The NADPH‐linked acetoacetyl‐CoA reductase from Zoogloea ramigera

Abstract

The NADPH-linked acetoacetyl-CoA reductase (R)-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), from the bacterium Zoogloea ramigera, involved in the formation of D-3-hyroxybutyryl-CoA for poly(D-3-hydroxybutyrate)biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieving in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an .alpha.4 homotetramer of four 25-kDa subunits. It has a Km of 2 .mu.M and a kcat/Km of 1.8 .times. 108 M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 .mu.M. K is 10-10 M for the dehydrogenation. Kinetic studies of the back reaction revealed a sequential mechanism involving a ternary complex. The stereospecificity of the hydride-equivalent transfer was demonstrated using NMR techniques to be 4S (B side). Using the fingerprint method proposed by Wierenga et al. [(1986) J. Mol. Biol. 187, 101-107], we identified a 28-residue stretch (residues 3-31) as a possible NADPH fold. Finally the specificity of the reductase was examined using 3-oxo-acyl-CoA analogs and analogs lacking the adenosine 3'',5''-bisphosphate moiety of CoA. Only the straight-chain C5 analog (3-oxo-propionyl-CoA) was found to be an alternative substrate (40%) for the reductase.