Biochemical Characterization and Substrate Specificity Mechanism of an Exolytic Hyaluronate Lyase SinHL from <i>Streptococcus iniae</i> QMA0131

Hyaluronate lyases are widely used for the degradation of hyaluronic acid (HA) and preparation of HA oligosaccharides. Herein, a putative PL8 hyaluronate lyase SinHL (128.3 kDa) from <i>Streptococcus iniae</i> QMA0131 was expressed in <i>Escherichia coli</i> and biochemically characterized. SinHL is a strictly HA-specific and exolytic hyaluronate lyase that does not degrade CSs and can produce a desired low-molecular-weight HA from high-molecular-weight HA (HMW-HA, 1.52 × 10⁶ Da) under controlled reaction times. The correlation between the 26 residues within the -4 to +2 subsites of the catalytic cleft and enzyme activity was confirmed by alanine-scanning mutagenesis. The carbohydrate-binding module of SinHL facilitated its activity and preference for HMW-HA without alteration of substrate specificity. Molecular dynamics simulations revealed that the narrower catalytic cleft and the smaller cavity adjacent to the catalytic residue Y558 only allowed the glycosidic oxygen O4 of HA to approach Y558, which may determine the substrate specificity for HA of SinHL.