Biochemistry
Article
acid; in addition, we report the enzymatic synthesis of
hydroxymalonyl-CoA by RtMCS (the biosynthesis of hydrox-
ymalonyl-CoA by MatB from Streptomyces coelicolor was first
reported in 2011 by Keatinge-Clay and co-workers18). A
previous report on the promiscuity of cinnamyl-CoA ligase
from S. coelicolor (CCL) was limited to cinnamic acid
derivatives;12 in this study, we report the biosynthesis of
phenylpropanoyl-CoA and its derivatives, as well as short alkyl-
CoAs such as butanoyl-, pentanoyl-, and hexanoyl-CoAs by
CCL.
4-Coumarate-CoA ligase from Nicotiana tabacum (Nt4CL)
and benzoate-CoA ligase from Rhodopseudomonas palustri
(RpBZL) are two previously reported acid-CoA ligases19 that
use cinnamic acid and benzoic acid substrates (and their
derivatives), respectively. In addition to cinnamate acid
derivatives, we found that Nt4CL could also use 3-phenyl-
propanoic acid (and its derivatives), medium- to long-chain
saturated alkylic acids (hexanoic, heptanoic, octanoic, nonanoic,
and decanoic acids), and naphthalenecarboxylic acids and
quinolinecarboxylic acids as substrates for the biosynthesis of
the corresponding acyl-CoA thioesters. Apart from a wide range
of benzoic acid derivatives, we report the biosynthesis of short-
chain saturated and unsaturated alkyl-CoAs (such as propanoyl-
CoA, pentanoyl-CoA, and trans-2-butenoyl-CoA or crotonoyl-
CoA, respectively) by RpBZL, providing novel enzymatic
routes for polyketide biosynthesis efforts.
A promiscuous phenylacetate-CoA ligase (PCL) was
previously reported from the fungus Penicillium chrysogenum
by Janssen and co-workers.17 In this study, we report the
substrate range of a functionally orthologous PCL from S.
coelicolor (20% identical amino acid sequence): apart from the
previously reported catalysis of phenylacetic acid (and its
derivatives) and saturated aliphatic acids, we report the
biosynthesis of unsaturated alkyl-CoAs and two novel
malonyl-CoA derivatives by PCL (3-thiophenemalonyl-CoA
and phenylmalonyl-CoA). It is noteworthy that given the broad
substrate range of RtMCS toward malonic acid derivatives, it
could not catalyze the production of 3-thiophenemalonyl-CoA
and phenylmalonyl-CoA. This study highlights the utility and
importance of determining substrate promiscuities beyond
conventional substrate pools and provides an additional tool(s)
for the establishment of precursor-directed combinatorial
polyketide biosynthesis.
dichlorophenyl)propanoic acid, 3-(3,5-difluorophenyl)-
propanoic acid, 3-(4-fluorophenyl)propanoic acid, 3-(4-
methoxyphenyl)propanoic acid, 3-(4-hydroxyphenyl)-2-hydrox-
ypropanoic acid, and 3-(3,4-dihydroxyphenyl)propanoic acid;
benzoate type, benzoic acid, 2-fluorobenzoic acid, 3-fluoroben-
zoic acid, 4-fluorobenzoic acid, 2-chlorobenzoic acid, 3-
chlorobenzoic acid, 4-chlorobenzoic acid, 2-bromobenzoic
acid, 3-bromobenzoic acid, 4-bromobenzoic acid, 2-iodobenzoic
acid, 3-iodobenzoic acid, 4-iodobenzoic acid, 2-aminobenzoic
acid, 4-aminobenzoic acid, 2-acetoxybenzoic acid (acetylsalicylic
acid), 2-hydroxy-3,5-diiodobenzoic acid, 2-hydroxybenzoic acid
(salicylic acid), 2-methoxybenzoic acid, 2-methylbenzoic acid
(o-toluic acid), 2,3-dihydroxybenzoic acid, 2,4-dihydroxyben-
zoic acid, 2,6-difluorobenzoic acid, 2,6-dimethylbenzoic acid,
2.5-dihydroxybenzoic acid, 3-aminobenzoic acid, 3,4-dimethox-
ybenzoic acid (veratric acid), 3,4,5-trihydroxybenzoic acid
(gallic acid), 3,5-dihydroxy-4-methoxybenzoic acid, 3,5-dihy-
droxybenzoic acid, 3,5-dimethylbenzoic acid, and 4-amino-2-
hydroxybenzoic acid (p-aminosalicylic acid); phenylacetate
type, phenylacetic acid, phenoxyacetic acid, 2,4-dichlorophe-
