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

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.

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.

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.

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.

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 (⧫).

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.

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.

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.