The first glycosynthase derived from an inverting glycoside hydrolase

Reducing end xylose-releasing exooligoxylanase (Rex, EC 3.2.1.156) is an inverting GH that hydrolyzes xylooligosaccharides (>= X-3) to release X-1 at their reducing end. The wild-type enzyme exhibited the Hehre resynthesis hydrolysis mechanism, in which alpha-X2F was hydrolyzed to X-2 and HF in the presence of X-1 as an acceptor molecule. However, the transglycosidation product (X-3) 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 X-3 from alpha-X2F and X-1. Among them, D263C showed the highest level of X-3 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 X-3. X-2 was formed from the early stage of the reaction of the D263C mutant, indicating that a portion of the X-3 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.