Discovery of Novel Endolytic Hyaluronate Lyases Belonging to a New Subfamily of GH56 and Enhancement of Catalytic Activity through Catalytic Cleft Engineering

Abstract Hyaluronate lyases have been widely used to degrade hyaluronic acid (HA) and prepare HA oligosaccharides. To date, no hyaluronate lyase has been reported in glycoside hydrolase family 56 (GH56). Herein, SpskHL from Sporothrix schenckii 1099-18 was first identified as a GH56 endolytic hyaluronate lyase, and then its four homologues were discovered. These five microbial GH56 enzymes cleaved HA, yielding unsaturated HA oligosaccharides with ΔHA2 as the end product, forming a new GH56 subfamily of endolytic hyaluronate lyase. Through alanine-scanning mutagenesis of 15 residues in the catalytic cleft of SpskHL and combining three positive mutations, triple-site mutant SpskHLM3 (Y120A/N125A/H189A) increased activity by 126%. Grafting of the triple-site mutation onto four other hyaluronate lyases increased their activities by 74%–155%. Molecular dynamics simulations suggested that all five triple-site mutants had an expanded catalytic cleft, a larger cavity near the catalytic residue E, and a shorter catalytic OE2-O4 distance, which might contribute to their enhanced activities.