The importance of PHB-synthase substrate specificity in polyhydroxyalkanoate synthesis by<i>Alcaligenes eutrophus</i>

Abstract

Alcaligenes eutrophus can accumulate poly-3-hydroxybutyrate (PHB) or polyhydroxyalkanoate (PHA) containing only 3-hydroxybutyrate (HB) and 3-hydroxyvalerate (HV) units. Granule-associated PHB-synthase was active with d(−)-3-hydroxybutyryl-CoA and d(−)-3-hydroxyvaleryl-CoA of the range of d(−)- and l(+)-3-hydroxyacyl-CoA substrates tested (C4–C10). In carbon-limited cultures, PHB-synthase was predominantly soluble, becoming granule-associated on transition to nitrogen limitation. Granule-associated PHB-synthase increased in activity at least up to pH 10.0 and Km values of 0.68 mM and 1.63 mM were determined for the C4 and C5 substrates, respectively, at pH 8.5. The soluble PHB-synthase, which was unstable, showed equal activity in the range pH 8.0–10.0, had a Km value for d(−)-3-hydroxybutyryl-CoA of 0.72 mM and an Mr of 160,000. PHB does not measurably turn over under steady-state polymer-accumulating conditions.