糖鎖を丸ごと付加し、生成物を分解しない酵素

グライコシンターゼ（Endo-M-N175Q）は、Endo-Mの活性中心付近を部位特異的に変異させ、糖転移活性は残したまま糖加水分解活性を抑制した、山本・梅川らによって開発された酵素です。糖供与体としてオキサゾリン誘導体を用いることで効率良く付加転移し、得られた糖転移生成物は分解されにくく、蓄積する特徴があります。これらの特徴は、糖鎖工学の有用なツールとして応用が期待されています。

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

M. Umekawa, C. Li, T. Higashiyama, W. Huang, H. Ashida, K. Yamamoto, L-X. Wang, J. Biol. Chem. 2010, 285, 511-521. DOI: 10.1074/jbc.M109.059832

Endo-M, an endo-β-N-acetylglucosaminidase from Mucor 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.