noxyacetate, 4-fluorophenoxyacetic acid, 4-hydroxyphenylacetic
acid, phenylpyruvic acid, 4-methoxyphenylacetate, and 2-
hydroxyphenylacetic acid; naphthalene, pyridine, and quinoline
type, 1-naphthalenecarboxylic acid, 2-naphthalenecarboxylic
acid, pyridine-2-carboxylic acid (picolinic acid), pyridine-3-
carboxylic acid (nicotinic acid), 2-chloropyridine-3-carboxylic
acid, pyrazine carboxylic acid, pyridine-4-carboxylic acid
(isonicotinic acid), 2-quinolinecarboxylic acid (quinaldic
acid), 2-quinoxalinecarboxylic acid, 3-quinolinecarboxylic acid,
4-quinolinecarboxylic acid, and isoquinoline-1-carboxylic acid;
saturated aliphatic type, acetic acid, propanoic acid, butanoic
acid, valeric acid (pentanoic acid), hexanoic acid, heptanoic
acid, octanoic acid, nonanoic acid, decanoic acid, acetoacetic
acid, pimelic acid, pyruvic acid, and succinic acid; unsaturated
aliphatic type, 2-hexenoic acid, 2-methyl-2-butenoic acid (tiglic
acid), 2-methyl-2-propenoic acid (methacrylic acid), 2-
pentenoic acid, 2-propenoic acid (acrylic acid), 3-butenoic
acid, 3-hexenoic acid, 3-methyl-2-butenoic acid (3,3-dimethy-
lacrylic acid), 3-methyl-4-pentenoic acid, 3-pentenoic acid, 4-
pentenoic acid, 5-hexenoic acid, and trans-2-butenoic acid
(crotonic acid); malonic type, malonic acid, methylmalonic
acid, phenylmalonic acid, 3-thienylmalonic acid, allylmalonic
acid, benzylmalonic acid, butylmalonic acid, cyclopentylmalonic
acid, ethylmalonic acid, hydroxymalonic acid, isopropylmalonic
acid, and shikimic acid.
The chemical structures of all substrates tested are shown in
Figure S1 of the Supporting Information. The coupling
enzymes adenylate kinase (AK), pyruvate kinase (PK), and
lactic dehydrogenase (LDH) and 5,5′-dithiobis(2-nitrobenzoic
acid) (DTNB) were purchased from Sigma-Aldrich Co. All
reagents were the highest quality grade commercially available.
Cloning, Expression, and Protein Purification of
Phenylacetate-CoA Ligase (PCL) from S. coelicolor
A3(2). The gene encoding PCL (GI|1099823) was amplified
via polymerase chain reaction (PCR) from genomic DNA
isolated from S. coelicolor A3(2) (ATCC) using Platinum Pfx
DNA polymerase (Invitrogen). The PCR mixture (100 μL)
contained 1 ng of plasmid DNA, 10 μL of 10× Pfx
amplification buffer, 1 mM MgSO4, dNTPs (0.4 mM each),
40 pmol of each primer (forward primer 5′-CGAAGGAGTC-
CATATGAGCAGCGAGCCGACGACCGGGACGGCC-3′
and reverse primer 5′-GTCCGTGCTCTCGAGT-
CACGCGCCCCGCTGGTCCCACACCCG-3′), and 5 units
MATERIALS AND METHODS
■
The carboxylic acid substrates for acid-CoA ligases were
purchased from Sigma-Aldrich Co. (St. Louis, MO), Tokyo
Chemical Industry (TCI) Co., Extrasynthese Co., and Lier
Chemical Co. (Sichuan, China). A total of 123 carboxylic acids
were tested: cinnamate type, cinnamic acid, 2-fluorocinnamic
acid, 3-fluorocinnamic acid, 4-fluorocinnamic acid, α-fluorocin-
namic acid, 2-chlorocinnamic acid, 3-chlorocinnamic acid, 4-
chlorocinnamic acid, 4-methylcinnamic acid, α-methylcinnamic
acid, 2-hydroxycinnamic acid, 4-hydroxycinnamic acid, 2-
methoxycinnamic acid, 4-methoxycinnamic acid, 2,4-dimethox-
ycinnamic acid, 3,4-dimethoxycinnamic acid, 3-chloro-4-me-
thoxycinnamic acid, 3-methoxy-4-hydroxycinnamic acid (ferulic
acid), 4-hydroxy-3,5-dimethoxycinnamic acid, and 4-nitro-
cinnamic acid; phenylpropanoate type, 3-phenylpropanoic
acid, 2-hydroxy-3-phenylpropanoic acid, 3-(2-bromophenyl)-
propanoic acid, 3-(2-methoxyphenyl)propanoic acid, 3-(3-
chloro-4-methoxyphenyl)propanoic acid, 3-(3-chlorophenyl)-
propanoic acid, 3-(3-methoxyphenyl)propanoic acid, 3-(3,5-
4569
dx.doi.org/10.1021/bi300425j | Biochemistry 2012, 51, 4568−4579