Polysaccharide degradation in an Antarctic bacterium: Discovery of glycoside hydrolases from remote regions of the sequence space

Glycoside hydrolases (GHs) are enzymes involved in the degradation of oligosaccharides and polysaccharides. The sequence space of GHs is rapidly expanding due to the increasing number of available sequences. This expansion paves the way for the discovery of novel enzymes with peculiar structural and functional properties. This work is focused on two GHs, Ps_GH5 and Ps_GH50, from the genome of the Antarctic bacterium Pseudomonas sp. ef1. These enzymes are in an unexplored region of the sequence space of their respective GH families, not allowing a reliable sequence-based function prediction. For this reason, a computational pipeline was developed that combines deep learning “dynamic docking” on AlphaFold 3D models with physics-based molecular dynamics simulations to infer their substrate specificity. From in silico screening of a repertoire of potential oligosaccharides, only xylooligosaccharides for Ps_GH5 and galactooligosaccharides for Ps_GH50 emerged as catalytically competent substrates. Biochemical characterization agrees with computational simulations indicating that Ps_GH5 is an endo-β-xylanase, and Ps_GH50 is active mainly on small galactooligosaccharides. In conclusion, this study identifies two novel GHs subfamilies placed in remote regions of the sequence space and highlights the efficacy of substrate specificity prediction by computational approaches in the discovery of new enzymes.