Crystal structure and mechanism of human carboxypeptidase O : insights into its specific activity for acidic residues
García Guerrero, María del Carmen (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Garcia-Pardo, Javier (Institut Català de Nanociència i Nanotecnologia)
Berenguer De La Cuesta, Esther (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Fernandez-Alvarez, Roberto (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Barfi, Gifty B. (Andrews University. Department of Biology)
Lyons, Peter J. (Andrews University. Department of Biology)
Avilés, Francesc Xavier (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Huber, Robert (Technische Universität München)
Lorenzo Rivera, Julia (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)
Reverter i Cendrós, David (Universitat Autònoma de Barcelona. Departament de Bioquímica i de Biologia Molecular)

Fecha:	2018
Resumen:	Human metallocarboxypeptidase O (hCPO) is a recently discovered digestive enzyme localized to the apical membrane of intestinal epithelial cells. Unlike pancreatic metallocarboxypeptidases, hCPO is glycosylated and produced as an active enzyme with distinctive substrate specificity toward C-terminal (C-t) acidic residues. Here we present the crystal structure of hCPO at 1. 85-Å resolution, both alone and in complex with a carboxypeptidase inhibitor (NvCI) from the marine snail Nerita versicolor. The structure provides detailed information regarding determinants of enzyme specificity, in particular Arg275, placed at the bottom of the substrate-binding pocket. This residue, located at "canonical" position 255, where it is Ile in human pancreatic carboxypeptidases A1 (hCPA1) and A2 (hCPA2) and Asp in B (hCPB), plays a dominant role in determining the preference of hCPO for acidic C-t residues. Site-directed mutagenesis to Asp and Ala changes the specificity to C-t basic and hydrophobic residues, respectively. The single-site mutants thus faithfully mimic the enzymatic properties of CPB and CPA, respectively. hCPO also shows a preference for Glu over Asp, probably as a consequence of a tighter fitting of the Glu side chain in its S1' substrate-binding pocket. This unique preference of hCPO, together with hCPA1, hCPA2, and hCPB, completes the array of C-t cleavages enabling the digestion of the dietary proteins within the intestine. Finally, in addition to activity toward small synthetic substrates and peptides, hCPO can also trim C-t extensions of proteins, such as epidermal growth factor, suggesting a role in the maturation and degradation of growth factors and bioactive peptides.
Ayudas:	Ministerio de Economía y Competitividad BFU2015-66417-P Agencia Estatal de Investigación BIO2016-78057-R Ministerio de Ciencia e Innovación BES-2011-044872 Ministerio de Educación, Cultura y Deporte FPU12/06137
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Lengua:	Anglès
Documento:	Article ; recerca ; Versió acceptada per publicar
Materia:	Carboxypeptidase ; Protein digestion ; Crystal structure ; Acidic protease
Publicado en:	Proceedings of the National Academy of Sciences of the United States of America, Vol. 115, Issue 17 (April 2018) , p. E3932-E3939, ISSN 1091-6490

DOI: 10.1073/pnas.1803685115
PMID: 29636417

Postprint 29 p, 862.1 KB

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Documentos de investigación > Documentos de los grupos de investigación de la UAB > Centros y grupos de investigación (producción científica) > Ciencias > Institut Català de Nanociència i Nanotecnologia (ICN2)
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