Unusual structural features of the bacteriophage-associated hyaluronate lyase (hylp2)

Abstract

Hyaluronate lyases are a class of endoglycosaminidase enzymes, which are of considerable complexity and heterogeneity. Their primary function is to degrade hyaluronan and certain other glycosaminoglycans and facilitate the spread of disease. Among hyaluronate lyases, the bacteriophage-associated enzymes are unique as they have the lowest molecular mass, very low amino acid sequence homology with bacterial hyaluronate lyases, and exhibit absolute specificity for one type of glycosaminoglycan, i.e. hyaluronan. Despite such unique characteristics significant details on structural features of these lyases are not available. The Streptococcus pyogenes bacteriophage 10403 contains a gene, hylP2, which encodes for hyaluronate lyase (HylP2) in this organism. HylP2 was cloned, overexpressed, and purified to homogeneity. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa, under physiological conditions. Limited proteolysis and guanidine hydrochloride denaturation studies demonstrated that the N-terminal region of the protein is flexible, whereas the C-terminal portion has a compact conformation. The enzyme shows sequential unfolding, with the N-terminal unfolding first followed by the simultaneous unfolding and dissociation of the stabilized trimeric C-terminal domain. We isolated a functionally active C-terminal fragment (Ser128-Lys 337) of the protein that was stabilized in a trimeric configuration. Comparative functional studies with full-length protein, N:C complex, and isolated C-terminal domain demonstrated that the active site of HylP2 is present in the C-terminal portion of the enzyme, and the N-terminal portion modulates the substrate specificity and enzymatic activity of the C-terminal domain. © 2006 by The American Society for Biochemistry and Molecular Biology, Inc.