The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli

Eur J Biochem. 1988 May 16;174(1):177-82. doi: 10.1111/j.1432-1033.1988.tb14079.x.


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 alpha 4 homotetramer of four 25-kDa subunits. It has a Km of 2 microM and a kcat/Km of 1.8 X 10(8) M-1 s-1 for acetoacetyl-CoA; it is inhibited by acetoacetyl-CoA above 10 microM. 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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Alcohol Oxidoreductases / genetics
  • Alcohol Oxidoreductases / isolation & purification*
  • Cloning, Molecular
  • Enzyme Activation / drug effects
  • Escherichia coli / enzymology*
  • Genetic Vectors
  • NADP / pharmacology*
  • Oxidation-Reduction / drug effects
  • Pseudomonadaceae / enzymology*
  • Pseudomonadaceae / genetics
  • Substrate Specificity


  • NADP
  • Alcohol Oxidoreductases
  • acetoacetyl-CoA reductase