Mechanistic characterization of members of the amidohydrolase superfamily

The amidohydrolase superfamily is a functionally diverse group of enzymes found in every organism sequenced to date. The landmark for this superfamily is the conservation of a (beta/alpha)8-barrel structural fold. Isoaspartyl dipeptidase (IAD) from Escherichia coli catalyzes the hydrolytic cleavage of beta-aspartyl dipeptides. Structural studies of the wild-type enzyme demonstrate that the active site consists of a binuclear metal center. Bell-shaped pH-rate profiles are observed for all four metal-substituted forms of the wild-type enzyme and the site-directed mutants, E77Q and Y137F. Structural analysis of IAD with the bound substrate and site-directed mutagenesis shows the importance of the side chains of residues Glu-77, Tyr-137, Arg-169, Arg-233, Asp- 285, and Ser-289 in the substrate binding and hydrolysis. The reaction mechanism for the hydrolysis of dipeptides by IAD is initiated by the polarization of the amide bond via complexation to the beta-metal and the hydrogen bond to Tyr-137. Asp-385 participates in the activation of the bridging hydroxide for nucleophilic attack at the peptide carbon center. The lately protonated Asp-285 donates the proton to the alpha-amino group of the leaving group, causing the collapse of the tetrahedral intermediate and cleavage of the carbon-nitrogen bond. N-formimino-L-glutamate iminohydrolase (HutF) from Pseudomonas aeruginosa acts in the deimination of the fourth intermediate of the histidine degradation pathway, N-formimino-L-glutamate. An amino acid sequence alignment between HutF and other members of the amidohydrolase superfamily containing mononuclear metal centers suggests that the residues Glu-235, His-269, and Asp-320 are involved in substrate binding and deimination. Site-directed mutagenesis of Glu-235, His-269, and Asp-320, in conjunction with the analysis of the four metalsubstituted enzyme forms and pH-rate profiles provides valuable information toward the proposal of a mechanism for deimination of N-formimino-L-glutamate by HutF. This information suggests that the reaction is initiated by the activation of the hydrolytic water through base catalysis via His-269. The enhanced nucleophile attacks the formimino carbon center. In a concerted reaction, Asp-320 deprotonates the hydroxide nucleophile, and His-269 donates a proton to the terminal amino of the iminium group resulting in the collapse of the tetrahedral intermediate, the cleavage of the carbon-nitrogen bond and the release of the products.