Characterization of hyaluronic acid specific hyaluronate lyase (HylP) from Streptococcus pyogenes.
Streptococcus pyogenes is associated with a wide variety of mucosal and invasive infections that claim human life. The conversion from non pathogenic to toxigenic strain of S. pyogenes are thought to be mediated by bacteriophage infection in several cases. The hyaluronic acid (HA) degrading enzyme Hyaluronate lyase (HL) is proposed to be one of the key bacteriophage-encoded virulence factors. In the present work, HL of S. pyogenes bacteriophage H4489A (HylP) was expressed in Escherichia coli, purified and their structural and functional properties were studied. The enzyme exists in an extended trimeric conformation whose function is influenced by calcium ions. The collagenous Gly-X-Y motif of the enzyme influences stability and interact with calcium ions suggesting its role in the enzyme regulation The HylP shows sequential unfolding through the N-terminal domain. The primary catalytic residues of the enzyme seem to be in the first pocket consisting of Asp170 and Tyr182; however the enzyme activity is considerably reduced with mutation in the second pocket consisting of Glu295 and Tyr298. The catalytic residues span between the regions containing 135-308 amino acids where both the catalytic pocket has a prominent positively charged residue. The net positive potential of the cleft may help in recruiting the negatively charged polymeric HA. Interestingly, unlike other phage HLs, HylP is inhibited by l-ascorbic through non competitive manner.
CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India.
Sudhir Kumar
SK
Singh
CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India.
Soniya
S
Malhotra
CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India; Academy of Scientific and Innovative Research, CSIR-Central Drug Research Institute, Sector 10, Jankipuram Extension, Sitapur Road, Lucknow 226 031, Uttar Pradesh, India. Electronic address: sohail@cdri.res.in.
Md Sohail
MS
Akhtar
D001435:Bacteriophages / Q000201:enzymology* / Q000235:genetics; D002384:Catalysis; D004926:Escherichia coli / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic; D006801:Humans; D006820:Hyaluronic Acid / Q000737:chemistry / Q000378:metabolism; D011133:Polysaccharide-Lyases / Q000096:biosynthesis / Q000737:chemistry / Q000235:genetics*; D017434:Protein Structure, Tertiary; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D013379:Substrate Specificity
D006820:Hyaluronic Acid; D011133:Polysaccharide-Lyases; C100002:hyaluronate lyase
Bacteriophage; Enzyme catalysis; Hyaluronate lyase; Hyaluronic acid