Enterococcus faecalis acetoacetyl-coenzyme A thiolase/3-hydroxy-3-methylglutaryl-coenzyme A reductase, a dual-function protein of isopentenyl diphosphate biosynthesis

Abstract

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, DeltaH(a) and K(m) 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 K(i) 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.

FIG. 1.

Intermediates and enzymes of the mevalonate pathway for isopentenyl diphosphate biosynthesis.

FIG. 2.

Substrates and products of the reaction catalyzed by HMG-CoA reductase (reaction 3). The putative enzyme-bound intermediates mevaldyl-CoA and mevaldehyde are shown in brackets.

FIG. 3.

SDS-PAGE of the expressed mvaE gene product purified by nickel affinity chromatography. S, standards of the indicated mass. Lane 1, early fractions of E. faecalis acetoacetyl-CoA thiolase/HMG-CoA reductase. Numbers indicate molecular sizes in kilodaltons.

FIG. 4.

The mvaE gene product is expressed as a fusion protein in enterococci. Western blots of the indicated enterococcal lysates and of the purified mvaE gene product are shown. Numbers indicate molecular sizes in kilodaltons.

FIG. 5.

Effect of temperature on activity. Assays were conducted at the indicated temperatures under otherwise standard conditions. (A) Reaction 2, thiolysis of acetoacetyl-CoA; (B) reaction 3, reductive deacylation of HMG-CoA to mevalonate. The insets are selected data shown as Arrhenius plots.

FIG. 6.

Effect of hydrogen ion concentration on activity. All assays were conducted in a solution containing 50 mM sodium acetate, 50 mM glycine, 50 mM Tris, 50 mM 2-(N-morpholino)ethanesulfonic acid at the indicated pH under otherwise standard conditions. (A) Reaction 1, synthesis of acetoacetyl-CoA (•), and reaction 2, thiolysis of acetoacetyl-CoA (○); (B) reaction 3, reductive deacylation of HMG-CoA to mevalonate (▪), and reaction 4, reduction of mevaldehyde to mevalonate (□); (C) reaction 5, oxidative acylation of mevaldehyde to HMG-CoA (◊), and reaction 6, oxidative acylation of mevalonate to HMG-CoA (⧫).

FIG. 7.

Acetoacetyl-CoA thiolase proceeds via a ping-pong mechanism. (A) Acetoacetyl-CoA thiolysis (reaction 2). Assays employed the indicated concentrations of CoA and either 11 (•), 23 (○), 34 (▪), or 45 μM (□) acetoacetyl-CoA. Inset: the Y intercepts were plotted versus the reciprocal of acetoacetyl-CoA concentration. (B) CoA competes with acetyl-CoA during synthesis of acetoacetyl-CoA. The reaction employed 0 (•), 30 (○), or 60 mM (□) CoA, the indicated concentrations of acetyl-CoA, and otherwise standard conditions.

FIG. 8.

Inhibition by a statin drug of reaction 3, the reductive deacylation of HMG-CoA to mevalonate. Assays were conducted in the presence of 0 (○) or 500 μM (•) fluvastatin at the indicated concentrations of HMG-CoA under otherwise standard conditions. All reactions were initiated by adding NADPH.

FIG. 9.

Effect of DEPC and subsequent addition of hydroxylamine hydrochloride on HMG-CoA reductase activity. (A) Effect on reaction 3, the reductive deacylation of HMG-CoA to mevalonate. Treatment with DEPC and with hydroxylamine hydrochloride was conduced on ice. Samples contained fusion protein in a solution containing 250 mM KCl, 10% (vol/vol) glycerol, 250 mM K _xPO₄, pH 6.5. DEPC in ethanol was added to a concentration of 0.8 (⧫) or 8.0 mM (•). A control contained ethanol but no DEPC (○). After 40 min, hydroxylamine hydrochloride, pH 6.5, was added to a concentration of 700 mM (arrow). Ten-microliter portions removed at the indicated times were assayed for the ability to catalyze reaction 3, the reductive deacylation of HMG-CoA to mevalonate. A_o and A are the specific activities at zero time and at the indicated times, respectively. (B) Effect on reaction 4, the reductive deacylation of mevaldehyde to mevalonate. Reaction conditions were as described above but at pH 7.0 and using 8 mM DEPC and an ethanol control (□). Data for reaction 4 (▪) are shown. Also shown are data for reaction 3 (•), included to establish that reaction with DEPC had indeed occurred.

FIG. 10.

E. faecalis HMG-CoA reductase histidine 756 and its cognates. Amino acid sequences from characterized class II HMG-CoA reductases were aligned with CLUSTALW (www.expasy.ch).