The NADPH-linked acetoacetyl-CoA reductase from Zoogloea ramigera. Characterization and mechanistic studies of the cloned enzyme over-produced in Escherichia coli.
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 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.
Ploux
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Department of Chemistry, Massachusetts Institute of Technology, Cambridge.
Masamune
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S
Walsh
C T
CT
D000429:Alcohol Oxidoreductases / Q000235:genetics / Q000302:isolation & purification*; D003001:Cloning, Molecular; D004789:Enzyme Activation / Q000187:drug effects; D004926:Escherichia coli / Q000201:enzymology*; D005822:Genetic Vectors; D009249:NADP / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D011548:Pseudomonadaceae / Q000201:enzymology* / Q000235:genetics; D013379:Substrate Specificity
D009249:NADP; D000429:Alcohol Oxidoreductases; C023744:acetoacetyl-CoA reductase