Biochemical characterization of malate synthase G of P. aeruginosa
1 Division of Gene Technology, Department of Biosystems, Katholieke Universiteit Leuven, Kasteelpark Arenberg 21, box 2462, Leuven, BE-3001, Belgium
2 Laboratory for Pharmacotechnology and Biopharmacy, Katholieke Universiteit Leuven, Campus Gasthuisberg O&N 2, Box 921, Herestraat 49, 3000 Leuven, Belgium
BMC Biochemistry 2009, 10:20 doi:10.1186/1471-2091-10-20Published: 24 June 2009
Malate synthase catalyzes the second step of the glyoxylate bypass, the condensation of acetyl coenzyme A and glyoxylate to form malate and coenzyme A (CoA). In several microorganisms, the glyoxylate bypass is of general importance to microbial pathogenesis. The predicted malate synthase G of Pseudomonas aeruginosa has also been implicated in virulence of this opportunistic pathogen.
Here, we report the verification of the malate synthase activity of this predicted protein and its recombinant production in E. coli, purification and biochemical characterization. The malate synthase G of P. aeruginosa PAO1 has a temperature and pH optimum of 37.5°C and 8.5, respectively. Although displaying normal thermal stability, the enzyme was stable up to incubation at pH 11. The following kinetic parameters of P. aeruginosa PAO1 malate synthase G were obtained: Km glyoxylate (70 μM), Km acetyl CoA (12 μM) and Vmax (16.5 μmol/minutes/mg enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source.
The implication of the glyoxylate bypass in the pathology of various microorganisms makes malate synthase G an attractive new target for antibacterial therapy. The purification procedure and biochemical characterization assist in the development of antibacterial components directed against this target in P. aeruginosa.