title: Catalytic properties and sensitivity to tentoxin of Chlamydomonas reinhardtii ATP synthases changed in codon 83 of atpB by site-directed mutagenesis.
abstract: The participation of the amino acid beta83 in determining the sensitivity of chloroplast ATP synthases to tentoxin was reported previously. We have changed codon 83 of the Chlamydomonas reinhardtii atpB gene by site-directed mutagenesis to further examine the role of this amino acid in the response of the ATP synthase to tentoxin and in the mechanism of ATP synthesis and hydrolysis. Amino acid beta83 was changed from Glu to Asp (betaE83D) and to Lys (betaE83K), and the highly conserved tetrapeptide betaT82-E83-G84-L85 (DeltaTEGL) was deleted. Mutant strains were produced by particle gun transformation of atpB deletion mutants cw15DeltaatpB and FUD50 with the mutated atpB genes. The transformants containing the betaE83D and betaE83K mutant genes grew well photoautotrophically. The DeltaTEGL transformant did not grow photoautotrophically, and no CF1 subunits were detected by immunostaining of Western blots using CF1 specific antibodies. The rates of ATP synthesis at clamped DeltapH with thylakoids isolated from cw15 and the two mutants, betaE83D and betaE83K, were similar. However, only the phosphorylation activity of the mutant betaE83D was inhibited by tentoxin with 50% inhibition attained at 4 microM. These results confirm that amino acid beta83 is critical in determining the response of ATP synthase to tentoxin. The rates of the latent Mg-ATPase activity of the CF1s isolated from cw15, betaE83D, and betaE83K were similar and could be enhanced by heat, alcohols, and octylglucoside. As in the case of the membrane-bound enzyme, only CF1 from the betaE83D mutant was sensitive to tentoxin. A lower alcohol concentration was required for optimal stimulation of the ATPase of the betaE83K-CF1 than that of CF1 from the other two strains. Moreover, the optimal activity of the betaE83K-CF1 was also lower. These results suggest that introduction of an amino acid with a positively charged side chain in position 83 in the "crown" domain affects the active conformation of the CF1-ATPase.
authors.affiliation: Doris and Bertie Black Center for Bioenergetics in Life Sciences, Ben Gurion University of the Negev, Beer Sheva 84105, Israel.
authors.forename: D
authors.initials: D
authors.lastname: Hu
authors.forename: H R
authors.initials: HR
authors.lastname: Fiedler
authors.forename: T
authors.initials: T
authors.lastname: Golan
authors.forename: M
authors.initials: M
authors.lastname: Edelman
authors.forename: H
authors.initials: H
authors.lastname: Strotmann
authors.forename: N
authors.initials: N
authors.lastname: Shavit
authors.forename: S
authors.initials: S
authors.lastname: Leu
mesh_terms: D025181:ATP Synthetase Complexes; D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D016825:Chlamydomonas reinhardtii / Q000201:enzymology*; D003062:Codon; D000431:Ethanol / Q000494:pharmacology; D005960:Glucosides / Q000494:pharmacology; D000432:Methanol / Q000494:pharmacology; D008969:Molecular Sequence Data; D009097:Multienzyme Complexes / Q000235:genetics*; D016297:Mutagenesis, Site-Directed; D009183:Mycotoxins / Q000494:pharmacology*; D010456:Peptides, Cyclic / Q000494:pharmacology*; D017856:Phosphotransferases (Phosphate Group Acceptor) / Q000235:genetics*; D016133:Polymerase Chain Reaction; D017384:Sequence Deletion
chemical_list: D003062:Codon; D005960:Glucosides; D009097:Multienzyme Complexes; D009183:Mycotoxins; D010456:Peptides, Cyclic; C018619:octyl-beta-D-glucoside; D000431:Ethanol; D025181:ATP Synthetase Complexes; D017856:Phosphotransferases (Phosphate Group Acceptor); D017301:Ca(2+) Mg(2+)-ATPase; C003384:tentoxin; D000432:Methanol