Amidation of Bioactive Peptides: The Structure of the Lyase Domain of the Amidating Enzyme

Many neuropeptides and peptide hormones require amidation of their carboxy terminal for full biological activity. The enzyme peptidyl-a-hydroxyglycine a-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-a-hydroxyglycine to generate the a-amidated peptide and glyoxylate. Here we report the X-ray crystal structure of the PAL catalytic core (PALcc) alone and in complex with the nonpeptidic substrate a-hydroxyhippuric acid. The structures show that PAL folds as a six-bladed beta-propeller. The active site is formed by a Zn(II) ion coordinated by three histidine residues; the substrate binds to this site with its a-hydroxyl group coordinated to the Zn(II) ion. The structures also reveal a tyrosine residue (Tyr(654)) at the active site as the catalytic base for hydroxyl deprotonation, an unusual role for tyrosine. A reaction mechanism is proposed based on this structural data and validated by biochemical analysis of site-directed PALcc mutants.