A Synthetic Gene Library Yields a Previously Unknown Glycoside Phosphorylase That Degrades and Assembles Poly-β-1,3-GlcNAc, Completing the Suite of β-Linked GlcNAc Polysaccharides.

The considerable utility of glycoside phosphorylases (GPs) has led to substantial efforts over the past two decades to expand the breadth of known GP activities. Driven largely by the increase of available genomic DNA sequence data, the gap between the number of sequences in the carbohydrate active enzyme database (CAZy DB) and its functionally characterized members continues to grow. This wealth of sequence data presented an exciting opportunity to explore the ever-expanding CAZy DB to discover new GPs with never-before-described functionalities.

N-Glycan Degradation Pathways in Gut- and Soil-Dwelling Actinobacteria Share Common Core Genes.

N-Glycosylation is a fundamental protein modification found in both eukaryotes and archaea. Despite lacking N-glycans, many commensal and pathogenic bacteria have developed mechanisms to degrade these isoforms for a variety of functions, including nutrient acquisition and evasion of the immune system. Although much is known about many of the enzymes responsible for N-glycan degradation, the enzymes involved in cleaving the N-glycan core have only recently been discovered.

Development and Application of a High-Throughput Functional Metagenomic Screen for Glycoside Phosphorylases.

Glycoside phosphorylases (GPs) catalyze the reversible phosphorolysis of glycosidic bonds, releasing sugar 1-phosphates. To identify a greater range of these under-appreciated enzymes, we have developed a high-throughput functional screening method based on molybdenum blue formation. In a proof-of-principle screen focused on cellulose-degrading GPs we interrogated ∼23,000 large insert (fosmid) clones sourced from microbial communities inhabiting two separate environments and identified seven novel GPs from carbohydrate active enzyme family GH94 and one from GH149.

N-acetylglucosaminidases from CAZy family GH3 are really glycoside phosphorylases, thereby explaining their use of histidine as an acid/base catalyst in place of glutamic acid.

CAZy glycoside hydrolase family GH3 consists primarily of stereochemistry-retaining β-glucosidases but also contains a subfamily of β-N-acetylglucosaminidases. Enzymes from this subfamily were recently shown to use a histidine residue within a His-Asp dyad contained in a signature sequence as their catalytic acid/base residue. Reasons for their use of His rather than the Glu or Asp found in other glycosidases were not apparent.