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Journal of Bacteriology, July 2008, p. 4888-4893, Vol. 190, No. 14
0021-9193/08/$08.00+0     doi:10.1128/JB.00454-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Morelia, Michoacán, México,¹ Departamento de Bioquímica, Instituto Nacional de Cardiología, México D.F., México,² Department of Biochemistry, Biophysics and Molecular Biology, Iowa State University, Ames, Iowa³

Received 2 April 2008/ Accepted 1 May 2008

Biotin-containing 3-methylcrotonyl coenzyme A (MC-CoA) carboxylase (MCCase) and geranyl-CoA (G-CoA) 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 (affinity constant for non-Michaelis-Menten kinetics behavior) values of MCCase for 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 G-CoA. In contrast, GCCase catalyzed the carboxylation of both G-CoA and MC-CoA. GCCase also showed sigmoidal kinetic behavior for G-CoA and bicarbonate but showed Michaelis-Menten kinetics for MC-CoA and the cosubstrate ATP. The apparent K_0.5 values of GCCase were 8.8 µM and 1.2 µM for G-CoA and bicarbonate, respectively, and the apparent K_m values of GCCase were 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 the MCCase and GCCase enzymes play important roles in the leucine and acyclic terpene catabolic pathways.

Published ahead of print on 9 May 2008.

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