title: Mutational analysis of the amino acid residues essential for the cis and trans cleavage activity of the potato virus Y 50-kDa protease.
abstract: To examine the proteolytic activities of various truncated derivatives of the potato virus Y (PVY) 50-kDa protease, the derivatives were expressed in Escherichia coli in polyprotein forms fused with coat protein (CP). For the intermolecular cleavage reaction, the truncated proteases were expressed together with the substrate protein containing the polymerase-CP junction. The activity was evaluated by the amount of the mature CP released from the precursor by the intra- and intermolecular cleavage occurring in E. coli. By this experiment, we identified the moiety responsible for the proteolytic activity of the 50-kDa protease to be a 26-kDa polypeptide mapped to the C-terminal half of the protease. Introduction of His234-->Tyr, Asp269-->Asn, or Cys339-->Gly substitution in the putative catalytic triad of the protease abolished its activity. However, the mutated protease with Cys339-->Ser replacement retained a reduced proteolytic activity.
authors.affiliation: Department of Biotechnology, Faculty of Agriculture, University of Tokyo, Japan.
authors.forename: Y
authors.initials: Y
authors.lastname: Yoshida
authors.forename: M
authors.initials: M
authors.lastname: Hidaka
authors.forename: H
authors.initials: H
authors.lastname: Masaki
authors.forename: T
authors.initials: T
authors.lastname: Uozumi
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000235:genetics / Q000378:metabolism*; D001483:Base Sequence; D002384:Catalysis; D010450:Endopeptidases / Q000235:genetics / Q000378:metabolism*; D004926:Escherichia coli; D006868:Hydrolysis; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D017800:Potyvirus / Q000201:enzymology* / Q000235:genetics; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D000596:Amino Acids; D009838:Oligodeoxyribonucleotides; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses