5vqe

Publication Abstract from PubMed

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 beta-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-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, encoding a retaining beta-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 N-acetylglucosaminide. In summary, we present a high-throughput screening approach for identifying beta-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.

Structural and mechanistic analysis of a beta-glycoside phosphorylase identified by screening a metagenomic library.,Macdonald SS, Patel A, Larmour VLC, Morgan-Lang C, Hallam SJ, Mark BL, Withers SG J Biol Chem. 2018 Jan 9. pii: RA117.000948. doi: 10.1074/jbc.RA117.000948. PMID:29317495^[1]

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine.