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Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.

ABSTRACT: Endo-M, an endo-beta-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.

SUBMITTER: Umekawa M

PROVIDER: S-EPMC2804199 | biostudies-literature | 2010 Jan

REPOSITORIES: biostudies-literature

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Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.

Umekawa Midori M Li Cishan C Higashiyama Takayuki T Huang Wei W Ashida Hisashi H Yamamoto Kenji K Wang Lai-Xi LX

The Journal of biological chemistry 20091030 1

Endo-M, an endo-beta-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 acti ...[more]

PMID: 19880511

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Project description:Endo-β-N-acetylglucosaminidase (ENGase), which catalyzes hydrolysis of N-linked oligosaccharides, is a useful tool for analyzing oligosaccharide contents of glycoproteins. However, there are only a few known ENGases that can catalyze the hydrolysis of human complex type oligosaccharides, and although commercially available, they are expensive. Here, we report the cloning of two ENGase encoding cDNAs from the basidiomycete fungus Coprinopsis cinerea, Endo-CC1 and Endo-CC2. We successfully expressed recombinant His6-tagged Endo-CC1 and Endo-CC2 in Escherichia coli and purified them for enzymatic characterization. Both Endo-CC1 and Endo-CC2 showed hydrolytic activity on high-mannose and complex type oligosaccharides. Since Endo-CC1 could be prepared more easily than Endo-CC2 from E. coli cultures, we examined the enzymatic properties of Endo-CC1 in detail. Our results showed that Endo-CC1 acted on both N-linked high-mannose type and sialobiantennary type complex oligosaccharides of glycoproteins RNase B and human transferrin, respectively, but not on the sialotriantennary type complex oligosaccharide of glycoprotein fetuin. Examination of the transglycosylation activity of Endo-CC1 revealed that the wild-type Endo-CC1 could not transfer the sialobiantennary type complex oligosaccharide onto the deglycosylated RNase B. To obtain an Endo-CC1 mutant with desired transglycosylation activity, we performed mutation analysis of the active site residue Asn 180 (N180), known to be important for catalysis, by individually replacing it with the remaining 19 amino acid residues. Transglycosylation analyses of these mutants led us to identify one mutant, namely Endo-CC1N180H, which exhibited the desired transglycosylation activity. Taken together, we suggest that Endo-CC1 would potentially be a valuable tool for analyzing oligosaccharides on glycoproteins, as large quantities of it could be made available more easily and less expensively than the currently used enzyme, Endo-M.

Project description:In the endoplasmic reticulum-associated degradation system of plant and animal cells, high-mannose type free N-glycans (HMT-FNGs) are produced from misfolded glycoproteins prior to proteasomal degradation, and two enzymes, cytosolic peptide:N-glycanase (cPNGase) and endo-β-N-acetylglucosaminidase (endo-β-GlcNAc-ase), are involved in the deglycosylation. Although the physiological functions of these FNGs in plant growth and development remain to be elucidated, detailed characterization of cPNGase and endo-β-GlcNAc-ase is required. In our previous work, we described the purification, characterization, and subcellular distribution of some plant endo-β-GlcNAc-ases and preliminarily reported the gene information of rice endo-β-GlcNAc-ase (Endo-Os). Furthermore, we analyzed the changes in gene expression of endo-β-GlcNAc-ase during tomato fruit maturation and constructed a mutant line of Arabidopsis thaliana, in which the two endo-β-GlcNAc-ase genes were knocked-out based on the Endo-Os gene. In this report, we describe the purification, characterization, amino acid sequence, and gene cloning of Endo-Os in detail. Purified Endo-Os, with an optimal pH of 6.5, showed high activity for high-mannose type N-glycans bearing the Manα1-2Manα1-3Manβ1 unit; this substrate specificity was almost the same as that of other plant endo-β-GlcNAc-ases, suggesting that Endo-Os plays a critical role in the production of HTM-FNGs in the cytosol. Electrospray ionization-mass spectrometry analysis of the tryptic peptides revealed 17 internal amino acid sequences, including the C terminus; the N-terminal sequence could not be identified due to chemical modification. These internal amino acid sequences were consistent with the amino acid sequence (UniProt ID: Q5W6R1) deduced from the Oryza sativa cDNA clone AK112067 (gene ID: Os05g0346500). Recombinant Endo-Os expressed in Escherichia coli using cDNA showed the same enzymatic properties as those of native Endo-Os.

Dataset Information

Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.

Publications

Efficient glycosynthase mutant derived from Mucor hiemalis endo-beta-N-acetylglucosaminidase capable of transferring oligosaccharide from both sugar oxazoline and natural N-glycan.

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