Mechanistic studies on class I polyhydroxybutyrate (PHB) synthase from Ralstonia eutropha: Class I and III synthases share a similar catalytic mechanism

The Class I and III polyhydroxybutyrate (PHB) synthases from Ralstonia eutr opha and Chromatium vinosum, respectively, catalyze the polymerization of b eta -hydroxybutyryl-coenzyme A (HBCoA) to generate PWB. These synthases hav e different molecular weights, subunit composition, and kinetic properties. Recent studies with the C. vinosum synthase suggested that it is structura lly homologous to bacterial lipases and allowed identification of active si te residues important for catalysis [Jia, Y., Kappock, T. J., Frick, T., Si nskey, A. J,, and Stubbe, J. (2000) Biochemistry 39, 3927-3936]. Sequence a lignments between the Class I and III synthases revealed similar residues i n the ii, eutropha synthase. Site-directed mutants of these residues were p repared and examined using HBCoA and a terminally saturated trimer of HBCoA (sT-CoA) as probes. These studies reveal that the R. eutropha synthase pos sesses an essential catalytic dyad (C3 19-H508) in which the C319 is involv ed in covalent catalysis. A conserved Asp, D480, was shown not to be requir ed for acylation of C319 by sT-CoA and is proposed to function as a general base catalyst to activate the hydroxyl of HBCoA for ester formation. Studi es of the [H-3]sT-CoA with wild-type and mutant synthases reveal that 0.5 e quiv of radiolabel is covalently bound per monomer of synthase, suggesting that a dimeric form of the enzyme is involved in elongation. These studies, in conjunction with search algorithms for secondary structure, suggest tha t the Class I and III synthases are mechanistically similar and structurall y homologous, despite their physical and kinetic differences.