Characterization of Glycosynthase Mutants Derived from Glycoside Hydrolase Family 10 Xylanases
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Four xylanases belonging to glycoside hydrolase family 10—<I>Thermotoga maritima</I> XylB (TM), <I>Clostridium stercorarium</I> XynB (CS), <I>Bacillus halodurans</I> XynA (BH), and <I>Cellulomonas fimi</I> Cex (CF)—were converted to glycosynthases by substituting the nucleophilic glutamic acid residues with glycine, alanine, and serine. The glycine mutants exhibited the highest levels of glycosynthase activity with all four enzymes. All the glycine mutants formed polymeric β-1,4-linked xylopyranose as a precipitate during reaction with α-xylobiosyl fluoride. Two glycine mutants (TM and CF) recognized X<SUB>2</SUB> as an effective acceptor molecule to prohibit the formation of the polymer, while the other two (CS and BH) did not. The difference in acceptor specificity is considered to reflect the difference in substrate affinity at their +2 subsites. The results agreed with the structural predictions of the subsite, where TM and CF exhibit high affinity at subsite 2, suggesting that the glycosynthase technique is useful for investigating the affinity of +subsites.
- Agricultural and Biological Chemistry
Agricultural and Biological Chemistry 70(5), 1210-1217, 2006-05-23
Japan Society for Bioscience, Biotechnology, and Agrochemistry