<i>Enterococcus faecalis</i>
            Acetoacetyl-Coenzyme A Thiolase/3-Hydroxy-3-Methylglutaryl-Coenzyme A Reductase, a Dual-Function Protein of Isopentenyl Diphosphate Biosynthesis

Hedl, Matija; Sutherlin, Autumn; Wilding, E. Imogen; Mazzulla, Marie; McDevitt, Damien; Lane, Pamela; Burgner, John W.; Lehnbeuter, Kevin R.; Stauffacher, Cynthia V.; Gwynn, Michael N.; Rodwell, Victor W.

doi:10.1128/jb.184.8.2116-2122.2002

Cited by 62 publications

(59 citation statements)

References 26 publications

(30 reference statements)

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“…Although the mevalonate pathway is also essential for human subjects, there are two classes of HMG-CoA reductase that differ both with respect to their structure and their inhibition characteristics (4). The K i values for statin drug inhibition of the class I HMG-CoA reductases of eukaryotes are nanomolar (2) but are millimolar for the class II HMG-CoA reductases of S. aureus (28) and E. faecalis (11). Significant differences also characterize the crystal structures of the class I human enzyme (12) and the class II enzyme of Pseudomonas mevalonii (13).…”

Section: Discussionmentioning

confidence: 98%

“…The first and third reactions of the mevalonate pathway in E. faecalis, the synthesis of acetoacetyl-CoA and the formation of mevalonate, are catalyzed by a single bifunctional enzyme (11). Since HMG-CoA synthase catalyzes the second reaction of the pathway, we investigated the coupled conversion of acetyl-CoA to mevalonate.…”

Section: Resultsmentioning

confidence: 99%

“…An "x" indicates a nonconserved residue. Glu95 (8), Cys129 (17), and His264 (16) Of the enzymes of the mevalonate pathway in enterococci, only S. aureus HMG-CoA reductase (28) and the acetoacetyl-CoA thiolase/HMG-CoA reductase of E. faecalis (11) have been expressed and characterized. We report here the cloning, expression, purification, and characterization of the first bacterial HMG-CoA synthase.…”

Section: Discussionmentioning

confidence: 99%

“…An unusual feature of the mevalonate pathway of Enterococcus faecalis is that a dual-function polypeptide, acetoacetylCoA thiolase/HMG-CoA reductase (11,27), catalyzes the first and third reactions of the pathway (Fig. 1).…”

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confidence: 99%

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Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Sutherlin¹,

Hedl²,

Sanchez-Neri³

et al. 2002

J Bacteriol

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Biosynthesis of the isoprenoid precursor isopentenyl diphosphate (IPP) proceeds via two distinct pathways.Sequence comparisons and microbiological data suggest that multidrug-resistant strains of gram-positive cocci employ exclusively the mevalonate pathway for IPP biosynthesis. Bacterial mevalonate pathway enzymes therefore offer potential targets for development of active site-directed inhibitors for use as antibiotics. We used the PCR and Enterococcus faecalis genomic DNA to isolate the mvaS gene that encodes 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) synthase, the second enzyme of the mevalonate pathway. mvaS was expressed in Escherichia coli from a pET28 vector with an attached N-terminal histidine tag. The expressed enzyme was purified by affinity chromatography on Ni 2؉ -agarose to apparent homogeneity and a specific activity of 10 mol/min/mg. Analytical ultracentrifugation showed that the enzyme is a dimer (mass, 83.9 kDa; s 20,w , 5.3). Optimal activity occurred in 2.0 mM MgCl 2 at 37 o C. The ⌬H a was 6,000 cal. The pH activity profile, optimum activity at pH 9.8, yielded a pK a of 8.8 for a dissociating group, presumably Glu78. The stoichiometry per monomer of acetyl-CoA binding was 1.2 ؎ 0.2 and that of covalent acetylation was 0.60 ؎ 0.02. The K m for the hydrolysis of acetyl-CoA was 10 M. Coupled conversion of acetyl-CoA to mevalonate was demonstrated by using HMG-CoA synthase and acetoacetyl-CoA thiolase/HMG-CoA reductase from E. faecalis.

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Section: Discussionmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

mentioning

confidence: 99%

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Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Sutherlin¹,

Hedl²,

Sanchez-Neri³

et al. 2002

J Bacteriol

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“…Hence inhibitors of the mevalonate pathway should work against these Gram-positive pathogens. 133,134) 3. Bacterial fatty acid synthesis Fatty acid biosynthesis is essential for bacterial growth.…”

Section: New Targetsmentioning

confidence: 99%

Antibiotic Resistance in Bacteria and Its Future for Novel Antibiotic Development

Yoneyama

Katsumata

2006

Bioscience, Biotechnology, and Biochemistry

236

196

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Multienzyme mevalonate pathway bioreactor

Sutherlin

Rodwell²

2004

Biotech & Bioengineering

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The five-carbon metabolic intermediate isopentenyl diphosphate constitutes the basic building block for the biosynthesis of all isoprenoids in all forms of life. Two distinct pathways lead from amphibolic intermediates to isopentenyl diphosphate. The Gram-positive cocci and certain other pathogenic bacteria employ exclusively the mevalonate pathway, a set of six enzyme-catalyzed reactions that convert 3 mol of acetyl-CoA to 1 mol each of carbon dioxide and isopentenyl diphosphate. The survival of the Gram-positive cocci requires a fully functional set of mevalonate pathway enzymes. These enzymes therefore represent potential targets of inhibitors that might be employed as antibiotics directed against multidrug-resistant strains of certain bacterial pathogens. A rapid throughput, bioreactor-based assay to assess the effects of potential inhibitors on several enzymes simultaneously should prove useful for the survey of candidate inhibitors. To approach this goal, and as a proof of concept, we employed enzymes from the Gram-positive pathogen Enterococcus faecalis. Purified recombinant enzymes that catalyze the first three reactions of the mevalonate pathway were immobilized in two kinds of continuous flow enzyme bioreactors: a classical hollow fiber bioreactor and an immobilized plug flow bioreactor that exploited a novel method of enzyme immobilization. Both bioreactor types employed recombinant acetoacetyl-CoA thiolase, HMG-CoA synthase, and HMG-CoA reductase from E. faecalis to convert acetyl-CoA to mevalonate, the central intermediate of the mevalonate pathway. Reactor performance was monitored continuously by spectrophotometric measurement of the concentration of NADPH in the reactor effluent. Additional potential applications of an Ni(++) affinity support bioreactor include using recombinant enzymes from extremophiles for biosynthetic applications. Finally, linking a Ni(++) affinity support bioreactor to an HPLC-mass spectrometer would provide an experimental and pedagogical tool for study of metabolite flux and pool sizes of intermediates to model regulation in intact cells.

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

Cited by 62 publications

References 26 publications

Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Enterococcus faecalis 3-Hydroxy-3-Methylglutaryl Coenzyme A Synthase, an Enzyme of Isopentenyl Diphosphate Biosynthesis

Antibiotic Resistance in Bacteria and Its Future for Novel Antibiotic Development

Multienzyme mevalonate pathway bioreactor

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