Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis

Abstract

P. citronellolis contained 4 different acyl-Co A carboxylases, including acetyl-, propionyl-, 3-methylcrotonyl- and geranyl-CoA carboxylases, when grown on the appropriate C sources. Acetyl-CoA carboxylase activity in crude extracts was stimulated approximately 40-fold by inclusion of 0.4-0.5 M ammonium sulfate in the assay. Unexpectedly high levels of propionyl-CoA carboxylase activity, also stimulated by ammonium sulfate, were found in the acetate-grown cells. That these acetyl- and propionyl-CoA carboxylase activities were due to different enzymes was shown by their resolution during purification by a procedure that stabilized acetyl-CoA carboxylase as a complex and separated propionyl-CoA carboxylase into 2 required protein fractions. Propionate- or valine-grown cells contained a propionyl-CoA carboxylase activity that was strongly inhibited by ammonium sulfate in the assay and may represent an inducible form of the enzyme. Geranyl- and 3-methylcrotonyl-CoA carboxylases that catalyze the carboxylation of the 3-methyl groups of homologous acyl-CoA acceptors, were induced by growth on the monoterpenes, citronnelic or geranoic acid; only 3-methylcrotonyl-CoA carboxylase was induced by growth on leucine or isovaleric acid. Induction of either carboxylase was associated with the appearance of similar high-MW, biotin-containing proteins as measured by gel filtration. These 2 carboxylases are probably distinct enzymes since 3-methylcrotonyl-CoA carboxylase from isovalerate-grown cells does not carboxylate geranyl-CoA, while geranyl-CoA carboxylase will carboxylate both acyl-CoA homologues. P. citronellolis appears to be a useful system for studying the structural aspects of pairs of homologous acyl-CoA carboxylases.