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The NADPH-linked acetoacetyl-CoA reductase from <em>Zoogloea ramigera</em>.
- Source :
-
European Journal of Biochemistry . 5/16/88, Vol. 174 Issue 1, p177-182. 6p. - Publication Year :
- 1988
-
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-3hydroxybutyrate) 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 acetoacetyI-CoA above 10 µM. 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. [ABSTRACT FROM AUTHOR]
Details
- Language :
- English
- ISSN :
- 00142956
- Volume :
- 174
- Issue :
- 1
- Database :
- Academic Search Index
- Journal :
- European Journal of Biochemistry
- Publication Type :
- Academic Journal
- Accession number :
- 13797203
- Full Text :
- https://doi.org/10.1111/j.1432-1033.1988.tb14079.x