Evolution of Phosphorylases from N‑Acetylglucosaminide Hydrolases in Family GH3

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