title: Random mutagenesis of the gene for the beta-subunit of F1-ATPase from Escherichia coli.
abstract: ATP synthesis by oxidative phosphorylation in Escherichia coli occurs in catalytic sites on the beta-subunits of F1-ATPase. Random mutagenesis of the beta-subunit combined with phenotypic screening is potentially important for studies of the catalytic mechanism. However, when applied to haploid strains, this approach is hampered by a preponderance of mutants in which assembly of F1-ATPase in vivo is defective, precluding enzyme purification. Here we mutagenized plasmids carrying the uncD (beta-subunit) gene with hydroxylamine or N-methyl-N'-nitro-N-nitrosoguanidine and isolated, by phenotypic screening and complementation tests, six plasmids carrying mutant uncD alleles. When the mutant plasmids were used to transform a suitable uncD- strain, assembly of F1-ATPase in vivo occurred in each case. Moreover, in one case (beta Gly-223----Asp) F1-ATPase assembly proceeded although it had previously been reported that this mutation, when present on the chromosome of a haploid strain, prevented assembly of the enzyme in vivo. Therefore, this work demonstrates an improved approach for random mutagenesis of the F1-beta-subunit. Six new mutant uncD alleles were identified: beta Cys-137----Tyr; beta Gly-142----Asp; beta Gly-146----Ser; beta Gly-207----Asp; beta-Gly-223----Asp; and a double mutant beta Pro-403----Ser,Gly-415----Asp which we could not separate. The first five of these lie within or very close to the predicted catalytic nucleotide-binding domain of the beta-subunit. The double mutant lies outside this domain; we speculate that the region around residues beta 403-415 is part of an alpha-beta intersubunit contact surface. Membrane ATPase and ATP-driven proton pumping activities were impaired by all six mutations. Purified F1-ATPase was obtained from each mutant and shown to have impaired specific ATPase activity.
authors.affiliation: Department of Biochemistry, University of Rochester Medical Center, New York 14642.
authors.forename: F A
authors.initials: FA
authors.lastname: Kironde
authors.forename: D
authors.initials: D
authors.lastname: Parsonage
authors.forename: A E
authors.initials: AE
authors.lastname: Senior
mesh_terms: D000483:Alleles; D004926:Escherichia coli / Q000201:enzymology*; D006898:Hydroxylamines; D008769:Methylnitronitrosoguanidine; D009154:Mutation; D010957:Plasmids / Q000187:drug effects; D006180:Proton-Translocating ATPases / Q000235:genetics*
chemical_list: D006898:Hydroxylamines; D008769:Methylnitronitrosoguanidine; D006180:Proton-Translocating ATPases