Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT |
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Authors: | Altenbach Denise Nüesch Eveline Ritsema Tita Boller Thomas Wiemken Andres |
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Institution: | Botanisches Institut der Universit?t Basel, Zurich Basel Plant Science Center, Hebelstrasse 1, CH-4056 Basel, Switzerland. denise.altenbach@unibas.ch |
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Abstract: | The active center of the glycoside hydrolase family 32 contains the three characteristic motifs (N/S)DPNG, RDP, and EC. We replaced the N-terminal region including the (N/S)DPNG motif of barley 6-SFT (sucrose:fructan 6-fructosyltransferase) by the corresponding region of Festuca 1-SST (sucrose:sucrose 1-fructosyltransferase). The chimeric enzyme, expressed in Pichia, retained the specificity of 6-SFT. Attempts to replace a larger piece at the N-terminus including also the RDP motif failed. A point mutation introduced in the RDP motif of 1-SST abolished enzymatic activity. Interestingly, point mutations of the EC-motif resulted in an enzyme which had lost the capability to form 1-kestose and glucose from sucrose but still accepted 1-kestose, producing fructose and sucrose as well as nystose. |
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Keywords: | Fructosyltransferase Invertase Enzyme specificity Fructan Pichia pastoris |
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