CLONING AND CHARACTERIZATION OF THE GENE ENCODING 1-CYCLOHEXENYLCARBONYL COENZYME-A REDUCTASE FROM STREPTOMYCES-COLLINUS

We report the cloning of the gene encoding the 1-cyclohexenylcarbonyl coenzyme A reductase (ChcA) of Streptomyces collinus, an enzyme putati vely involved in the final reduction step in the formation of the cycl ohexyl 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 sim iliarity extending along almost the entire length of the protein was o bserved with members of the short-chain alcohol dehydrogenase superfam ily. The S. collinus chcA gene was overexpressed in Escherichia coli b y 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 a nd electrophoretic mobility on sodium dodecyl sulfate-polyacrylamide g els as the enoyl-coenzyme A reductase protein prepared from S. collinu s. The enzyme demonstrated the ability to catalyze, in vitro, three of the reductive steps involved in the formation of cyclohexanecarboxyli c acid. An S. collinus chcA mutant, constructed by deletion of a genom ic region comprising the 5' end of chcA, lost the ChcA activity and th e ability to synthesize either cyclohexanecarboxylic acid or ansatrien in. These results suggest that chc4 encodes the ChcA that is involved in catalyzing multiple reductive steps in the pathway that provides th e cyclohexanecarboxylic acid from shikimic acid.