Characterization of Escherichia coli ATP synthase beta-subunit mutations using a chromosomal deletion strain.
(1) We constructed Escherichia coli strain JP17 with a deletion in the ATP synthase beta-subunit gene. JP17 is completely deficient in ATP synthase activity and expresses no beta-subunit. Expression of normal beta-subunit from a plasmid restores haploid levels of ATP synthase in membranes. JP17 was shown to be efficacious for studies of beta-subunit mutations. Site-directed mutants were studied directly in JP17. Randomly generated chromosomal mutants were identified by PCR and DNA sequencing, cloned, and expressed in JP17. (2) Eight novel mutations occurring within the putative catalytic nucleotide-binding domain were characterized with respect to their effects on catalysis and structure. The mutations beta C137S, beta G152D, beta G152R, beta E161Q, beta E161R, and beta G251D each impaired catalysis without affecting enzyme assembly or oligomeric structure and are of interest for future studies of catalytic mechanism. The mutations beta D301V and beta D302V, involving strongly conserved carboxyl residues, caused oligomeric instability of F1. However, growth characteristics of these mutants suggested that neither carboxyl side chain is critical for catalysis. (3) The mutations beta R398C and beta R398W rendered ATP synthase resistant to aurovertin, giving strong support to the view that beta R398 is a key residue in the aurovertin-binding site. Neither beta R398C or beta R398W impaired catalysis significantly.
Lee
R S
RS
Department of Biochemistry, University of Rochester School of Medicine and Dentistry, New York 14642.
Pagan
J
J
Wilke-Mounts
S
S
Senior
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AE
D001313:Aurovertins / Q000378:metabolism; D001483:Base Sequence; D002384:Catalysis; D002872:Chromosome Deletion; D002876:Chromosomes, Bacterial; D004926:Escherichia coli / Q000201:enzymology / Q000235:genetics* / Q000378:metabolism; D005798:Genes, Bacterial; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D006180:Proton-Translocating ATPases / Q000737:chemistry / Q000235:genetics*; D011955:Receptors, Drug / Q000235:genetics
D001313:Aurovertins; D011955:Receptors, Drug; D006180:Proton-Translocating ATPases