Production of recombinant NADPH: 2-ketopropyl-coenzyme M oxidoreductase/carboxylase.

摘要

The metabolism of aliphatic epoxides uses the cofactor coenzyme M (2-mercaptoethanesulfonic acid) as a nucleophile for epoxide ring opening and as the carrier of intermediates in epoxide metabolism. In the propylene-oxidizing soil bacteria Xanthobacter autotrophicus strain Py2, 2KPC is formed from epoxypropane by nucleophilic addition of CoM followed by dehydrogenation of the initial adduct. The substrate 2-KPC is then cleaved and carboxylated in an NADPH-dependent reaction catalyzed by 2-ketopropyl-CoM carboxylase/oxidoreductase (2-KPCC), yielding CoM and acetoacetate. Biochemical, genetic, and structural studies of 2-KPCC demonstrate that this enzyme is a member of the NADPH: disulfide oxidoreductase family of enzymes. In order to test tenets of the proposed catalytic mechanism of 2-KPCC, xecC, the gene encoding 2-KPCC was cloned and expressed in E. coli. The expression of soluble and active 2-KPCC depended on a highly tunable arabinose promoter and on co-expressing the enzyme, 2-KPCC, with the product protein of xecB.