5VQE

Beta-glucoside phosphorylase BglX bound to 2FGlc

5VQE の概要

• エントリーDOI: 10.2210/pdb5vqe/pdb
• 関連するPDBエントリー: 5VQD
• 分子名称: Beta-glucoside phosphorylase BglX, 2-deoxy-2-fluoro-alpha-D-glucopyranose (3 entities in total)
• 機能のキーワード: beta-glucoside phosphorylase, cazy family 3 gh3 bglx 2fglc 2, 4-dinitrophenyl 2-deoxy-2-fluoro-beta-d-glucopyranoside, hydrolase
• 由来する生物種: unidentified
• タンパク質・核酸の鎖数: 1
• 化学式量合計: 64856.27

構造登録者

Patel, A.,Mark, B.L. (登録日: 2017-05-08, 公開日: 2018-01-17, 最終更新日: 2024-10-23)

主引用文献

Macdonald, S.S.,Patel, A.,Larmour, V.L.C.,Morgan-Lang, C.,Hallam, S.J.,Mark, B.L.,Withers, S.G.
Structural and mechanistic analysis of a beta-glycoside phosphorylase identified by screening a metagenomic library.
J. Biol. Chem., 293:3451-3467, 2018

PubMed Abstract: 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.

PubMed: 29317495
DOI: 10.1074/jbc.RA117.000948

実験手法

X-RAY DIFFRACTION (1.889 Å)