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.

FIG. 1.

Participation of GCCase and MCCase in the acyclic monoterpene and leucine catabolic pathways of P. aeruginosa PAO1 (1, 10). AtuD, citronellyl-CoA dehydrogenase; AtuC/AtuF, GCCase; AtuE, isohexenylglutaconyl-CoA hydratase; LiuA, isovaleryl-CoA dehydrogenase; LiuB/LiuD, MCCase; LiuC, 3-methylglutaconyl-CoA hydratase; LiuE, 3-hydroxy-3-methyl-glutaryl-CoA (also proposed as 3-hydroxy-3-isohexenylglutaryl-CoA lyase).

FIG. 2.

Electrophoretic analysis of the recombinant heterologous expression of P. aeruginosa MCCase and GCCase subunits. (A) Coomassie blue-stained SDS-PAGE analysis of assembled gels of recombinant proteins expressed in E. coli and purified using affinity chromatography. Lanes: 1, AtuF-His; 2, AtuC-His; 3, LiuD-His; 4, LiuB-His expressed from pTrcHis-2A vector; and 5, AtuC-His/AtuF-S expressed from pCDFDuet-1 coexpression vector. Molecular mass makers are shown on the left; the protein bands corresponding to AtuC/AtuF are indicated with arrowheads. (B) Western blot analysis of purified recombinant proteins probed with avidin-HRP conjugate to detect biotin-containing proteins. Lanes: 1, LiuB-His; 2, LiuD-His; 3, AtuF-His; 4, AtuC-His; 5, AtuC-His/AtuF-S; and 6, extract from PAO1 culture grown on citronellol. (C) Western blot analysis of purified recombinant proteins probed first with anti-AtuC-His polyclonal antibody and then with anti-mouse-HRP as a secondary antibody. Lanes are the same as in panel B.

FIG. 3.

Kinetic behavior of recombinantly produced P. aeruginosa MCCase and GCCase enzymes. (A and B) Carboxylase activities of recombinant LiuB/LiuD (A) and AtuC/AtuF (B) proteins, purified and reconstituted as described in Materials and Methods. (C and D) ATP (C) and bicarbonate (D) concentration dependence of the MCCase and GCCase activities. Data given are the average of three determinations; standard deviations of the given values are shown in panel A, and the averages of two determinations with variations of less than 5% of the given values are shown in panels B, C, and D.