3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli.

摘要: Prior work from this laboratory characterized eukaryotic (hamster) and eubacterial (Pseudomonas mevalonii) 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductases. We report here the characterization of an HMG-CoA reductase third domain, archaea. halobacterium Haloferax volcanii was initially partially purified extracts H. volcanii. Subsequently, a portion lovastatin (formerly called mevinolin) resistance marker mev subcloned into Escherichia coli expression vector pT7-7. While no activity detectable following in E. coli, could be recovered after were exposed to 3 M KCl. Following purification electrophoretic homogeneity, specific expressed enzyme, 24 microU/mg, equaled that homogeneous hamster or P. mevalonii reductase. Activity optimal at pH 7.3. Kms 66 microM (NADPH) 60 [(S)-HMG-CoA]. (R)-HMG-CoA inhibited competitively with (S)-HMG-CoA. also catalyzed reduction mevaldehyde [optimal 6.0; Vmax 11 microU/mg; 32 (NADPH), 550 [(R,S)-mevaldehyde]] oxidative acylation 8.0; 2.1 350 (NADP+), 300 (CoA), 470 [(R,S)-mevaldehyde]]. These properties are comparable those reductases, suggesting similar catalytic mechanism.