Characterization of two members of malic enzymes from Enterococcus faecalis

MARTÍN ESPARIZ; VICTOR BLANCATO; CHRISTIAN MAGNI

San Miguel de Tucumán, Tucumán, Argentina

Congreso; 45th Annual Meeting Argentine Society for Biochemistry and Molecular Biology; 2009

Sociedad Argentina de Investigación en Bioquímica y Biología Molecular

Enterococci are an ubiquitous microorganisms that compose the natural flora of a variety of fermented foods and also are capable of causing disease as opportunistic pathogens. E. faecalis contains two different members of the malic enzyme family, EF3316 (EfCitM, a putative oxaloacetate (OAA) decarboxylase) and EF1206 (EfMaeE, a putative malate oxidoreductase (OAA-decarboxylating)). citM is transcribed from the citrate fermentation operon while maeE is located near maeP implicated in malate utilization. It is widely accepted that both fermentative pathways contribute to pH homeostasis in bacteria. In this work, we described the purification of the recombinant enzymes expressed in E. coli. Our in vitro studies showed that i) EfCitM is an OAA decarboxylase with maximal activity at pH 4.4, a K m,OAA of 0.47 ±0.066 mM, and a k cat of 5 ±1.0 s-1. No malate activity was detected. ii) EfMaeE catalyzed the oxidative decarboxylation of malate at an optimum pH of 8.5 with a Km,malate of 0.56 ±0.035 mM, and a kcat of 0.29 ±0.03 min-1. Interesting, we found OAA decarboxylase activity for EfMaeE at low pH. Considering the high optimum pH for malic activity of EfMaeE, this enzyme would not have a role in pH homeostasis in E. faecalis. Nevertheless, the contribution of the OAA decarboxylase activity of both enzymes to the intracellular alkalinization of E. faecalis will be analyzed in future works.