Isolation and characterization of an acyl-coenzyme A carboxylase from an erythromycin-producing Streptomyces erythreus

Abstract

Streptomyces erythreus produces erythromycin presumably from methylmalonyl-coenzyme A, (CoA) which might be generated by carboxylation of propionyl-CoA. A biotin-containing enzyme which carboxylates acetyl-CoA, propionyl-CoA, and butyryl-CoA was purified to near homogeneity from S. erythreus using DEAE-cellulose, affinity chromatography on monomeric avidin-Sepharose, and blue Sepharose. The enzyme carboxylates propionyl-CoA (100%) with a K m of 0.09 m m and V of 0.86μmol/mg/min, acetyl-CoA (16%) with a K m of 0.17 m m and V of 0.08μmol/mg/min, and butyryl-CoA (7.7%) with a K m of 0.67 m m and V of 0.044 μmol/mg/min. The native enzyme has a molecular weight of 537,000 and consists of two types of subunits with molecular weights of 67,000 and 61,000, respectively, indicating an octameric α 4 β 4 type of structure. Biotin is associated with the large subunit (α). The enzyme has a pH optimum between 7.5 and 7.8. It is stimulated (three- to fourfold) by K +, Rb + and Cs + but not by Na + or Li + and is inhibited by high concentrations of NH 4 + and C1 −. Neither citrate nor free CoA stimulated the enzyme. The enzyme was shown to be stereospecific and generated only S-methylmalonyl-CoA from the carboxylation of propionyl-CoA. The present case appears to be the first enzyme possibly involved in erythromycin production to be isolated in homogeneous form.