3-Hydroxy-3-methylglutaryl-coenzyme A reductase from Haloferax volcanii: purification, characterization, and expression in Escherichia coli.
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 from the third domain, the archaea. HMG-CoA reductase of the halobacterium Haloferax volcanii was initially partially purified from extracts of H. volcanii. Subsequently, a portion of the H. volcanii lovastatin (formerly called mevinolin) resistance marker mev was subcloned into the Escherichia coli expression vector pT7-7. While no HMG-CoA reductase activity was detectable following expression in E. coli, activity could be recovered after extracts were exposed to 3 M KCl. Following purification to electrophoretic homogeneity, the specific activity of the expressed enzyme, 24 microU/mg, equaled that of homogeneous hamster or P. mevalonii HMG-CoA reductase. Activity was optimal at pH 7.3. Kms were 66 microM (NADPH) and 60 microM [(S)-HMG-CoA]. (R)-HMG-CoA and lovastatin inhibited competitively with (S)-HMG-CoA. H. volcanii HMG-CoA reductase also catalyzed the reduction of mevaldehyde [optimal activity at pH 6.0; Vmax 11 microU/mg; Kms 32 microM (NADPH), 550 microM [(R,S)-mevaldehyde]] and the oxidative acylation of mevaldehyde [optimal activity at pH 8.0; Vmax 2.1 microU/mg; Kms 350 microM (NADP+), 300 microM (CoA), 470 microM [(R,S)-mevaldehyde]]. These properties are comparable to those of hamster and P. mevalonii HMG-CoA reductases, suggesting a similar catalytic mechanism.
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.
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Bischoff
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Rodwell
D000214:Acyl Coenzyme A / Q000378:metabolism; D000215:Acylation / Q000187:drug effects; D000447:Aldehydes; D002352:Carrier Proteins / Q000235:genetics; D004926:Escherichia coli / Q000235:genetics; D005798:Genes, Bacterial; D006216:Halobacteriaceae / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D006903:Hydroxymethylglutaryl CoA Reductases / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D007700:Kinetics; D008148:Lovastatin / Q000494:pharmacology; D008565:Membrane Proteins / Q000235:genetics; D026901:Membrane Transport Proteins; D008798:Mevalonic Acid / Q000031:analogs & derivatives / Q000378:metabolism; D008970:Molecular Weight; D009249:NADP / Q000378:metabolism; D011189:Potassium Chloride / Q000494:pharmacology; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000302:isolation & purification; D013237:Stereoisomerism
D000214:Acyl Coenzyme A; D000447:Aldehydes; D002352:Carrier Proteins; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008565:Membrane Proteins; D026901:Membrane Transport Proteins; D011993:Recombinant Fusion Proteins; C077949:Mev protein, Cricetulus griseus; C008047:3-hydroxy-3-methylglutaryl-coenzyme A; D009249:NADP; D011189:Potassium Chloride; D008148:Lovastatin; D006903:Hydroxymethylglutaryl CoA Reductases; D008798:Mevalonic Acid