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Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation

Ritsema, Tita ; Verhaar, Auke ; Vijn, Irma ; Smeekens, Sjef

In: Plant Molecular Biology, 2005, vol. 58, no. 5, p. 597-607

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    Titre
    • Using Natural Variation to Investigate the Function of Individual Amino Acids in the Sucrose-Binding Box of Fructan:Fructan 6G-Fructosyltransferase (6G-FFT) in Product Formation
    Auteur
    • Ritsema, Tita. Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584, Utrecht, CH, The Netherlands
    • Verhaar, Auke. Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584, Utrecht, CH, The Netherlands
    • Vijn, Irma. Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584, Utrecht, CH, The Netherlands
    • Smeekens, Sjef. Department of Molecular Plant Physiology, University Utrecht, Padualaan 8, 3584, Utrecht, CH, The Netherlands
    Type de document
    • Postprint
    Langue
    • Anglais
    Publié dans
    • Plant Molecular Biology, 2005, vol. 58, no. 5, p. 597-607. Kluwer Academic Publishers
    Autre version électronique
    Identifiant OAI-PMH
    • oai:doc.rero.ch:321170
    Summary
    Enzymes of the glycosyl hydrolase family 32 are highly similar with respect to primary sequence but catalyze divergent reactions. Previously, the importance of the conserved sucrose-binding box in determining product specificity of onion fructan:fructan 6G-fructosyltransferase (6G-FFT) was established [Ritsema etal., 2004, Plant Mol. Biol. 54: 853-863]. Onion 6G-FFT synthesizes the complex fructan neo-series inulin by transferring fructose residues to either a terminal fructose or a terminal glucose residue. In the present study we have elucidated the molecular determinants of product specificity by substitution of individual amino acids of the sucrose binding box with amino acids that are present on homologous positions in other fructosyltransferases or vacuolar invertases. Substituting the presumed nucleophile Asp85 of the β-fructosidase motif resulted in an inactive enzyme. 6G-FFT mutants S87N and S87D did not change substrate or product specificities, whereas mutants N84Y and N84G resulted in an inactive enzyme. Most interestingly, mutants N84S, N84A, and N84Q added fructose residues preferably to a terminal fructose and hardly to the terminal glucose. This resulted in the preferential production of inulin-type fructans. Combining mutations showed that amino acid 84 determines product specificity of 6G-FFT irrespective of the amino acid at position 87