Characterization of a Malic Enzyme and a Soluble Oxaloacetate Decarboxylase encoded by Two Paralogous Genes in Enterococcus faecalis.

ESPARIZ, M.; REPISO, G.; BLANCATO, V.; MORTERA, P.; ALARCON, S.; MAGNI, C.

FEBS journal

wiley

Año: 2011 vol. 278 p. 2140 - 2151

1742-4658

<!-- /* Font Definitions */ @font-face {font-family:Calibri; mso-font-alt:"Segoe UI"; mso-font-charset:0; mso-generic-font-family:swiss; mso-font-pitch:variable; mso-font-signature:-520092929 1073786111 9 0 415 0;} /* Style Definitions */ p.MsoNormal, li.MsoNormal, div.MsoNormal {mso-style-parent:""; margin-top:0cm; margin-right:0cm; margin-bottom:10.0pt; margin-left:0cm; line-height:115%; mso-pagination:widow-orphan; font-size:11.0pt; font-family:Calibri; mso-fareast-font-family:Calibri; mso-bidi-font-family:"Times New Roman"; mso-fareast-language:EN-US;} @page Section1 {size:612.0pt 792.0pt; margin:70.85pt 3.0cm 70.85pt 3.0cm; mso-header-margin:36.0pt; mso-footer-margin:36.0pt; mso-paper-source:0;} div.Section1 {page:Section1;} --> The Enterococcus faecalis genome contains two paralogous genes, maeE and citM, coding for proteins that show high homology with members of the malic enzyme family. The putative proteins MaeE (41 kDa) and CitM (42 kDa) share a high degree of homology between them (47 % identities and 68 % conservative substitutions). However, the genetic context of each gene suggested that maeE is associated with malate utilization whereas citM is linked to the citrate fermentation pathway. In the present work, we focus on the biochemical characterization and physiological contribution of these enzymes in E. faecalis. With this aim, the recombinant versions of both proteins were expressed in Escherichia coli, affinity purified and finally their kinetic parameters determined. This approach allowed us to establish that MaeE is a malate oxidative decarboxylating enzyme and CitM is a soluble oxaloacetate decarboxylase. Moreover, our genetic studies in E. faecalis showed that the citrate fermentation phenotype is not affected by citM deletion. On the other hand, maeE gene disruption originated a malate fermentation deficient strain indicating that MaeE is responsible for malate metabolism in E. faecalis. Lastly, it was demonstrated that malate fermentation in E. faecalis is associated with cytoplasmic and extracellular alkalinization which clearly contributes to pH homeostasis in neutral or mild acidic conditions.