title: Biochemical characterization and structural prediction of a novel cytosolic leucyl aminopeptidase of the M17 family from Schizosaccharomyces pombe.
abstract: A new leucyl aminopeptidase activity has been identified in the fission yeast Schizosaccharomyces pombe. The enzyme, which has been purified and named leucyl aminopeptidase yspII (LAP yspII), had a molecular mass of 320 and 54 kDa by gel filtration and SDS/PAGE, respectively, suggesting a homohexameric structure. The enzyme cleaved synthetic aminoacyl-4-nitroanilides at an optimum of pH 8.5, and preferred leucine and methionine as N-terminal amino acids. A clear dependence on Mn2+ concentration for activity was found, and an apparent association constant of 0.33 mM was calculated for the metal ion. Bestatin behaved as a competitive inhibitor of LAP yspII (K(i) = 0.14 microM), while chelating agents such as chloroquine, EDTA and 1,10-phenanthroline also reduced enzyme activity. A MALDI-MS analysis, followed by sequencing of two of the resulting peptides, showed that LAP yspII undoubtedly corresponds to the putative aminopeptidase C13A11.05 identified in the S. pombe genome project. The protein exhibited nearly 40% sequence identity to fungal and mammalian aminopeptidases belonging to the M17 family of metallopeptidases. Catalytic residues (Lys292 and Arg366), as well as those involved in coordination with the cocatalytic metal ions (Lys280, Asp285, Asp303, Asp362 and Glu364) and those forming the hydrophobic pocket for substrate binding (Met300, Asn360, Ala363, Thr390, Leu391, Ala483 and Met486), were perfectly conserved among all known aminopeptidases. The S. pombe enzyme is predicted to be formed two clearly distinguished domains with a well conserved C-terminal catalytic domain showing a characteristic topology of eight beta-sheets surrounded by alpha-helical segments in the form of a saddle.
authors.affiliation: Laboratorio de Bioquímica y Biología Molecular, Centro de Química del Instituto de Ciencias, Universidad Autónoma de Puebla, Puebla, Mexico. pherrera@siu.buap.mx
authors.forename: Irma
authors.initials: I
authors.lastname: Herrera-Camacho
authors.forename: Nora H
authors.initials: NH
authors.lastname: Rosas-Murrieta
authors.forename: Arturo
authors.initials: A
authors.lastname: Rojo-Domínguez
authors.forename: Lourdes
authors.initials: L
authors.lastname: Millán
authors.forename: Julio
authors.initials: J
authors.lastname: Reyes-Leyva
authors.forename: Gerardo
authors.initials: G
authors.lastname: Santos-López
authors.forename: Paz
authors.initials: P
authors.lastname: Suárez-Rendueles
mesh_terms: D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D020134:Catalytic Domain; D002413:Cations, Divalent / Q000378:metabolism; D003600:Cytosol / Q000201:enzymology*; D004789:Enzyme Activation; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D007930:Leucine / Q000031:analogs & derivatives / Q000494:pharmacology; D007931:Leucyl Aminopeptidase / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D008345:Manganese / Q000378:metabolism; D008956:Models, Chemical; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008970:Molecular Weight; D011485:Protein Binding; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D017434:Protein Structure, Tertiary; D012568:Schizosaccharomyces / Q000201:enzymology*; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity
chemical_list: D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008345:Manganese; D007931:Leucyl Aminopeptidase; D007930:Leucine; C012211:ubenimex