (Fig. S1B†).
amplify correct fragments when mutants resulted from
In the in vitro studies, we were able to express six of the seven a homologous recombination, but not from the wild type or
putative ACL genes in E. coli and to purify the proteins for mutants resulted from a random insertion of the construct
enzyme activity assay and kinetic studies. The enzyme activity (Fig. S2 and S3†).
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subsequent asymmetric reduction of the b-keto ester with
borane dimethyl sulde complex in the presence of a commer-
cial oxazaborolidine catalyst provides the beta hydroxy esters
2a,b in 20–40% yield with a 90–92% enantiomeric excess of the
R isomer.27 Hydrolysis of the methyl esters to the carboxylic
acids can be accomplished in almost quantitative yield (3a,b) in
the presence of LiOH. Selective formation of thioesters 4a,b in
the presence of the free alcohol is accomplished in 70–80%
yield through carbodiimide-mediated coupling with SNAC
in the presence of stoichiometric DCC and catalytic DMAP
(see ESI† for details).
Production and analysis of metabolites in mutant strains
L. enzymogenes OH11 and its mutants were grown in 1/10 TSB
for 1 day, and an aliquot of 200 L was spread on a solid NYGA
plate (bacteriological peptone, 5 g Lꢀ1; yeast extract, 3 g Lꢀ1
;
glycerꢁol, 20 g Lꢀ1, and agar 15 g Lꢀ1). The plates were incubated
at 28 C for 2 days. To extract the metabolites, the solid media
were collected and extracted with methanol. The methanol
extract was dried with a rotavapor (Buchi, Rotavapor R-200) to
afford the crude extract, which was dissolved in 2 mL methanol
containing 0.05% TFA. A 20 mL aliquot of each of the extracts
was analyzed by HPLC (1220 Innity LC, Agilent Technologies)
using a reversed-phase column (Cosmosil 5C18-AR-II, 4.6 ID ꢂ
250 mm). Water/0.01 M TFA (solvent A) and acetonitrile/
methanol ¼ 1 : 1 (solvent B) were used as the mobile phases
with a ow rate of 1.0 mL minꢀ1. The HPLC program was as
follows: 57% B in A in the rst 5 min, 57–100% B in 5–32 min,
100% B in 32–40 min, back to 57% B at 41 min, and maintained
to 48 min. The metabolites were detected at 280 nm on a UV
detector. LCQ-MS was used to verify the mass of the peak of
WAP-8294A2 and analogs.
Feeding of ACL6 deletion mutant with 3-hydroxy-7-
methyloctanoyl SNAC
ACL6 in-frame deletion mutant was grown in 1/10 TSB for 1 day,
and an aliquot of 1 mL was inoculated into two 1 liter NYGB
liquid cultures. The culture was fed with the fatty acyl-SNAC
dissolved in DMSO under pulse feeding on 2 following days
(0.12 mmol Lꢀ1 and 0.06 mmol Lꢀ1) following a reported
procedure.28 Metabolites were extracted from the supernatant
with ethyl acetate : methanol : acetic acid ¼ 80 : 15 : 5. The
organic phase was dried under reduced pressure (rotary evap-
orator) to afford the crude extract. The extract was dissolved in
2 mL methanol containing 0.05% TFA. A 20 mL aliquot of each
of the extracts was analyzed by HPLC as described above.
Generation of gene deletion mutants
To construct the ACL6 in-frame deletion vector, primer exten-
sion PCR reactions were performed to generate a 705 bp frag-
ment from both the upstream and downstream regions of ACL6
gene. Homologous recombination at both regions would delete
the entire ACL6 gene without interference with other genes. The
primers used for these experiments are listed in the Table S1.†
The PCR amplied fragment was then cloned into the conjugal
vector pJQ200SK as an XhoI/BamHI fragment to produce
pJQ200SK-ACL-6. To conjugally transfer pJQ200SK-ACL-6 into L.
enzymogenes OH11, the procedure was identical to the previ-
ously described.11 The correct single crossover mutant was
selected by diagnostic PCR (Fig. S3†). Individual colonies of
conrmed single crossover mutants were subjected to liquid
cultures containing 5% (W/V) sucrose to select for the loss of the
vector through a second homologous recombination. The single
crossover mutants were grown in 1/10 TSB medium for 14
hours. Then the individual cultures were re-inoculated into 1/10
TSB (with 1 : 100 ratio) containing 5% sucrose and 25 mg mLꢀ1
kanamycin medium. Aliquots (50 mL) were removed every
3 hours from this liquid culture and spread onto 1/10 TSA plates
(5% sucrose, 25 mg mLꢀ1 kanamycin). Single colonies were
picked up from the plates and allowed to grow in 1/10 TSB (5%
sucrose, 25 mg mLꢀ1 kanamycin) liquid medium for another 2–5
days. To conrm the double crossover mutant, another diag-
nostic PCR was performed using genomic DNA extracted from
the single colonies as template (Fig. S3†).
