The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera

Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli


Correspondence to C. T. Walsh, Department of Biological Chemistry and Molecular Pharmacology, Harvard University Medical School, 25 Shattuck Street, Boston, Massachusetts, USA 02115


The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC, from the bacterium Zoogloea ramigera, involved in the formation of d-3-hydroxybutyryl-CoA for poly(d-3-hydroxybutyrate) biosynthesis, has been purified from an over-producing Escherichia coli strain. The purification was achieved in two steps, yielding an electrophoretically homogeneous enzyme of high specific activity (608 U/mg). The enzyme is an α4 homotetramer of four 25-kDa subunits. It has a Km of 2 μM and a kcat/Km of 1.8 × 108 M−1 s−1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 μM. Km 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.