Mutational analysis of the active center of plant fructosyltransferases: Festuca 1-SST and barley 6-SFT.
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.
Altenbach
Denise
D
Botanisches Institut der Universität Basel, Zurich Basel Plant Science Center, Hebelstrasse 1, CH-4056 Basel, Switzerland. denise.altenbach@unibas.ch
Nüesch
Eveline
E
Ritsema
Tita
T
Boller
Thomas
T
Wiemken
Andres
A
D001665:Binding Sites; D004252:DNA Mutational Analysis; D031751:Festuca / Q000201:enzymology*; D006602:Hexosyltransferases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D010843:Pichia / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013379:Substrate Specificity; D013395:Sucrose / Q000378:metabolism; D014312:Trisaccharides / Q000378:metabolism; D001426:Bacterial Proteins
D006602:Hexosyltransferases; D010940:Plant Proteins; D011993:Recombinant Fusion Proteins; D013395:Sucrose; D014312:Trisaccharides; C072433:1-kestose; C097181:sucrose - fructan 6-fructosyltransferase; C021451:inulosucrase; D001426:Bacterial Proteins