Substitution of a single amino acid switches the tentoxin-resistant thermophilic F1-ATPase into a tentoxinsensitive enzyme.

In contrast to the homologous bacterial and mitochondrial enzymes the chloroplast F-1-ATPase (CF1) is strongly affected by the phytopathogenic inhibitor tentoxin. Based on structural information obtained from crystals of a CF1-tentoxin co-complex (Groth, G. (2002) Proc. Natt. 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.