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J. Bacteriol., Jan 1996, 19-23, Vol 178, No. 1
Copyright © 1996, American Society for Microbiology

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

KM Bischoff and VW Rodwell
Department of Biochemistry, Purdue University, West Lafayette, Indiana 47907-1153, USA.

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.


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