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Journal of Bacteriology, April 2002, p. 2116-2122, Vol. 184, No. 8
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.8.2116-2122.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Enterococcus faecalis Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis{dagger}

Matija Hedl,1 Autumn Sutherlin,1 E. Imogen Wilding,2 Marie Mazzulla,2 Damien McDevitt,2 Pamela Lane,2 John W. Burgner, II,3 Kevin R. Lehnbeuter,1 Cynthia V. Stauffacher,3 Michael N. Gwynn,2 and Victor W. Rodwell1*

Departments of Biochemistry,1 Biological Sciences, Purdue University, West Lafayette, Indiana 47907,3 Microbial, Musculoskeletal, and Proliferative Diseases Center of Excellence and Drug Discovery, GlaxoSmithKline Pharmaceuticals, Collegeville, Pennsylvania 194262

Received 31 October 2001/ Accepted 22 January 2002

Many bacteria employ the nonmevalonate pathway for synthesis of isopentenyl diphosphate, the monomer unit for isoprenoid biosynthesis. However, gram-positive cocci exclusively use the mevalonate pathway, which is essential for their growth (E. I. Wilding et al., J. Bacteriol. 182:4319-4327, 2000). Enzymes of the mevalonate pathway are thus potential targets for drug intervention. Uniquely, the enterococci possess a single open reading frame, mvaE, that appears to encode two enzymes of the mevalonate pathway, acetoacetyl-coenzyme A thiolase and 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. Western blotting revealed that the mvaE gene product is a single polypeptide in Enterococcus faecalis, Enterococcus faecium, and Enterococcus hirae. The mvaE gene was cloned from E. faecalis and was expressed with an N-terminal His tag in Escherichia coli. The gene product was then purified by nickel affinity chromatography. As predicted, the 86.5-kDa mvaE gene product catalyzed both the acetoacetyl-CoA thiolase and HMG-CoA reductase reactions. Temperature optima, {Delta}Ha and Km values, and pH optima were determined for both activities. Kinetic studies of acetoacetyl-CoA thiolase implicated a ping-pong mechanism. CoA acted as an inhibitor competitive with acetyl-CoA. A millimolar Ki for a statin drug confirmed that E. faecalis HMG-CoA reductase is a class II enzyme. The oxidoreductant was NADP(H). A role for an active-site histidine during the first redox step of the HMG-CoA, reductase reaction was suggested by the ability of diethylpyrocarbonate to block formation of mevalonate from HMG-CoA, but not from mevaldehyde. Sequence comparisons with other HMG-CoA reductases suggest that the essential active-site histidine is His756. The mvaE gene product represents the first example of an HMG-CoA reductase fused to another enzyme.

* Corresponding author. Mailing address: Department of Biochemistry, Purdue University, West Lafayette, IN 47907-1153. Phone: (765) 494-1608. Fax: (765) 494-7897. E-mail: vrodwell{at}purdue.edu.

{dagger} Journal paper 16671 from the Purdue Agricultural Experiment Station.

Journal of Bacteriology, April 2002, p. 2116-2122, Vol. 184, No. 8
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.8.2116-2122.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



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