A Novel Δ³,Δ²-Enoyl-CoA Isomerase Involved in the Biosynthesis of the Cyclohexanecarboxylic Acid-Derived Moiety of the Polyketide Ansatrienin A^,

Abstract

The side chain of the antifungal polyketide ansatrienin A produced by Streptomyces collinus contains a cyclohexanecarboxylic acid (CHC) derived moiety. This CHC in the coenzyme A activated form (CHC-CoA) is derived from shikimic acid via a pathway in which the penultimate step is the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA. We have purified a 28 kDa 2-cyclohexenylcarbonyl-CoA isomerase (ChcB) from S. collinus and cloned and sequenced the corresponding chcB gene. The predicted amino acid sequence of ChcB showed moderate sequence identity to members of the hydratase/isomerase superfamily of enzymes. The recombinant ChcB was overexpressed in Escherichia coli and purified to homogeneity using metal chelate chromatography. Kinetic analysis demonstrated that recombinant ChcB had wide substrate specificity and could catalyze a double bond isomerization using 2-cyclohexenylcarbonyl-CoA (K(m) 116 +/- 68 microM, k(cat)( )()3.7 +/- 1.0 min(-)(1)), trans-3-hexenyl-CoA (K(m) 39 +/- 10 microM, k(cat)( )()12.8 +/- 1 min(-)(1)), and vinylacetyl-CoA (K(m) 156 +/- 34 microM, k(cat)( )()29 +/- 3 min(-)(1)) as substrates. ChcB activity in cell extracts of S. collinus SP1, an insertionally disrupted chcB mutant, was shown to decrease by more than 99% (as compared to the wild-type strain) using all three of these substrates. The S. collinus SP1 strain, unlike the wild-type strain, could not produce omega-cyclohexyl fatty acids but was still able to grow efficiently on methyl oleate as a sole carbon source. These observations demonstrate that the S. collinus ChcB is required for catalyzing the isomerization of 2-cyclohexenylcarbonyl-CoA to 1-cyclohexenylcarbonyl-CoA during CHC-CoA biosynthesis but not for degradation of unsaturated fatty acids. The chcB gene does not appear to be associated with the ansatrienin biosynthetic gene cluster, which has previously been shown to contain at least one gene known to be essential for CHC-CoA biosynthesis. This finding represents a notable exception to the general rule regarding the clustering of polyketide biosynthetic pathway genes.

A New Mechanism for Anaerobic Unsaturated Fatty Acid Formation inStreptococcus pneumoniae

“…This protein is part of a multifunctional phenylacetic acid degradation complex and shows 35% identity and 51% similarity to FabM. ChcB is a novel ⌬3,⌬2-enoyl-CoA isomerase involved in the biosynthesis of the cyclohexanecarboxylic acid moiety of the polyketide ansatrienin A (34). ChcB has 27% identity and 41% similarity to FabM.…”

Enhancement and Selective Production of Phoslactomycin B, a Protein Phosphatase IIa Inhibitor, through Identification and Engineering of the Corresponding Biosynthetic Gene Cluster

“…The sequence analysis revealed the existence of the complete set of highly conserved CHC-CoA biosynthetic genes (plmJK, plmL, chcA, plmM) (28,33) as well as plmI (encoding a 3-deoxy-Darabino-heptulosonate-7-phosphate synthase) responsible for formation of the CHC-CoA starter unit. As in S. collinus, the chcB gene encoding the ⌬ 2,3 -enoyl CoA isomerase responsible for catalyzing the penultimate step in the CHC-CoA pathway does not appear to be located close to the other CHC-CoA biosynthetic genes or within the antibiotic biosynthetic gene cluster (36). A total of six large ORFs encoding the core PLM PKS were also identified (Fig.…”

Identification of a cyclohexylcarbonyl CoA biosynthetic gene cluster and application in the production of doramectin

“…Furthermore, no increase in this activity is seen when either of these species is transformed with pAC12, suggesting that the gene responsible for catalyzing this step in the pathway is not clustered with the CHC biosynthetic genes. This hypothesis has been confirmed with the purification S. collinus ChcB and the cloning of the corresponding gene 14 . With the exception of ChcA and ChcB, the exact roles of AnsJ-AnsM in CHC biosynthesis remains to be determined.…”