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J. Biol. Chem., Vol. 283, Issue 8, 4469-4479, February 22, 2008

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Mutants of Mucor hiemalis Endo-β-N-acetylglucosaminidase Show Enhanced Transglycosylation and Glycosynthase-like Activities^*

Midori Umekawa, Wei Huang, Bing Li, Kiyotaka Fujita^¶, Hisashi Ashida, Lai-Xi Wang¹, and Kenji Yamamoto²

From the Division of Integrated Life Science, Graduate School of Biostudies, Kyoto University, Kitashirakawa, Sakyo-ku, Kyoto 606-8502, Japan, the Institute of Human Virology and Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine, Baltimore, Maryland 21201, and the ^¶Department of Biochemical Science and Technology, Faculty of Agriculture, Kagoshima University, Kagoshima 890-0065, Japan

Endo-β-N-acetylglucosaminidase from Mucor hiemalis (Endo-M), a family 85 glycoside hydrolase, acts on the β1,4 linkage of N,N'-diacetylchitobiose moiety in the N-linked glycans of glycoproteins and catalyzes not only the hydrolysis reaction but also the transglycosylation reaction that transfers the releasing sugar chain to an acceptor other than water to form a new glycosidic linkage. The transglycosylation activity of Endo-M holds a great promise for the chemo-enzymatic synthesis and glyco-engineering of glycoproteins, but the inherent hydrolytic activity for product hydrolysis and low transglycosylation have hampered its broad applications. This paper describes the site-directed mutagenesis on residues in the putative catalytic region of Endo-M to generate mutants with superior transglycosylation activity. Two interesting mutants were discovered. The Y217F mutant was found to possess much enhanced transglycosylation activity and yet much diminished hydrolytic activity in comparison with the wild-type Endo-M. Kinetic analyses revealed that the K_m value of Y217F for an acceptor substrate 4-methylumbelliferyl-β-L-N-acetylglucosaminide was only one-tenth of that of the wild-type, implicating a much higher affinity of Y217F for the acceptor substrate than the wild-type. The other mutant, N175A, acts like a glycosynthase. It was found that mutation at Asn¹⁷⁵"knocked out" the hydrolytic activity, but the mutant was able to take the highly active sugar oxazolines (the transition state mimics) as donor substrates for transglycosylation. This is the first glycosynthase derived from endo-β-N-acetylglucosaminidases that proceed via a substrate-assisted mechanism. Our findings provide further insights on the substrate-assisted mechanism of GH85. The usefulness of the novel glycosynthase was exemplified by the efficient synthesis of a human immunodeficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.

Received for publication, August 27, 2007 , and in revised form, December 4, 2007.

^* This work was supported in part by the 21st Century Centers of Excellence Program of the Japan Society for the Promotion of Science (to the Graduate School of Biostudies and Institute for Virus Research, Kyoto University), by Core Research for Evolutional Science and Technology of the Japan Science and Technology Agency, and by National Institutes of Health Grants R01 GM 073717 and R01 GM080374 (to L. X. W.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ To whom correspondence may be addressed: Institute of Human Virology and Dept. of Biochemistry and Molecular Biology, University of Maryland School of Medicine, 725 W. Lombard St., Baltimore, MD 21201. Tel.: 410-706-4982; Fax: 410-706-5068; E-mail: lwang3{at}ihv.umaryland.edu. ² To whom correspondence may be addressed. Tel.: 81-75-753-6277; Fax: 81-75-753-9228; E-mail: yamamotk{at}kais.kyoto-u.ac.jp.

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