Crystal Structure of a Bacteriophage-associated Hyaluronate Lyase

Division of Molecular and Structural Biology, