First genetic evidence of oxaloacetate decarboxylase involvement in citrate metabolism in firmicutes

REPIZO, G BLANCATO MAGNI C

Potrero de los Funes

Congreso; XLV Reunión Anual de SAIB; 2011

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Citrate metabolism in E. faecalis involves the conversion of citrate to pyruvate by the sequential action of citrate lyase and oxaloacetate decarboxylase (OAD) enzymes. Our previous reports indicated that the gene cluster involved in citrate degradation in E. faecalis encodes an OAD membrane complex homologous to that reported for Klebsiella pneumoniae. In K. pneumoniae the complex is constituted by three subunits (Kpn-alpha, beta and gamma), whereas in E. faecalis a four subunit complex was found (Ef-alpha, beta, delta and eta) in which the extra Ef-eta subunit may have functional properties analogous to the Kpn-gamma subunit. Our analysis indicated that alpha, delta and eta form a soluble complex that is capable of interacting with the membrane-bound beta subunit in a dynamic way. Moreover, construction of E. faecalis mutant strains for the different OAD subunits was performed. Diminished growth of the mutants in citrate supplemented media indicated that the activity of OAD is essential for citrate utilization. Also, the nature of the interaction between the subunits was analyzed. We observed that the complex was stable at acidic pH, but its integrity was compromised at incrementing basic pHs. Furthermore, analysis of single and double mutant strains allowed identifying interacting partners. In summary, we propose that the new eta subunit mediates the interaction between the remaining subunits of the complex.