Complete inhibition of the tentoxin-resistant F1-ATPase from Escherichia coli by the phytopathogenic inhibitor tentoxin after substitution of critical residues in the alpha - and beta -subunit.

Substitution of critical residues in the alpha- and beta-subunit can turn the typically resistant ATP synthase from the bacterium Escherichia coli into an enzyme showing high sensitivity to the phytopathogenic inhibitor tentoxin, which usually affects only certain sensitive plant species. In contrast to recent results obtained with the thermophilic F(1) (Groth, G., Hisabori, T., Lill, H., and Bald, D. (2002) J. Biol. Chem. 277, 20117-20119), substitution of a critical serine in the beta-subunit (betaSer(59)), which is supposed to provide an important intermolecular hydrogen bond in the binding site, was not sufficient on its own for conferring tentoxin sensitivity to the E. coli F(1) complex. Superimposition of the chloroplast F(1)-tentoxin inhibitor complex on a homology model of the E. coli F(1) complex provided detailed information on the critical residues in the alpha-subunit of the binding cleft and allowed us to model the binding site according to the steric requirements of the inhibitor. Substitution of the highly conserved residue alphaLeu(64) seems to be most important for allowing access of the inhibitor to the binding site. Combining this substitution with either additional replacements in the alpha-subunit (Q49A, L95A, E96Q, I273M) or the replacement of Ser(59) in the beta-subunit enhanced the sensitivity to the inhibitor and resulted in a complete inhibition of the E. coli F(1)-ATPase by the plant-specific inhibitor tentoxin.