Purification and Characterization of Citrate Synthase from Methylobacterium extorquens--a Methylotrophic Producer of Polyhydroxybutyrate

L. L. Belova,¹ A. P. Sokolov,¹ I. G. Morgunov,¹ and Yu. A. Trotsenko^1,2

¹Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, 142292 Russia; fax: (7-095) 923-36-02; E-mail: trotsenko@ibpm.serpukhov.su

²To whom correspondence should be addressed.

Submitted August 2, 1996; revision submitted September 19, 1996.

Citrate synthase (citrate oxaloacetate-lyase, CoA-acetylating; EC 4.1.3.7, CS) was isolated and purified to homogeneity from a methylotrophic producer of polyhydroxybutyrate (PHB), Methylobacterium extorquens 15. The purification procedure includes streptomycin sulfate treatment of cell-free extract, ammonium sulfate fractionation, two steps of hydrophobic chromatography, and ion-exchange chromatography. The specific activity of the final enzyme preparation was 24 U/mg protein. The enzyme has apparent molecular weight 260 kD and consists of four 66-kD subunits. The enzyme shows a sigmoid saturation curve with CoASA (h = 1.3). Kinetic parameters are: K_m = 84 µM for CoASA; K_m = 12 µM for oxaloacetate; V_max = 29.7 µmoles/min per mg protein. KCl at concentrations up to 80 mM activate the CS. ATP exerts a significant inhibitory effect on the enzyme activity, whereas NAD(P)H, isocitrate, alpha-ketoglutarate, ADP, acetoacetyl-CoA, glyoxylate, and glutamate have no influence. A possible role of the CS in coordinated control of CoASA transformation through the tricarboxylic acid cycle and PHB biosynthesis in this methylotroph is discussed.

KEY WORDS: citrate synthase, methylotroph, Methylobacterium extorquens, polyhydroxybutyrate.

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