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Mutational analysis of the active center of plant fructosyltransferases: Festuca 1‐SST and barley 6‐SFT
Author(s) -
Altenbach Denise,
Nüesch Eveline,
Ritsema Tita,
Boller Thomas,
Wiemken Andres
Publication year - 2005
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/j.febslet.2005.07.034
Subject(s) - festuca , center (category theory) , biology , botany , chemistry , poaceae , crystallography
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.