Characterization of an Acyl Coenzyme a Carboxylase from Rhizobium etli

Abstract

Biotin, as the prosthetic group of biotin-dependent carboxylases, is essential for all organisms. To further define biotin-dependent metabolic pathways in Rhizobium (Encarnacion et al 1995; Dunn et al 1996, 1997; Dunn 1998), we are characterizing an acyl CoA carboxylase from R. etli CE3. This enzyme was previously shown to be capable of carboxylating propionyl CoA (to form methylmalonyl CoA) and acetyl CoA (to form malonyl CoA; Dunn et al 1998) The acyl CoA carboxylase was purified 123-fold by a combination of chromatographic steps. The partially purified enzyme displayed its highest activity using propionyl CoA (C3:0) as a substrate, but was also able to carboxylate acetyl CoA (C2:0) and butyryl CoA (C4:0) at much lower rates. The apparent and values of the enzyme for propionyl CoA were 0.05 mM and 1448 nmol/min/mg protein, respectively, and the corresponding values using acetyl CoA as a substrate were 0.63 mM and 43 nmol/min/mg protein. These data show that the enzyme has a strong kinetic preference for propionyl CoA. The values for ATP and bicarbonate were 0.09 and 0.71 mM, respectively, with propionyl CoA as a substrate. Of several potential effectors tested, compounds producing significant inhibition of the propionyl CoA or acetyl CoA carboxylating activities of the acyl CoA carboxylase included ATP, AMP, palmitate, stearate, valerate, methylmalonyl CoA and malonyl CoA. KC1 markedly activated the enzyme Analysis of the enzymes subunit and total molecular mass indicate an structure. The subunit (78 kDa) contains the biotin moiety. Although the kinetic preference of the acyl CoA carboxylase is for propionyl CoA, and may serve as part of an anaplerotic pathway using this substrate (Dunn 1998), we failed to detect a separate acetyl CoA carboxylating activity in R. etli apart from that catalyzed by the acyl CoA carboxylase. This indicates that the acyl CoA carboxylase, using acetyl CoA as a substrate, may be the sole enzyme responsible for catalyzing the first step in fatty acid synthesis, and thus play an indispensible role in the cell.