Abstract
In contrast to the homologous bacterial and mitochondrial enzymes the chloroplast F(1)-ATPase (CF(1)) is strongly affected by the phytopathogenic inhibitor tentoxin. Based on structural information obtained from crystals of a CF(1)-tentoxin co-complex (Groth, G. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 3464-3468) we have replaced residues betaSer(66) and alphaArg(132) in the alpha(3)beta(3)gamma subcomplex of the thermophilic F(1)-ATPase from Bacillus PS3 by the corresponding residues of the chloroplast ATPase to confer tentoxin sensitivity to the thermophilic enzyme. The mutation alphaArg(132) --> Pro, proposed to relieve steric constraints on tentoxin binding, did not have any significant effect. However, mutation betaSer(66) --> Ala, predicted to provide a crucial hydrogen bond with the inhibitor, resulted in tentoxin inhibition of ATP hydrolysis comparable with the situation found with the chloroplast enzyme.
Highlights
From the ‡Department of Plant Biochemistry, HeinrichHeine Universitat, D-40225 Dusseldorf, Germany, the §Chemical Resources Laboratory, Tokyo Institute of Technology, Nagatsuta 4259, Midori-ku, Yokohama 226-8503, Japan, the ¶Yoshida ATP System Project, Exploratory Research for Advanced Technology (ERATO), Japan Science and Technology Corporation (JST), 5800-3 Nagatsuta-cho, Midori-ku, Yokohama 226-0026, Japan, and the ʈDepartment of Structural Biology, Faculty of Earth and Life Science, Vrije Universiteit Amsterdam, De Boelelaan 1087, 1081 HV Amsterdam, The Netherlands
In membrane-bound F0F1-ATP synthase, both ATP synthesis and ATP hydrolysis are inhibited by tentoxin [1], with the soluble F1 subcomplex, which is not capable of ATP synthesis; ATP hydrolysis is inhibited [2]
Superimposition of the chloroplast F1-ATPase (CF1) and TF1 Structures at the Tentoxin Binding Site—A superimposition of the structure of the CF1-tentoxin complex [13] with the structure of the ␣33 subcomplex of TF1 [11], which is shown in Fig. 1, B and C, revealed that the positions of residues ␣Leu65, ␣Val75, and ␣Leu238, which probably form important hydrophobic contacts with the inhibitor, are essentially conserved as well as the position of the crucial residue Asp83 in the  subunit (Fig. 1B)
Summary
99, 3464 –3468) we have replaced residues Ser66 and ␣Arg132 in the ␣33␥ subcomplex of the thermophilic F1-ATPase from Bacillus PS3 by the corresponding residues of the chloroplast ATPase to confer tentoxin sensitivity to the thermophilic enzyme. Mutation Ser66 3 Ala, predicted to provide a crucial hydrogen bond with the inhibitor, resulted in tentoxin inhibition of ATP hydrolysis comparable with the situation found with the chloroplast enzyme.
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