title: Proteolytic activity of plum pox virus-tobacco etch virus chimeric NIa proteases.
abstract: Plasmids encoding chimeric NIa-type proteases made of sequences from the potyviruses plum pox virus (PPV) and tobacco etch virus (TEV) have been constructed. Their proteolytic activity on the large nuclear inclusion protein (NIb)-capsid protein (CP) junction of each virus was assayed in Escherichia coli cells. The amino half of the protease seemed to be involved neither in the enzymatic catalysis nor in substrate recognition. In spite of the large homology among the PPV and TEV NIa-type proteases, the exchange of fragments from the carboxyl halves of the molecules usually caused a drastic decrease in the enzymatic activity. Inactive chimeric proteases did not interfere with cleavage by PPV wild type protease expressed from a second plasmid. The results suggest that the recognition and catalytic sites of the NIa proteases are closely interlinked and, although residues relevant for the correct interaction with the substrate could be present in other parts of the protein, a main determinant for substrate specificity should lie in a region situated, approximately, between positions 30 and 90 from the carboxyl end. This region includes the conserved His at position 360 of PPV or 355 of TEV, which has been postulated to interact with the Gln at position -1 of the cleavage sites.
authors.affiliation: Centro de Biología Molecular (CSIC-UAM), Universidad Autónoma de Madrid, Spain.
authors.forename: J A
authors.initials: JA
authors.lastname: García
authors.forename: S
authors.initials: S
authors.lastname: Laín
mesh_terms: D001483:Base Sequence; D002678:Chimera; D003001:Cloning, Molecular; D004279:DNA, Viral / Q000235:genetics; D010450:Endopeptidases; D004926:Escherichia coli / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D015345:Oligonucleotide Probes; D010942:Plant Viruses / Q000201:enzymology* / Q000235:genetics; D015183:Restriction Mapping; D014764:Viral Proteins / Q000235:genetics / Q000378:metabolism*
chemical_list: D004279:DNA, Viral; D015345:Oligonucleotide Probes; D014764:Viral Proteins; D010450:Endopeptidases; C059992:nuclear inclusion protein a, mosaic viruses