Expression of seven ACL genes and purication of the
enzymes
To construct expression vectors, the coding region of each of
the seven ACL genes was amplied by PCR using genomic DNA
of L. enzymogenes OH11 as template. The primers are listed in
Table S1.† Each of the PCR fragments was digested and cloned
into pANT841. The cloned gene in each of the constructs was
sequenced, and the result showed that all the ACL sequences
were correct. The ACL genes were released from pANT841 and
cloned into the expression vector pET28a. The pET28a
constructs were introduced into E. coli BL21(DE3). Individual
colonies were inoculated in 3 mL of liquid LB medium con-
taining kanamycin (50 mg mLꢀ1) and incubated in a shaker
(250 rpm) at 37 ꢁC overnight. The culture was then added to
50 mL fresh LB medium and incubated in a shaker (250 rpm)
at 37 ꢁC until the cell density (OD600) reached 0.6. To induce the
expression of the ACL genes, IPTG (0.1 mM) was added to the
cultures, and the cells were allowed to grow at the same
conditions for another 3 h. To extract proteins, the cells were
harvested and resuspended in 2 mL of Tris–HCl buffer (250 mM
NaCl, 100 mM LiCl, 50 mM Tris, pH 7.8). The cell suspension
was treated with lysozyme (1 mg mLꢀ1) and sonicated ve times
(3 second sonication with 2 second pause) on ice. The soluble
fraction of protein extracts was loaded onto a Ni-NTA column,
which was previously calibrated with Tris buffer containing
10 mM imidazole. The column was washed three times with Tris
Synthesis of (R)-3-hydroxy fatty acids and (R)-3-hydroxy fatty
acyl SNAC
To generate the initial carbon backbone, we generated the buffer containing 20 mM imidazole, and the His6-tagged
dianion of methyl acetoacetate with LDA then subsequent protein was eluted twice with 200 mL of Tris buffer containing
alkylation with an appropriate bromoalkane to create the target 250 mM imidazole. The eluted proteins were checked by SDS-
carbon skeleton in roughly 20–50% yields (1a,b) (Fig. 4).26
A
PAGE (Fig. S4†). The fractions containing pure ACLs were
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collected and dialyzed against Tris buffer containing 15% Lysobacter enzymogenes strain OH11. This research was sup-
glycerol.
ported in part by the NIH (R01AI097260), NSFC (31329005), and
a University of Nebraska-Lincoln Redox Biology Center pilot
grant.
In vitro assay of acyl-CoA ligase activity
Enzymatic reactions to generate fatty acyl-CoA were set up using
the following conditions: 10 mM ACL, 0.2 mM CoA, 10 mM ATP,
20 mM fatty acid in DMSO, 5 mM MgCl2, 0.2% (w/v) triton, 200
mM DTT, and 100 mM Tris–HCl pH 7.8. All reactions were
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ꢁ
incubated at 30 C overnight. Samples were analyzed by HPLC
(1220 Innity LC, Agilent Technologies) using a reversed-phase
column (Phenomenex, 4.6 ID ꢂ 150 mm). Water containing 25
mM ammonium acetate and 0.5% acetic acid (solvent A) and
acetonitrile containing 0.5% acetic acid (solvent B) were used as
the mobile phases with a ow rate of 1.0 mL minꢀ1. The HPLC
program was as follows: 10% B in A in the rst 3 min, 10–90% B
in 3–15 min, 90% B in 15–25 min, back to 10% B at 26 min, and
maintained to 30 min.29 The metabolites were detected at 260
nm on a UV detector. MS (Finnigan mat, LCQ) was used to verify
the mass of the reaction products. LC-MS/MS was used to
further conrm the product. An Agilent LC-1200 (Santa Clara,
CA) was connected to a 2.1 ꢂ 100 mm Symmetry ODS column
from Waters (Milford, MA) and a Triple Quadrupole Mass
Spectrometer model 4000 QTrap from ABSciex (Framingham,
MA) operating in either single quadrupole (Q1), enhanced mass
spectrum (EMS), MS/MS or multiple reaction monitoring
(MRM) modes. The samples were injected onto the column and
eluted with 98% mobile phase A (0.1% formic acid in water, J. T.
Baker) to 60% B (0.1% formic acid in acetonitrile, Acros
Organics) over 15 minutes, followed by 5 minutes of 98% B and
5 min of 98% A, all at a ow rate of 0.25 mL minꢀ1
.
Kinetic analysis of acyl-CoA ligase activity
The EnzChek Pyrophosphate Assay Kit (Molecular Probes™)
was used to measure the activities of ACLs. A standard curve (y
¼ 0.0156x + 0.0004, R2 ¼ 0.99603) for the pyrophosphate assay
was generated using pyrophosphate standard as a source of PPi.
To measure the activity of ACLs, the following reagents were
combined in 0.1 mL reaction volumes: 0.4 mM ATP, 0.4 mM
CoA, 0.4 mM free fatty acids in DMSO, 0.2 mM 2-amino-6-
mercapto-7-methyl-purine ribonucleoside (MESG), 1 U purine
nucleoside phosphorylase (PNP), 0.01 U pyrophosphatase, and
various ACLs with nal concentration of 15 mM. Aer incu-
ꢁ
bating at 22 C for 30 minutes, the absorbance at 360 nm was
measured and corrected for background absorbance. The
substrates tested were chemically synthesized (R)-3-hydroxy-7-
methyloctanoic acid (corresponding to the fatty acyl chain in
WAP 8294-A2) and (R)-3-hydroxyloctanoic acid (corresponding
to the fatty acyl chain in WAP 8294-A1). The kinetic parameters
of ACLs were determined by initial velocity experiments using
the coupled assay. These data were globally tted to Michaelis–
Menten equation, yielding Km values for the substrates.
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Acknowledgements
We thank Dr Javier Seravalli and Prof. James Takacs for tech- 24 Y. H. Lee, C. Gallant, H. Guo, Y. Li, C. A. Wang and
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