title: Transglycosidase-like activity of Mucor hiemalis endoglycosidase mutants enabling the synthesis of glycoconjugates using a natural glycan donor.
abstract: Glycan conversion of glycoprotein via the transglycosylation activity of endo-β-N-acetylglucosaminidase is a promising chemoenzymatic technology for the production of glycoproteins including bio-medicines with a homogeneous glycoform. Although Endo-M is a key enzyme in this process, its product undergoes rehydrolysis, which leads to a lower yield, and limits the practical application of this enzyme. We developed several Endo-M mutant enzymes including N175Q with glycosynthase-like activity and/or transglycosidase-like activity. We found that the Endo-M N175H mutant showed glycosynthase-like activity comparable to N175Q as well as transglycosidase-like activity superior to N175Q. Using a natural sialylglycopeptide as a donor substrate, N175H readily transferred the sialo-glycan onto an N-acetylglucosamine residue attached to bovine ribonuclease B (RNase B), yielding a nonnative sialoglycosylated RNase B. These results demonstrate that use of Endo-M N175H is an alternative glycoengineering technique, which provides a relatively high yield of transglycosylation product and avoids the laborious synthesis of a sugar oxazoline as a donor substrate.
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, Japan.
authors.forename: Kouta
authors.initials: K
authors.lastname: Sakaguchi
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, Japan.
authors.forename: Toshihiko
authors.initials: T
authors.lastname: Katoh
authors.affiliation: Research Institute for Bioresources and Biotechnology, Ishikawa Prefectural University, Nonoichi, Ishikawa, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Yamamoto
mesh_terms: D001688:Biological Products / Q000378:metabolism; D002240:Carbohydrate Sequence; D006001:Glycoconjugates / Q000096:biosynthesis* / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase / Q000235:genetics* / Q000378:metabolism*; D009089:Mucor / Q000201:enzymology* / Q000235:genetics; D009154:Mutation; D011134:Polysaccharides / Q000378:metabolism*
chemical_list: D001688:Biological Products; D006001:Glycoconjugates; D006023:Glycoproteins; D011134:Polysaccharides; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase
keywords: Endo-M; chemoenzymatic synthesis; endo-β-N-acetylglucosaminidase; glycosynthase; transglycosidase; transglycosylation