Class I Polyhydroxyalkanoate Synthase from the Purple Photosynthetic Bacterium Rhodovulum sulfidophilum Predominantly Exists as a Functional Dimer in the Absence of a Substrate

[Image: see text] Polyhydroxyalkanoates (PHAs) are a family of biopolyesters that accumulate as carbon and energy storage compounds in a variety of micro-organisms. The marine purple photosynthetic bacterium Rhodovulum sulfidophilum is capable of synthesizing PHA. In this study, we cloned a gene encoding a class I PHA synthase from R. sulfidophilum (phaC(Rs)) and synthesized PhaC(Rs) using a cell-free protein expression system. The specific activity of PhaC(Rs) increased linearly as the (R)-3-hydroxybutyryl-coenzyme A (3HB-CoA) concentration increased and never reached a plateau, even at 3.75 mM 3HB-CoA, suggesting that PhaC(Rs) was not saturated because of low substrate affinity. Size exclusion chromatography and native polyacrylamide gel electrophoresis analyses revealed that PhaC(Rs) exists predominantly as an active dimer even in the absence of 3HB-CoA, unlike previously characterized PhaCs. The linear relationship between the PhaC(Rs) activity and 3HB-CoA concentrations could result from a low substrate affinity as well as the absence of a rate-limiting step during PHA polymerization because of the existence of predominantly active dimers.