Structural evidence for the involvement of the residues Ser187 and Tyr422 in substrate recognition in the 3-methylcrotonyl-coenzyme A carboxylase from Pseudomonas aeruginosa.

The enzyme 3-methylcrotonyl-CoA carboxylase from Pseudomonas aeruginosa (Pa-MCCase) is essential for the assimilation of leucine and acyclic monoterpenes. The structure of the Pa-MCCase was analysed by computational modelling to establish the molecular basis of substrate recognition. The active site is composed of two zones, which may play important roles in substrate recognition and catalysis. To further understand the interactions of the active site with the substrate, site-directed mutagenesis of the conserved residues S187 and R51 located in zone I, and F417, Y422 and G423 from zone II of the Pa-MCCase was carried out. The residue substitutions S187A and Y422D completely abolished the Pa-MCCase activity, whereas substitutions R51A, F417Y and G423A indicated that these residues are not essential. Interestingly, the residues R47, R51 and S187 form a well-defined pocket that may play important roles in substrate coupling to the Co-A motif. At zone one, mutation S187A was essential, but mutant R51A retained activity, suggesting that the R51 function could be relegated to neighbouring positive residues. Residue Y422 instead of contributing to substrate discrimination, it may participate in deprotonation of methyl group on MC-CoA, because it is located at adequate distances from the 3-methylcrotonyl-chain and carboxybiotin groups in the Pa-MCCase carboxylation site.