title: Substrate specificity of the 3-methylcrotonyl coenzyme A (CoA) and geranyl-CoA carboxylases from Pseudomonas aeruginosa.
abstract: 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 degrees 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 microM, 13 microM, and 0.8 microM, 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 microM and 1.2 microM for G-CoA and bicarbonate, respectively, and the apparent K(m) values of GCCase were 10 microM for ATP and 14 microM 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.
authors.affiliation: Instituto de Investigaciones Químico-Biológicas, Universidad Michoacana de San Nicolás de Hidalgo, Ciudad Universitaria, Morelia, Michoacán, México.
authors.forename: J A
authors.initials: JA
authors.lastname: Aguilar
authors.forename: C
authors.initials: C
authors.lastname: Díaz-Pérez
authors.forename: A L
authors.initials: AL
authors.lastname: Díaz-Pérez
authors.forename: J S
authors.initials: JS
authors.lastname: Rodríguez-Zavala
authors.forename: B J
authors.initials: BJ
authors.lastname: Nikolau
authors.forename: J
authors.initials: J
authors.lastname: Campos-García
mesh_terms: D000214:Acyl Coenzyme A / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D001639:Bicarbonates / Q000378:metabolism; D019735:Carbon-Carbon Ligases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D003001:Cloning, Molecular; D004795:Enzyme Stability; D004926:Escherichia coli; D015870:Gene Expression; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D053858:Metabolic Networks and Pathways; D008954:Models, Biological; D011550:Pseudomonas aeruginosa / Q000201:enzymology*; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013696:Temperature
chemical_list: D000214:Acyl Coenzyme A; D001426:Bacterial Proteins; D001639:Bicarbonates; D011994:Recombinant Proteins; C033618:tiglyl-coenzyme A; D000255:Adenosine Triphosphate; D019735:Carbon-Carbon Ligases; C021573:methylcrotonoyl-CoA carboxylase; C020566:geranoyl-CoA carboxylase