Rapid chemoenzymatic synthesis of monodisperse hyaluronan oligosaccharides with immobilized enzyme reactors

被引:73
作者
Deangelis, PL [1 ]
Oatman, LC [1 ]
Gay, DF [1 ]
机构
[1] Univ Oklahoma, Hlth Sci Ctr, Oklahoma Ctr Med Glycobiol, Dept Biochem & Mol Biol, Oklahoma City, OK 73104 USA
关键词
D O I
10.1074/jbc.M306431200
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We describe the chemoenzymatic synthesis of a variety of monodisperse hyaluronan (beta4-glucuronic acid-beta3-N- acetylglucosamine ( HA)) oligosaccharides. Potential medical applications for HA oligosaccharides (similar to 10 - 20 sugars in length) include killing cancerous tumors and enhancing wound vascularization. Previously, the lack of defined oligosaccharides has limited the exploration of these sugars as components of new therapeutics. The Pasteurella multocida HA synthase, pmHAS, a polymerizing enzyme that normally elongates HA chains rapidly (similar to1-100 sugars/s), was converted by mutagenesis into two single-action glycosyltransferases (glucuronic acid transferase and N-acetylglucosamine transferase). The two resulting enzymes were purified and immobilized individually onto solid supports. The two types of enzyme reactors were used in an alternating fashion to produce extremely pure sugar polymers of a single length (up to HA20) in a controlled, stepwise fashion without purification of the intermediates. These molecules are the longest, non-block, monodisperse synthetic oligosaccharides hitherto reported. This technology platform is also amenable to the synthesis of medicant-tagged or radioactive oligosaccharides for biomedical testing. Furthermore, these experiments with immobilized mutant enzymes prove both that pmHAS-catalyzed polymerization is non-processive and that a monomer of enzyme is the functional catalytic unit.
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收藏
页码:35199 / 35203
页数:5
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