title: Pokeweed antiviral protein (PAP) mutations which permit E.coli growth do not eliminate catalytic activity towards prokaryotic ribosomes.
abstract: Pokeweed antiviral protein (PAP) has N-glycosidase activity towards both eukaryotic and prokaryotic ribosomes. This is in marked contrast with the A chains of type 2 ribosome inactivating proteins (RIPs) such as ricin and abrin, which inactivate only eukaryotic ribosomes. A recent report described spontaneous mutations in PAP that implicated specific amino acids to be involved in determining the activity of PAP towards prokaryotic ribosomes. As part of an ongoing study into RIP--ribosome interactions these mutations were specifically recreated in a PAP clone encoding the mature 262 amino acid PAP sequence. Mutants were tested for their N-glycosidase activity by analysing the integrity of eukaryotic and prokaryotic ribosomes after mutant protein expression. Mutations of F196Y and K211R, either individually or within the same clone, were active toward both classes of ribosome, indicating that these amino acid positions are not involved in differentiating ribosomal substrates. Mutation R68G led to a protein that appeared to be inactive towards prokaryotic ribosomes, but also very poorly active towards eukaryotic ribosomes. This mutation is currently under further investigation.
authors.affiliation: Department of Biological Sciences, University of Warwick, Coventry, UK.
authors.forename: J A
authors.initials: JA
authors.lastname: Chaddock
authors.forename: J M
authors.initials: JM
authors.lastname: Lord
authors.forename: M R
authors.initials: MR
authors.lastname: Hartley
authors.forename: L M
authors.initials: LM
authors.lastname: Roberts
mesh_terms: D001483:Base Sequence; D002384:Catalysis; D003001:Cloning, Molecular; D004247:DNA; D004926:Escherichia coli / Q000235:genetics / Q000254:growth & development*; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D009154:Mutation; D009699:N-Glycosyl Hydrolases; D010940:Plant Proteins / Q000235:genetics* / Q000378:metabolism; D010944:Plants / Q000235:genetics; D054789:Ribosome Inactivating Proteins, Type 1; D012270:Ribosomes / Q000378:metabolism*
chemical_list: D010940:Plant Proteins; D054789:Ribosome Inactivating Proteins, Type 1; D004247:DNA; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C024275:pokeweed antiviral protein