Structural and mechanistic analysis of a β-glycoside phosphorylase identified by screening a metagenomic library

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-dinitrophenol from DNPGlc in the presence of phosphate, identified the gene bglP, 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 Nacetylglucosaminide. 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. GH3 β-glycoside phosphorylase from a metagenomic library 2 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.Carbohydrate active enzymes (CAZymes) are the biocatalysts responsible for the assembly, degradation and modification of glycans in biological systems 1 . They are also widely employed enzymes in industry, being used in brewing and food processing, animal feed preparation, industrial pulp and paper applications and increasingly in biofuel and bioproduct development [2][3][4][5] . While the use of CAZymes is cost-effective in glycan degradation, glycan assembly generally requires the use of expensive starting materials, such as nucleotide phosphosugars 6 . The high-cost of these materials makes de novo industrial-scale glycan synthesis difficult and usually non-viable.One class of CAZyme that offers a potential solution to the high costs typically associated with enzymatic glycan synthesis is that of the glycoside phosphorylases (GPs), which are increasingly being recognized and used for the biocatalysis and biotransformation of glycans 7-9 . These enzymes ordinarily carry out phosphorolysis by transferring a glycosyl moiety from the nonreducing end of a di-or polysaccharide substrate onto inorganic phosphate, thereby generating a sugar-1-phosphate 10 ( Figure 1A). GPs distinguish themselves from most CAZymes in that the hydrolytic free energy associated with the glycosidic e...

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Thioglycoligase derived from fungal GH3 β-xylosidase is a multi-glycoligase with broad acceptor tolerance

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