A Fungal Hyaluronate Lyase with a Distinct Catalytic Machinery Expands the Polysaccharide Lyase Landscape

Abstract Hyaluronic acid (HA) is a major glycosaminoglycan of the vertebrate extracellular matrix whose biological functions depend strongly on its molecular weight. Controlled depolymerization of HA into defined oligosaccharides is therefore of considerable biomedical and biotechnological interest. Although bacterial HA lyases have been extensively characterized, fungal HA-degrading enzymes remain poorly understood, and none has been structurally assigned to a defined CAZy family. Here, we identify and characterize PlLya44, an HA lyase secreted by the filamentous fungus Purpureocillium lilacinum. The enzyme was detected in the fungal secretome, produced in Pichia pastoris, and shown to efficiently depolymerize high molecular weight HA, generating mainly unsaturated oligosaccharides with both even- and odd-numbered products. Product analysis supports a predominantly processive endolytic mode of action distinct from the classical exo-processive mechanism of bacterial HA lyases. The crystal structure of PlLya44 uncovers an α-helical α6/α6 barrel fold characteristic of the (α/α)n toroid superfamily, harboring an elongated polarity-partitioned catalytic cleft. Structural comparisons and sequence similarity network analyses demonstrate that PlLya44 does not cluster with any currently defined polysaccharide lyase (PL) family, supporting its classification as the founding member of a distinct PL family. Ligand-bound structures, mutagenesis, ensemble refinement, and molecular dynamics reveal a reorganized catalytic architecture, reversing substrate orientation relative to previously characterized HA lyases. Together, these findings provide structural insight into a fungal HA lyase, uncover a previously unrecognized catalytic strategy for HA depolymerization, and establish PlLya44 as the founding member of a hitherto undescribed PL family, expanding the enzymatic landscape of glycosaminoglycan-active enzymes.