Two NADH-dependent (S)-3-hydroxyacyl-CoA dehydrogenases from polyhydroxyalkanoate-producing Ralstonia eutropha

Ralstonia eutropha H16 contains both NADH- and NADPH-dependent reduction activities to acetoacetyl-CoA, and the NADPH-dependent activity is mediated by PhaB paralogs with ( R )-stereospecificity providing ( R )-3-hydroxybutyryl (3HB)-CoA monomer for poly(( R )-3-hydroxybutyrate) synthesis. In contrast, the gene encoding the NADH-dependent enzyme has not been identified to date. This study focused on the NADH-dependent dehydrogenase with ( S )-stereospecificity in R. eutropha , as the ( S )-specific reduction of acetoacetyl-CoA potentially competed with the polyester biosynthesis via ( R )-3HB-CoA. The NADH-dependent reduction activity decreased to one-half when the gene for H16_A0282 (PaaH1), one of two homologs of clostridial NADH-3HB-CoA dehydrogenase, was deleted. The enzyme responsible for the remaining activity was partially purified and identified as H16_A0602 (Had) belonging to a different family from PaaH1. Gene disruption analysis elucidated that most of the NADH-dependent activity was mediated by PaaH1 and Had. The kinetic analysis using the recombinant enzymes indicated that PaaH1 and Had were both NADH-dependent 3-hydroxyacyl-CoA dehydrogenases with rather broad substrate specificity to 3-oxoacyl-CoAs of C 4 to C 8 . The deletion of had in the R. eutropha strain previously engineered for biosynthesis of poly(( R )-3-hydroxybutyrate- co -( R )-3-hydroxyhexanoate) led to decrease in the C 6 composition of the copolyester synthesized from soybean oil, suggesting the role of Had in ( S )-specific reduction of 3-oxohexanoyl-CoA with reverse β-oxidation direction. Crotonase (( S )-specific enoyl-CoA hydratase) in R. eutropha H16 was also partially purified and identified as H16_A3307.