Alternative strategy for converting an inverting glycoside hydrolase into a glycosynthase.
The tyrosine residue Y198 is known to support a nucleophilic water molecule with the general base residue, D263, in the reducing-end xylose-releasing exo-oligoxylanase (Rex). A mutation in the tyrosine residue changing it into phenylalanine caused a drastic decrease in the hydrolytic activity and a small increase in the F(-) releasing activity from alpha-xylobiosyl fluoride in the presence of xylose. In contrast, mutations at D263 resulted in the decreased F(-) releasing activity. As a result of the high F(-) releasing activity and low hydrolytic activity, Y198F of Rex accumulates a large amount of product during the glycosynthase reaction. We propose a novel method for producing a glycosynthase from an inverting glycoside hydrolase by mutating a residue that holds the nucleophilic water molecule with the general base residue while keeping the general base residue intact.
Honda
Yuji
Y
Ishikawa Prefectural University, 1-308, Suematsu, Nonoichi, Ishikawa.
Fushinobu
Shinya
S
Hidaka
Masafumi
M
Wakagi
Takayoshi
T
Shoun
Hirofumi
H
Taniguchi
Hajime
H
Kitaoka
Motomitsu
M
D019943:Amino Acid Substitution / Q000502:physiology; D006026:Glycoside Hydrolases / Q000235:genetics / Q000378:metabolism*; D016695:Glycosyltransferases / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008954:Models, Biological; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D050505:Mutant Proteins / Q000378:metabolism; D010649:Phenylalanine / Q000235:genetics; D013379:Substrate Specificity / Q000235:genetics; D013997:Time Factors; D014443:Tyrosine / Q000235:genetics / Q000502:physiology; D014995:Xylosidases / Q000235:genetics* / Q000378:metabolism*
D050505:Mutant Proteins; D014443:Tyrosine; D010649:Phenylalanine; D016695:Glycosyltransferases; D006026:Glycoside Hydrolases; D014995:Xylosidases; C510191:reducing-end xylose-releasing exo-oligoxylanase, Bacillus halodurans