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Biochemical characterization of a Rhizobium etli monovalent cation-stimulated acyl-coenzyme A carboxylase with a high substrate specificity constant for propionyl-coenzyme A

Programa de Ingeniería Metabólica, Centro de Investigación sobre Fijación de Nitrógeno, Universidad National Autónoma de México, A. P. 565-A, Cuernavaca, Morelos, Mexico

Correspondence
Michael F. Dunn
mike{at}cifn.unam.mx

Biotin has a profound effect on the metabolism of rhizobia. It is reported here that the activities of the biotin-dependent enzymes acetyl-coenzyme A carboxylase (ACC; EC 6.4.1.2) and propionyl-coenzyme A carboxylase (PCC; EC 6.4.1.3) are present in all species of the five genera comprising the Rhizobiaceae which were examined. Evidence is presented that the ACC and PCC activities detectable in Rhizobium etli extracts are catalysed by a single acyl-coenzyme A carboxylase. The enzyme from R. etli strain 12-53 was purified 478-fold and displayed its highest activity with propionyl-CoA as substrate, with apparent K_m and V_max values of 0·064 mM and 2885 nmol min^-1 (mg protein)^-1, respectively. The enzyme carboxylated acetyl-CoA and butyryl-CoA with apparent K_m values of 0·392 and 0·144 mM, respectively, and V_max values of 423 and 268 nmol min^-1 (mg protein)^-1, respectively. K⁺, or Cs⁺ markedly activated the enzyme, which was essentially inactive in their absence. Electrophoretic analysis indicated that the acyl-CoA carboxylase was composed of a 74 kDa biotin-containing subunit and a 45 kDa biotin-free subunit, and gel chromatography indicated a total molecular mass of 620 000 Da. The strong kinetic preference of the enzyme for propionyl-CoA is consistent with its participation in an anaplerotic pathway utilizing this substrate.