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Role of the ATP Synthase α-Subunit in Conferring Sensitivity to Tentoxin

Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K i ≈ 10 nM) site on the chloroplast F1 (CF1) strongly inhibits catalytic function, whereas binding to a second, lower...

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Published in:Biochemistry (Easton) 2001-06, Vol.40 (25), p.7542-7548
Main Authors: Tucker, Ward C, Du, Ziyun, Hein, Ray, Gromet-Elhanan, Zippora, Richter, Mark L
Format: Article
Language:English
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Summary:Tentoxin, produced by phytopathogenic fungi, selectively affects the function of the ATP synthase enzymes of certain sensitive plant species. Binding of tentoxin to a high affinity (K i ≈ 10 nM) site on the chloroplast F1 (CF1) strongly inhibits catalytic function, whereas binding to a second, lower affinity site (K d > 10 μM) leads to restoration and even stimulation of catalytic activity. Sensitivity to tentoxin has been shown to be due, in part, to the nature of the amino acid residue at position 83 on the catalytic β subunit of CF1. An aspartate in this position is required, but is not sufficient, for tentoxin inhibition. By comparison with the solved structure of mitochondrial F1 [Abrahams, J. P., Leslie, A. G. W., Lutter, R., and Walker, J. E. (1994) Nature 370, 621−628], Asp83 is probably located at an interface between α and β subunits on CF1 where residues on the α subunit could also participate in tentoxin binding. A hybrid core F1 enzyme assembled with β and γ subunits of the tentoxin-sensitive spinach CF1, and an α subunit of the tentoxin-insensitive photosynthetic bacterium Rhodospirillum rubrum F1 (RrF1), was stimulated but not inhibited by tentoxin [Tucker, W. C., Du, Z., Gromet-Elhanan, Z. and Richter, M. L. (2001) Eur. J. Biochem. 268, 2179−2186]. In this study, chimeric α subunits were prepared by introducing short segments of the spinach CF1 α subunit from a poorly conserved region which is immediately adjacent to β-Asp83 in the crystal structure, into equivalent positions in the RrF1 α subunit using oligonucleotide-directed mutagenesis. Hybrid enzymes containing these chimeric α subunits had both the high affinity inhibitory tentoxin binding site and the lower affinity stimulatory site. Changing β-Asp83 to leucine resulted in loss of both inhibition and stimulation by tentoxin in the chimeras. The results indicate that tentoxin inhibition requires additional α residues that are not present on the RrF1 α subunit. A structural model of a putative inhibitory tentoxin binding pocket is presented.
ISSN:0006-2960
1520-4995
DOI:10.1021/bi0105227