Isolation and Characterization of Acyl Coenzyme A Carboxylases from Mycobacterium tuberculosis and Mycobacterium bovis , Which Produce Multiple Methyl-Branched Mycocerosic Acids

Abstract

Mycobacterium tuberculosis H37Ra and M. bovis BCG produce multiple methyl-branched fatty acids called mycocerosic acids, presumably from methyl-malonyl coenzyme A (CoA). An acyl-CoA carboxylase was isolated from these organisms at a 30 to 50% yield by a purification procedure involving ammonium sulfate fractionation, gel filtration, and affinity chromatography with a monomeric avidin–Sepharose 4B-CL gel with d -biotin as the eluant. Sodium dodecyl sulfate electrophoresis and avidin binding indicate that each enzyme is probably composed of two dissimilar subunits with a covalently bound biotin in the larger subunit. The enzyme preparations from H37Ra and BCG had specific activities of 2.1 and 5.5 μmol min −1 mg −1 , respectively, when propionyl-CoA was the substrate. The enzymes from the two species displayed striking similarities in their kinetic parameters. They showed maximal activity at pH 8.0 when propionyl-CoA was the substrate, but displayed a relatively broad pH-activity profile when acetyl-CoA was the substrate. With both substrates, potassium phosphate buffer gave maximal activity. Apparent K m values for propionyl-CoA, ATP, Mg 2+ , and NaHCO 3 were 70 μM, 100 μM, 5.4 mM, and 2.2 mM, respectively. The enzyme also carboxylated acetyl-CoA and butyryl-CoA, and high-performance liquid chromatography showed the expected products of carboxylation. However, with these substrates, the K m was higher and the V max was lower than those of propionyl-CoA. The enzyme was shown to be stereospecific, synthesizing exclusively ( S )-methylmalonyl-CoA from propionyl-CoA. No other acyl-CoA carboxylase was observed during the purification procedure, indicating that the present carboxylase may provide malonyl-CoA for the synthesis of n -fatty acids as well as methylmalonyl-CoA for the synthesis of mycocerosic acids.