Substrate specificity of the 3-Methylcrotonyl-CoA and Geranyl-CoA Carboxylases from Pseudomonas aeruginosa

Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán; Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F., México; Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, IA. USA

^* To whom correspondence should be addressed. Email: jcgarcia{at}umich.mx.

	Abstract

Biotin-containing 3-methylcrotonyl-coenzyme A carboxylase (MCCase) and geranyl-coenzyme A carboxylase (GCCase) from Pseudomonas aeruginosa were expressed as His-tagged recombinant proteins in Escherichia coli. Both native and recombinant MCCase and GCCase showed pH and temperature optima of 8.5 and 37°C. The apparent K_0.5 values of MCCase for 3-methylcrotonyl-CoA (MC-CoA), ATP, and bicarbonate were 9.8 µM, 13 µM, and 0.8 µM, respectively. MCCase activity showed sigmoidal kinetics for all the substrates and did not carboxylate geranyl-CoA (G-CoA). In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed a sigmoidal kinetic behavior for G-CoA, and bicarbonate, but Michaelis-Menten kinetics for MC-CoA and the co-substrate ATP. The apparent kinetic constant values of GCCase were K₀₅ 8.8 µM and 1.2 µM for G-CoA and bicarbonate, respectively; K_m 10 µM for ATP, and 14 µM for MC-CoA. The catalytic efficiencies of GCCase for G-CoA and MC-CoA were 56 and 22, respectively, indicating that G-CoA is preferred over MC-CoA as a substrate. The enzymatic properties of GCCase suggest that it may substitute for MCCase in leucine catabolism and that both MCCase and GCCase enzymes play an important role in the leucine and acyclic terpenes catabolic pathways.