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-alpha-hydroxyglycine alpha-amidating lyase (PAL; EC 4.3.2.5) catalyzes the second and last step of this reaction, N-dealkylation of the peptidyl-alpha-hydroxyglycine to generate the alpha-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 alpha-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 alpha-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.
Chufán
Eduardo E
EE
Department of Biophysics and Biophysical Chemistry, Johns Hopkins School of Medicine, Johns Hopkins University, Baltimore, MD 21205, USA.
De
Mithu
M
Eipper
Betty A
BA
Mains
Richard E
RE
Amzel
L Mario
LM
D000409:Alanine / Q000378:metabolism; D019760:Amidine-Lyases / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D000818:Animals; D001120:Arginine / Q000737:chemistry; D001665:Binding Sites / Q000235:genetics; D016466:CHO Cells; D002118:Calcium / Q000378:metabolism; D006224:Cricetinae; D003412:Cricetulus; D018360:Crystallography, X-Ray; D005998:Glycine / Q000378:metabolism; D006626:Hippurates / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D057927:Hydrophobic and Hydrophilic Interactions; D007700:Kinetics; D008190:Lyases / Q000737:chemistry*; D008715:Methionine / Q000737:chemistry; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D010455:Peptides / Q000737:chemistry*; D011485:Protein Binding; D017510:Protein Folding; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D051381:Rats; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014162:Transfection; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D015032:Zinc / Q000737:chemistry
D006626:Hippurates; D010455:Peptides; C545353:alpha-hydroxyhippuric acid; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; D008715:Methionine; D008190:Lyases; D019760:Amidine-Lyases; C068521:peptidylamidoglycolate lyase; D015032:Zinc; D000409:Alanine; D002118:Calcium; D005998:Glycine