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

Abstract

In contrast to the homologous bacterial and mitochondrial enzymes the chloroplast F1-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. Natl. Acad. Sci. U. S. A. 99, 3464-3468) we have replaced residues βSer66 and αArg132 in the α3β3γ 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. The mutation αArg132 → Pro, proposed to relieve steric constraints on tentoxin binding, did not have any significant effect. However, mutation βSer66 → 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.