Characterization of the Enterococcus faecalis Oxaloacetate decarboxylase membrane-bound complex

BLANCATO V. S.; REPIZO G. D.; MAGNI C.

Villa Carlos Paz

Congreso; SAIB; 2008

Citrate metabolism in E. faecalis, as in other citrate-fermenting bacteria, involves the conversion of citrate to oxaloacetate and acetate by the enzyme citrate lyase. Then, oxaloacetate is decarboxylated to pyruvate by the enzyme oxaloacetate decarboxylase (OAD). This compound is subsequently degraded to different end products. Analysis of the gene cluster involved in the citrate degradation pathway in E. faecalis revealed the presence of all the enzymes required for this metabolism. Interestingly genes coding for two types of OADs were found, citM homologous to L. lactis soluble OAD and oad genes encoding a membrane-bound complex homologous to Klebsiella pneumoniae OAD. In this work, we report on the characterization of the OAD membrane complex. The expression of the biotinylated ƒÔƒ{subunit was detected by western blot in wild type cell extracts (grown in the presence of citrate) but not in a cit-deficient strain. The expression was restored in a complementant strain. Moreover, the ƒÔ-subunit was localized in the citosolic as well as in the membrane fraction of cell extracts, suggesting a dynamic complex. In order to confirm the expression of all the subunits, OAD complex was purified, and the recovered subunits were identified by MALDI-TOF.