Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities
Mutants of Mucor hiemalis endo-β-N-acetylglucosaminidase show enhanced transglycosylation and glycosynthase-like activities
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DOI：
10.1074/jbc.m707137200
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发表时间：
2008-02-22
影响因子：
4.8
通讯作者：
Yamamoto, Kenji
中科院分区：
文献类型：
--
作者：
Umekawa, Midori;Huang, Wei;Yamamoto, Kenji
毛霉内切-β-N-乙酰氨基葡萄糖苷酶(Endo-M)是一种85家族糖苷水解酶，作用于糖蛋白N-连接的糖链中N，N‘-二乙酰基壳二糖部分的β1，4键，不仅催化水解反应，还催化糖基化反应，将释放的糖链转移到水以外的受体上，形成新的糖苷键。Endo-M的转糖基化活性在糖蛋白的化学酶合成和糖工程中具有广阔的应用前景，但其固有的产物水解性和较低的转糖基化能力阻碍了它的广泛应用。本文描述了对Endo-M可能的催化区残基进行定点突变，以产生具有较高转糖基化活性的突变体。发现了两个有趣的突变体。与野生型内切酶相比，Y217F突变体的转糖基化活性显著增强，而水解性却显著降低。动力学分析表明，Y217F对受体底物4-methylumbel-liferyl-beta-D-N-acetylglucosaminide的K-m值仅为野生型的十分之一，表明Y217F对受体底物的亲和力远高于野生型。另一个突变体N175A的作用类似于糖合成酶。结果发现，Asn175位的突变“破坏”了水解酶的活性，但突变体能够将高活性的糖恶唑啉(模拟过渡态)作为转糖基化的供体底物。这是第一个由内切-β-N-乙酰氨基葡萄糖苷酶衍生的糖合酶，它通过底物辅助机制进行。我们的发现为GH85的底物辅助机制提供了进一步的见解。新型糖合成酶的有效性通过有效合成具有强大抗HIV活性的人类免疫缺陷病毒1型(HIV-1)糖肽来证明。
Endo-beta-N-acetylglucosaminidase from Mucor hiemalis(Endo-M), a family 85 glycoside hydrolase, acts on the beta 1,4 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-methylumbel-liferyl-beta-D-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-beta-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 deficiency virus, type 1 (HIV-1) glycopeptide with potent anti-HIV activity.