Cloning and characterization of the gene encoding 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus

Abstract

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putatively involved in the final reduction step in the formation of the cyclohexyl moiety of ansatrienin from shikimic acid. The cloned gene, with a proposed designation of chcA, encodes an 843-bp open reading frame which predicts a primary translation product of 280 amino acids and a calculated molecular mass of 29.7 kDa. Highly significant sequence similiarity extending along almost the entire length of the protein was observed with members of the short-chain alcohol dehydrogenase superfamily. The S. collinus chcA gene was overexpressed in Escherichia coli by using a bacteriophage T7 transient expression system, and a protein with a specific ChcA activity was detected. The E. coli-produced ChcA protein was purified and shown to have similar steady-state kinetics and electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide gels as the enoyl-coenzyme A reductase protein prepared from S. collinus. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxylic acid. An S. collinus chcA mutant, constructed by deletion of a genomic region comprising the 5 end of chcA, lost the ChcA activity and the ability to synthesize either cyclohexanecarboxylic acid or ansatrienin. These results suggest that chcA encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides the cyclohexanecarboxylic acid from shikimic acid.

Halohydrin Dehalogenases Are Structurally and Mechanistically Related to Short-Chain Dehydrogenases/Reductases

“…In the halohydrin dehalogenases, an arginine is observed at the position at which a lysine is found in the SDR family. However, replacement of the catalytic lysine by an arginine is not unique for halohydrin dehalogenases, since it has also occasionally been observed in other members of the SDR family (e.g., in sorbitol-6-phosphate 2-dehydrogenase of Streptococcus mutans [5] and 1-cyclohexenylcarbonyl coenzyme A reductase from Streptomyces collinus [34]). The observed sequence similarities between halohydrin dehalogenases and proteins of the SDR family strongly point to a common evolutionary origin.…”

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

“…A genomic library was constructed by using Supercos-1 cosmid vector as recommended in the manufacturer's protocol (Stratagene). About 2000 cosmid clones were probed by using the DNA of a digoxigenin-labeled chcA gene (28). Preparation of the digoxigenin probes and the subsequent hybridization and detection were performed as recommended in the manufacturer's protocol (Roche Applied Science).…”

“…The flanking region contained a series of ORFs that are highly homologous (Ͼ80% identity at the amino acid sequence level) with and have an organization similar to the primary metabolic genes identified through sequencing of the S. coelicolor genome (35), which aided in delimiting the PLM 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).…”

Analysis of two chromosomal regions adjacent to genes for a type II polyketide synthase involved in the biosynthesis of the antitumor polyketide mithramycin in Streptomyces argillaceus

“…This observation suggests that MtmTI might be inactive. However ChcA, the closest homologue to MtmTI, is a reductase that has been shown to be active, participating in the biosynthesis of the cyclohexyl moiety of the antifungal antibiotic ansatrienin A ± and it also lacks the tyrosine residue at the``conserved'' position in that region (Wang et al 1996). However, both ChcA and MtmTI have a tyrosine residue seven amino acids upstream of the lysine residue.…”