一种细菌糖基转移酶的结构与生化分析
Structural and biochemical analysis of a bacterial glycosyltransferase.
作者信息
Zhu Fan, Wu Ren, Zhang Hua, Wu Hui
机构信息
Department of Pediatric Dentistry, University of Alabama at Birmingham, Schools of Dentistry and Medicine, Birmingham, AL, USA.
出版信息
Methods Mol Biol. 2013;1022:29-39. doi: 10.1007/978-1-62703-465-4_3.
Abstract
Glycosyltransferases (GTs) are a large family of enzymes that specifically transfer sugar moieties to a diverse range of substrates. The process of bacterial glycosylation (such as biosynthesis of glycolipids, glycoproteins, and polysaccharides) has been studied extensively, yet the majority of GTs involved remains poorly characterized. Besides predicting enzymatic parameters of GTs, the resolution of three-dimensional structures of GTs can help to determine activity, donor sugar binding, and acceptor substrate binding sites. It also facilitates amino acid sequence-based structural modeling and biochemical characterization of their homologues. Here we describe a general procedure to accomplish expression and purification of soluble and active recombinant GTs. Enzymatic characterization, and crystallization of GTs, and data refinement for structural analysis are also covered in this protocol.
摘要
糖基转移酶（GTs）是一类庞大的酶家族，可将糖基特异性转移至多种不同的底物上。细菌糖基化过程（如糖脂、糖蛋白和多糖的生物合成）已得到广泛研究，但所涉及的大多数GTs的特性仍不清楚。除了预测GTs的酶学参数外，解析GTs的三维结构有助于确定其活性、供体糖结合位点和受体底物结合位点。这也有助于基于氨基酸序列的同源物结构建模和生化特性分析。在此，我们描述了一种实现可溶性和活性重组GTs表达与纯化的通用方法。本方案还涵盖了GTs的酶学特性分析、结晶以及用于结构分析的数据优化。
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