From the Departments of Chemistry and Biochemistry and.
the Genome Science and Technology Program.
J Biol Chem. 2018 Mar 2;293(9):3451-3467. doi: 10.1074/jbc.RA117.000948. Epub 2018 Jan 9.
Glycoside phosphorylases have considerable potential as catalysts for the assembly of useful glycans for products ranging from functional foods and prebiotics to novel materials. However, the substrate diversity of currently identified phosphorylases is relatively small, limiting their practical applications. To address this limitation, we developed a high-throughput screening approach using the activated substrate 2,4-dinitrophenyl β-d-glucoside (DNPGlc) and inorganic phosphate for identifying glycoside phosphorylase activity and used it to screen a large insert metagenomic library. The initial screen, based on release of 2,4-dinitrophenyl from DNPGlc in the presence of phosphate, identified the gene encoding a retaining β-glycoside phosphorylase from the CAZy GH3 family. Kinetic and mechanistic analysis of the gene product, BglP, confirmed a double displacement ping-pong mechanism involving a covalent glycosyl-enzyme intermediate. X-ray crystallographic analysis provided insights into the phosphate-binding mode and identified a key glutamine residue in the active site important for substrate recognition. Substituting this glutamine for a serine swapped the substrate specificity from glucoside to -acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying β-glycoside phosphorylases, which was robust, simple to implement, and useful in identifying active clones within a metagenomics library. Implementation of this screen enabled discovery of a new glycoside phosphorylase class and has paved the way to devising simple ways in which enzyme specificity can be encoded and swapped, which has implications for biotechnological applications.
糖苷磷酸化酶在组装有用聚糖方面具有很大的潜力，这些聚糖可用于从功能性食品和益生元到新型材料等各种产品。然而，目前鉴定出的磷酸化酶的底物多样性相对较小，限制了它们的实际应用。为了解决这个限制，我们开发了一种使用活性底物 2,4-二硝基苯-β-d-葡萄糖苷（DNPGlc）和无机磷酸盐的高通量筛选方法，用于鉴定糖苷磷酸化酶活性，并将其用于筛选大型插入宏基因组文库。最初的筛选基于在磷酸盐存在下从 DNPGlc 释放 2,4-二硝基苯，鉴定出编码 CAZy GH3 家族中保留的β-糖苷磷酸化酶的基因。对基因产物 BglP 的动力学和机制分析证实了涉及共价糖基-酶中间体的双取代乒乓机制。X 射线晶体学分析提供了对磷酸盐结合模式的深入了解，并确定了活性位点中一个关键的谷氨酰胺残基，该残基对底物识别很重要。将该谷氨酰胺替换为丝氨酸会将底物特异性从葡萄糖苷交换为乙酰葡萄糖胺苷。总之，我们提出了一种用于鉴定β-糖苷磷酸化酶的高通量筛选方法，该方法稳健、易于实施，并且可用于在宏基因组文库中鉴定活性克隆。该筛选的实施使发现了一种新的糖苷磷酸化酶类，并为设计可编码和交换酶特异性的简单方法铺平了道路，这对生物技术应用具有重要意义。
