Unusual structural features of the bacteriophage-associated hyaluronate lyase (hylp2).
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.
Mishra
Parul
P
Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow 226 001, India.
Akhtar
Md Sohail
MS
Bhakuni
Vinod
V
D001435:Bacteriophages / Q000378:metabolism*; D001665:Binding Sites; D020134:Catalytic Domain; D002845:Chromatography; D002918:Chymotrypsin / Q000737:chemistry; D002942:Circular Dichroism; D003432:Cross-Linking Reagents / Q000494:pharmacology; D019281:Dimerization; D004305:Dose-Response Relationship, Drug; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000378:metabolism; D005976:Glutaral / Q000737:chemistry; D006025:Glycosaminoglycans / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000378:metabolism; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008239:Lysine / Q000737:chemistry; D008970:Molecular Weight; D010957:Plasmids / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000737:chemistry* / Q000378:metabolism; D011487:Protein Conformation; D011489:Protein Denaturation; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D012694:Serine / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization; D013297:Streptococcus pyogenes / Q000378:metabolism; D013379:Substrate Specificity; D014364:Tryptophan / Q000737:chemistry
D003432:Cross-Linking Reagents; D006025:Glycosaminoglycans; D011994:Recombinant Proteins; D012694:Serine; D006639:Histidine; D014364:Tryptophan; D006026:Glycoside Hydrolases; D002918:Chymotrypsin; C117608:alpha-chymotrypsin; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase; D008239:Lysine; D005976:Glutaral