The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera

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

Authors

  • Olivier PLOUX,

    1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
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  • Satoru MASAMUNE,

    1. Department of Chemistry, Massachusetts Institute of Technology, Cambridge, Massachusetts
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  • Christopher T. WALSH

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

Abstract

The NADPH-linked acetoacetyl-CoA reductase, (R)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.36), 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.

Abbreviation
Nbs2

5,5′-dithiobis(2-nitrobenzoate)

Enzymes
 

(R)-3-Hydroxyacyl-CoA dehydrogenase acetoacetyl-CoA reductase (EC 1.1.1.36)

 

(S)-3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35)

 

glutathione reductase (EC 1.6.4.2)

 

acetoacetyl-CoA thiolase (EC 2.3.1.9)

Ancillary