Evolution of Phosphorylases from <i>N</i>-Acetylglucosaminide Hydrolases in Family GH3

Glycoside phosphorylases hold great potential as catalysts for the synthesis of valuable sugars, glycosides, and glycans or development energy-efficient microbial cell factories. However, number available phosphorylase specificities is rather limited. Here, we show that it possible to establish significant activity in GH3 glycoside hydrolases from Pseudomonas aeruginosa Bacillus subtilis. Single-site substitutions could introduce ability produce glycosyl phosphates, a combinatorial saturation study demonstrated this promiscuous side can be further optimized through various mutational paths. These findings suggest future endeavors start engineering templates. In addition, provide insights into elusive determinants natural phosphorylases.