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.

This article has been cited by other articles:

• GROCHOWSKI, L. L., WHITE, R. H. (2008). Promiscuous Anaerobes: New and Unconventional Metabolism in Methanogenic Archaea. Ann. N. Y. Acad. Sci. 1125: 190-214 [Abstract] [Full Text]  
• Matsumi, R., Manabe, K., Fukui, T., Atomi, H., Imanaka, T. (2007). Disruption of a Sugar Transporter Gene Cluster in a Hyperthermophilic Archaeon Using a Host-Marker System Based on Antibiotic Resistance. J. Bacteriol. 189: 2683-2691 [Abstract] [Full Text]  
• Koga, Y., Morii, H. (2007). Biosynthesis of Ether-Type Polar Lipids in Archaea and Evolutionary Considerations. Microbiol. Mol. Biol. Rev. 71: 97-120 [Abstract] [Full Text]  
• Hedl, M., Rodwell, V. W. (2004). Inhibition of the Class II HMG-CoA reductase of Pseudomonas mevalonii. Protein Sci. 13: 1693-1697 [Abstract] [Full Text]  
• Hedl, M., Tabernero, L., Stauffacher, C. V., Rodwell, V. W. (2004). Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductases. J. Bacteriol. 186: 1927-1932 [Full Text]  
• Tabernero, L., Rodwell, V. W., Stauffacher, C. V. (2003). Crystal Structure of a Statin Bound to a Class II Hydroxymethylglutaryl-CoA Reductase. J. Biol. Chem. 278: 19933-19938 [Abstract] [Full Text]  
• Sutherlin, A., Hedl, M., Sanchez-Neri, B., Burgner, J. W. II, Stauffacher, C. V., Rodwell, V. W. (2002). Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis. J. Bacteriol. 184: 4065-4070 [Abstract] [Full Text]  
• Hedl, M., Sutherlin, A., Wilding, E. I., Mazzulla, M., McDevitt, D., Lane, P., Burgner, J. W. II, Lehnbeuter, K. R., Stauffacher, C. V., Gwynn, M. N., Rodwell, V. W. (2002). Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis. J. Bacteriol. 184: 2116-2122 [Abstract] [Full Text]  
• Kon, T., Nemoto, N., Oshima, T., Yamagishi, A. (2002). Effects of a Squalene Epoxidase Inhibitor, Terbinafine, on Ether Lipid Biosyntheses in a Thermoacidophilic Archaeon, Thermoplasma acidophilum. J. Bacteriol. 184: 1395-1401 [Abstract] [Full Text]  
• Wilding, E. I., Kim, D.-Y., Bryant, A. P., Gwynn, M. N., Lunsford, R. D., McDevitt, D., Myers, J. E. Jr., Rosenberg, M., Sylvester, D., Stauffacher, C. V., Rodwell, V. W. (2000). Essentiality, Expression, and Characterization of the Class II 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase of Staphylococcus aureus. J. Bacteriol. 182: 5147-5152 [Abstract] [Full Text]  
• Wilding, E. I., Brown, J. R., Bryant, A. P., Chalker, A. F., Holmes, D. J., Ingraham, K. A., Iordanescu, S., So, C. Y., Rosenberg, M., Gwynn, M. N. (2000). Identification, Evolution, and Essentiality of the Mevalonate Pathway for Isopentenyl Diphosphate Biosynthesis in Gram-Positive Cocci. J. Bacteriol. 182: 4319-4327 [Abstract] [Full Text]  
• Profant, D. A., Roberts, C. J., Koning, A. J., Wright, R. L. (1999). The Role of the 3-Hydroxy 3-Methylglutaryl Coenzyme A Reductase Cytosolic Domain in Karmellae Biogenesis. Mol. Biol. Cell 10: 3409-3423 [Abstract] [Full Text]  
• Takahashi, S., Kuzuyama, T., Seto, H. (1999). Purification, Characterization, and Cloning of a Eubacterial 3-Hydroxy-3-Methylglutaryl Coenzyme A Reductase, a Key Enzyme Involved in Biosynthesis of Terpenoids. J. Bacteriol. 181: 1256-1263 [Abstract] [Full Text]