Poly-(R)-3-hydroxybutyrate (PHB) biosynthesis: Mechanistic studies on the biological Claisen condensation catalyzed by β-ketoacyl thiolase

Abstract

The organisms Zoogloea ramigera and Alcaligenes eutrophus, like many other bacteria, produce poly-(R)-3-hydroxybutyrate (PHB) from acetyl-CoA through the three-enzyme pathway involving thiolase, reductase and PHB synthase. Thiolase catalyzes condensation of two acetyl-CoA molecules to acetoacetyl-CoA via two steps: In the first half reaction the active site cysteine attacks acetyl-CoA to form an acetyl-S-enzyme intermediate which reacts, in the second half reaction, with the anion formerly formed from the second acetyl-CoA molecule by enzymic deprotonation to complete the condensation. An adequate supply of thiolase from either organism now made available through isolation of the encoding structural gene and construction of an overproduction vector has permitted extensive studies on the mechanism of the thiolase-catalyzed reaction. This article attempts to answer several basic questions of or comments on: (1) thiolase substrate specificity, (2) identification of the active site cysteine involved in acetyl enzyme formation in the first half reaction, (3) identification of the active site basic residue responsible for deprotonation of the second acetyl-CoA molecule, (4) timing of deprotonation vs. C-C bond formation both involved in the second half reactions, and (5) the energy profile of the forward and reverse reactions. © 1989 IUPAC