Efficient Glycosynthase Mutant Derived from Mucor hiemalis Endo-β-N-acetylglucosaminidase Capable of Transferring Oligosaccharide from Both Sugar Oxazoline and Natural N-Glycan

Abstract

Endo-M, an endo-␤-N-acetylglucosaminidase fromMucor hiemalis, is a family 85 glycoside hydrolase. This enzyme is unique in that it can transfer en bloc the oligosaccharide of various types of N-glycans onto different acceptors, and thereby it enzymatically generates diverse glycoconjugates. In this study, we performed mutational and kinetic studies focusing on a key catalytic asparagine 175 of Endo-M. We have shown that most of the Asn-175 mutants had significantly diminished hydrolysis activity but acted as glycosynthases capable of using synthetic sugar oxazoline for transglycosylation. Our results confirm the critical role of this asparagine residue in promoting the formation of an oxazolinium ion intermediate in the first step of the substrate-assisted catalysis. Interestingly, the N175Q mutant was found to possess dramatically enhanced glycosynthase-like activity with sugar oxazoline in comparison with N175A and a transglycosidase-like activity with "natural" N-glycan as well. These results also implicated the significance of amide side chain in the asparagine 175 of Endo-M for promoting oxazoline transglycosylation in the second step of the catalysis. The highly efficient syntheses of glycopeptides/glycoproteins by N175Q combined with synthetic sugar oxazolines or natural N-glycan substrates were exemplified. In addition, we also identified several previously unknown residues that seem to play a role in the catalysis of Endo-M. Endo-␤-N-acetylglucosaminidase (ENGase)3 (EC 3.2.1.96) is a glycoside hydrolase that acts on the ␤1,4-glycosidic linkage between the N,NЈ-diacetylchitobiose core of N-glycans. This type of enzymes is classified into two glycoside hydrolase (GH) families, GH18 and GH85, in the CAZY data base. GH18 includes bacterial endo-␤-N-acetylglucosaminidases, such as Endo-H (1), Endo-F 1 (2), Endo-F 2 , and Endo-F 3 (3) as well as many chitinases. On the other hand, GH85 is exclusively composed of endo-␤-N-acetylglucosaminidases from both prokaryotes and eukaryotes, such as Endo-M from Mucor hiemalis (4 -7), Endo-A from Arthrobacter protophormiae (8), Endo-D from Streptococcus pneumoniae (9), Endo-CE from Caenorhabditis elegans (10, 11), and ENGase from human (12). Notably, unlike GH18 enzymes, most of the GH85 enzymes possess transglycosylation activity, i.e. the ability to transfer oligosaccharide en bloc from a donor substrate to a GlcNAc-containing backbone as an acceptor to form new glycoconjugates (13)(14)(15)(16)(17)(18). This enzymatic method is one of the most promising approaches to synthesize homogeneous glycoproteins (19).Structural and mechanistic studies with some GH18 ENGases and GH18 chitinases (20) and GH20 ␤-N-acetylhexosaminidases (21) have indicated that the catalysis of these GH18 and GH20 enzymes proceeds in a substrate-assisted mechanism involving the participation of the 2-acetamide group in the substrate. In this mechanism (Scheme 1), a catalytic residue (Asp or Glu) acts as a general acid to protonate the glycosidic oxygen. Upon activation of the glycosidic bond, the 2...

Glycosynthase Mutants of Endoglycosidase S2 Show Potent Transglycosylation Activity and Remarkably Relaxed Substrate Specificity for Antibody Glycosylation Remodeling

“…These include a key asparagine residue for endoglycosidases Endo-A (Asn-171) (36), Endo-M (Asn-175) (37,38), and Endo-D (Asn-322) (24) of the GH85 family, or a key aspartic acid residue for the GH18 family endoglycosidases Endo-S (Asp-233) (25) and Endo-F3 (Asp-165) (32). Sequence alignment of Endo-S2 and Endo-S revealed that the Asp-184 of Endo-S2 was the residue equivalent to Asp-233 of Endo-S essential for promoting oxazolinium ion formation in hydrolysis (Fig.…”

Endo-F3 Glycosynthase Mutants Enable Chemoenzymatic Synthesis of Core-fucosylated Triantennary Complex Type Glycopeptides and Glycoproteins

“…We found that a quantitative conversion of the Fuc␣1,6GlcNAc-CD52 (8) to the glycopeptide (9) could be achieved within 2 h when a 3-fold excess of the glycan oxazoline (7) was used, demonstrating the efficiency of the Endo-F3 D165A-catalyzed transglycosylation. We have also tested several glycosynthases described previously, including the D233A mutant of Endo-S, the N322A and N322Q mutants of Endo-D, the N175Q mutant of Endo-M, and the N171A mutant of Endo-A (29,30,(32)(33)(34). We found that none of those glycosynthases were able to transfer triantennary complex glycans from the glycan oxazoline.…”

“…We have previously generated glycosynthases from endoglycosidases of both the GH85 and GH18 family by site-directed mutation at a key residue that is responsible for promoting the formation of the oxazolinium ion intermediate during hydrolysis, which proceeds in a substrate-assisted mechanism (29,30,(32)(33)(34). These include a key asparagine residue for the GH85 endoglycosidases Endo-A (Asn-171), Endo-M (Asn-275), and Endo-D (Asn-322) or a key aspartic acid residue for the GH18 family endoglycosidase Endo-S (Asp-233).…”

Biological Analysis of the Microbial Metabolism of Hetero-Oligosaccharides in Application to Glycotechnology

“…This enhanced transglycosylation activity together with its reduced product hydrolysis activity makes N175Q a valuable mutant for glycoconjugate synthesis. 57) …”

“…However, it was found that the rate of transglycosylation with mutant N175A was much lower than that of the wild-type Endo-M or mutant Y217F. Thus, to improve further the catalytic efficiency of the glycosynthase for practical use, we performed systematic mutagenesis at the critical Asn-175 site of Endo-M. 57) We tried to replace this residue with the other 19 natural amino acid residues, except for proline. Of these, N175Q showed extraordinarily high transglycosylation activity, with an initial rate about 9 times and 1.5 times higher than those of N175A and the wild-type enzyme respectively.…”