Abstract
Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (> or = X3) to release X1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X2 and HF in the presence of X1 as an acceptor molecule. However, the transglycosidation product (X3) was not detectable in the reaction. To convert reducing end xylose-releasing exooligoxylanase to glycosynthase, derivatives with mutations in the catalytic base (Asp-263) were constructed by saturation random mutagenesis. Nine amino acid residue mutants (Asp-263 to Gly, Ala, Val, Thr, Leu, Asn, Cys, Pro, or Ser) were found to possess glycosynthase activity forming X3 from alpha-X2F and X1. Among them, D263C showed the highest level of X3 production, and D263N exhibited the fastest consumption of alpha-X2F. The D263C mutant showed 10-fold lower hydrolytic activity than D263N, resulting in the highest yield of X3. X2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X3 formed by condensation was hydrolyzed before its release from the enzyme. To acquire glycosynthase activity from inverting enzymes, it is important to minimize the decrease in F(-)-releasing activity while maximizing the decrease in the hydrolytic activity. The present study expands the possibility of conversion of glycosynthases from inverting enzymes.
Highlights
In 1979, Hehre et al [6] reported that -amylase hydrolyzed -maltosyl fluoride, the opposite anomer of the glycosides to be hydrolyzed, into maltose and fluoride ions
The new glycoside is hydrolyzed with anomeric inversion at the same site on the enzyme before it is released from the active center, which is the normal reaction of an inverting Glycoside hydrolases (GH) [9, 10]
Because both the retaining and inverting GHs hydrolyze glycosides by two acidic catalytic residues as a general acid and a general base, we considered that removal of the general base from an inverting GH may result in production of a glycosynthase
Summary
In 1998, Withers and co-workers [12] reported that a mutant retaining enzyme, GH 1 -glucosidase from Agrobacterium sp., with a mutation at its nucleophilic residue (Glu-358), catalyzed synthesis of various -glucosides using ␣-glucosyl fluoride as a donor and various p-nitrophenyl--glycosides as acceptors [12]. We considered Rex to be a good model to analyze the Hehre resynthesis-hydrolysis mechanism as well as for the generation of a glycosynthase mutant due to its reaction specificity. Because it hydrolyzed xylotriose at the bond of the reducing end to form X1 from the reducing end and X2 from the non-reducing end, ␣-xylobiosyl fluoride (␣-X2F) and xylose are expected to be a suitable donor and acceptor, respectively.
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