Multiple acyl-coenzyme A carboxylases in Pseudomonas citronellolis

Abstract

Pseudomonas citronellolis was shown to contain four different acyl-coenzyme A carboxylases, including acetyl-, propionyl-, 3-methylcrotonyl-, and geranyl-CoA carboxylases, when grown on the appropriate carbon 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 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 two 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 which 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, citronellic 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-molecular-weight, biotin-containing proteins as measured by gel filtration. These two carboxylases are probably distinct enzymes since 3-methyl-crotonyl-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.

Carboxylases in Natural and Synthetic Microbial Pathways

“…Carboxylation of 3-methylcrotonyl-CoA by biotin-dependent methylcrotonyl-CoA carboxylase yields 3-methylglutaconyl-CoA (50, 51), which is then transformed into three molecules of acetyl-CoA (4, 63). However, in contrast to other acyl-CoA esters, which are usually carboxylated at the ␣ position, the CO 2 group is in this case introduced at the ␥ position (50,52,59). Recently, the genes encoding geranoyl-CoA carboxylase, a homolog of 3-methylcrotonyl-CoA carboxylase, were identified and functionally assigned (4,63).…”

Methylcrotonyl-CoA and geranyl-CoA carboxylases are involved in leucine/isovalerate utilization (Liu) and acyclic terpene utilization (Atu), and are encoded by liuB/liuD and atuC/atuF, in Pseudomonas aeruginosa

“…The resulting metabolite (3-methylcrotonyl-CoA) is degraded via the leucine/isovalerate utilization (Liu) pathway (Fig. 1), which involves another carboxylase (methylcrotonyl-CoA carboxylase, MCase) that is unable to utilize geranyl-CoA as a substrate, but is able to utilize carboxylate methylcrotonylCoA (Fall & Hector, 1977;Fall, 1981;Hector & Fall, 1976a, Fig. 1.…”

Substrate Specificity of the 3-Methylcrotonyl Coenzyme A (CoA) and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa

“…It has been suggested that the P. citronellolis GCCase carboxylates both G-CoA and MC-CoA, while MCCase carboxylates only MC-CoA (9,13,15). Although the genes that encode the enzymes of the acyclic terpene and leucine/isovalerate catabolic pathways in P. aeruginosa and P. citronellolis have been recently elucidated (1,10), there is still uncertainty regarding the bifunctionality of the carboxylases involved.…”