Expression and Characterization of ( R )-Specific Enoyl Coenzyme A Hydratase Involved in Polyhydroxyalkanoate Biosynthesis by Aeromonas caviae

Abstract

ABSTRACT Complementation analysis of a polyhydroxyalkanoate (PHA)-negative mutant of Aeromonas caviae proved that ORF3 in the pha locus (a 402-bp gene located downstream of the PHA synthase gene) participates in PHA biosynthesis on alkanoic acids, and the ORF3 gene is here referred to as phaJ Ac . Escherichia coli BL21(DE3) carrying phaJ Ac under the control of the T7 promoter overexpressed enoyl coenzyme A (enoyl-CoA) hydratase, which was purified by one-step anion-exchange chromatography. The N-terminal amino acid sequence of the purified hydratase corresponded to the amino acid sequence deduced from the nucleotide sequence of phaJ Ac except for the initial Met residue. The enoyl-CoA hydratase encoded by phaJ Ac exhibited ( R )-specific hydration activity toward trans -2-enoyl-CoA with four to six carbon atoms. These results have demonstrated that ( R )-specific hydration of 2-enoyl-CoA catalyzed by the translated product of phaJ Ac is a channeling pathway for supplying ( R )-3-hydroxyacyl-CoA monomer units from fatty acid β-oxidation to poly(3-hydroxybutyrate- co -3-hydroxyhexanoate) biosynthesis in A. caviae.