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 (Ser 128 -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.

Self-assembly of Bacteriophage-associated Hyaluronate Lyase (HYLP2) into an Enzymatically Active Fibrillar Film

“…5A. The fragmentation profiles of fibrils of HylP2 and native HylP2 were similar, and in both cases the protein was proteolyzed into two major protein fragments of 25 and 14 kDa, which corresponded to the C-and N-terminal domains respectively, of the protein as established earlier (20). To examine the minimum core portion of the protein involved in fibrillation, we further performed a cleavage of the fibrillar form of HylP2 as well as the native HylP2 with proteinase K. Proteinase K has been used for a number of other amyloid proteins for carrying out cleavage as it is a nonspecific protease and cleaves a protein at all noncompact flexible regions.…”

“…HylP2 is an ϳ110-kDa homotrimeric, ␣/␤ protein (20) that is devoid of disulfide bonds in its native folded state in the physiological medium. The first few residues from the N-terminal region of HylP2 are an ␣/␤ cap-like segment that is followed by ␣-helical coiled coils to residue 108.…”

“…1A), any change in the fluorescence emission maximum of this tryptophan residue could be correlated only with the folding or unfolding of the N-terminal region of HylP2. Under physiological conditions (pH 7.0), this tryptophan moiety is buried in the hydrophobic environment as the N-terminal region of the enzyme is in folded conformation (20). The intrinsic fluorescence spectra of the protein showed no significant differences for the wavelength of maximum emission ( max ϭ 330 nm), whereas on decreasing the pH from 7.0 to 4.0, a noticeable sigmoidal enhancement from 328 to 340 nm in the emission wavelength was observed.…”

“…HylP2-The cloning, overexpression, and purification of fulllength HylP2 was performed as described earlier (20).…”

“…We carried out biophysical characterization (20) and x-ray data analysis of HylP2 (Protein Data Bank code 2DP5), which demonstrated that the C-terminal domain of the protein predominantly consisted of ␤-sheets harboring the active site residues (20). To date there is no report on the presence of amyloid-like fibrillar structures in the bacteriophage of S. pyogenes, although the bacterium itself has been reported to possess amyloid-like M proteins on its surface, which help in spreading bacterial infection by facilitating the attachment of this Grampositive bacterium to host soft tissues (21).…”