Biochemical characterization of malate synthase G of P. aeruginosa.
BACKGROUND
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.


RESULTS
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 degrees 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 microM), Km acetyl CoA (12 microM) and Vmax (16.5 micromol/minutes/mg enzyme). In addition, deletion of the corresponding gene showed that it is a prerequisite for growth on acetate as sole carbon source.


CONCLUSIONS
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.
Roucourt
Bart
B
Department of Biosystems, Katholieke Universiteit Leuven, Belgium. bart.roucourt@cme.vib-kuleuven.be
Minnebo
Nikki
N
Augustijns
Patrick
P
Hertveldt
Kirsten
K
Volckaert
Guido
G
Lavigne
Rob
R
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D004795:Enzyme Stability; D006038:Glyoxylates / Q000378:metabolism; D007700:Kinetics; D008292:Malate Synthase / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000201:enzymology* / Q000235:genetics; D017384:Sequence Deletion; D013379:Substrate Specificity
D001426:Bacterial Proteins; D006038:Glyoxylates; D008292:Malate Synthase