The First Glycosynthase Derived from an Inverting Glycoside Hydrolase

Abstract

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 ␣-X 2 F 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 xylosereleasing 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 ␣-X 2 F and X 1 . Among them, D263C showed the highest level of X 3 production, and D263N exhibited the fastest consumption of ␣-X 2 F. 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.Glycoside hydrolases (GH) 2 are generally categorized into two types, retaining and inverting enzymes, based on changes in the anomeric configurations during the reactions (1-5). The reaction mechanisms of typical retaining and inverting enzymes are illustrated in Fig. 1. Both types of enzyme have two acidic catalytic residues acting as a general acid (a proton donor) and a general base (a nucleophile). In the retaining GH reaction, first, in concert, the general acid donates a proton to the glycosyl oxygen atom, and the nucleophile attacks the anomeric center, producing a covalent-bound intermediate of the nucleophile with Walden inversion. Next, the intermediate undergoes inverting hydrolysis. The reaction of the inverting GH is similar but differs in the reagent attacking the anomeric center; in this case, a water molecule activated by the catalytic base attacks the anomeric center to hydrolyze the glycoside with anomeric inversion.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. Later, various inverting enzymes were shown to hydrolyze the "wrong" glycosyl fluorides (7-10), suggesting that the reaction is common among inverting GHs. Instead of simple hydrolysis, the reaction mechanism consists of two steps. In the first step a new glycoside of the correct anomer forms from the wrong glycosyl fluoride and an acceptor by Walden inversion. The new glycoside is then hydrolyzed with anomeric inversion at the same site on the enzyme before it is release...

A Historical Perspective for the Catalytic Reaction Mechanism of Glycosidase; So As to Bring about Breakthrough in Confusing Situation

“…10). [67][68][69][70][71][72][73] William and Withers 69) have cited it as an example of the mechanism for the hydrolysis of ''wrong'' anomeric fluoride, meaning mistaken recognition of anomeric configuration, in the synthesis and hydrolysis of 4--xylobiose formed from -xylosyl fluoride 66) for inverting -xylosidase (Fig. 10A).…”

“…69,77,78) These schemes ( Fig. 11A and B) are accepted by many investigators, and are referred to in papers 71,73) and reviews, 81,82) but there is some confusion as to the processes. Probably, the schemes ( Fig.…”

“…70) These mutants produce -1,4-xylotriose from -xylobiosyl fluoride and -xylose, but they are intact inverting enzymes, and hence the reaction mechanism appears not to change basically. [71][72][73] It is ambiguous as to whether such mutants fall under the category of glycosynthase. Interesting papers [70][71][72][73]89) and reviews 81,82) on the synthesis of oligosaccharides by mutant enzymes have been published, but the reaction mechanism should be reexamined carefully.…”

Conversion of inverting glycoside hydrolases into catalysts for synthesizing glycosides employing a glycosynthase strategy

“…E-2. Mutation of the base residue (77) First, the base residue, D263, was mutated to convert Rex into glycosynthase following the same procedure used for the retaining GHs. A saturation mutagenesis library of Rex at position D263, regarded as D263X, was constructed and from this mutant library, 120 colonies were randomly selected and each mutant enzyme was puried by His-tag a‹nity column chromatography.…”

Increasing the synthesis/hydrolysis ratio of aminoacylase 1 by site-directed mutagenesis

“…In the absence of such strategies, available structure-function information may guide the improvement of library quality and hence decrease the library size. For processes in aqueous systems, optimization or even reversal of enzymatic synthesis over hydrolysis ratios has been achieved for reactions involving an acyl-enzyme intermediate using e.g., penicillin acylases or glycosyl enzymes by semi-random approaches [3,4]. Yet, optimization of hydrolytic enzymes not forming a covalent intermediate has remained a considerable challenge.…”