Purification and characterization of citrate synthase from Methylobacterium extorquens--a methylotrophic producer of polyhydroxybutyrate.
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 microM for CoASA; K(m) = 12 microM for oxaloacetate; Vmax = 29.7 mumoles/min per mg protein. KCl at concentrations up to 80 mM activates 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.
Belova
L L
LL
Institute of Biochemistry and Physiology of Microorganisms, Russian Academy of Sciences, Pushchino, Moscow Region, Russia.
Sokolov
A P
AP
Morgunov
I G
IG
Trotsenko YuA
D002853:Chromatography, Liquid; D002950:Citrate (si)-Synthase / Q000302:isolation & purification* / Q000378:metabolism; D004591:Electrophoresis, Polyacrylamide Gel; D006088:Gram-Negative Aerobic Bacteria / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration
D002950:Citrate (si)-Synthase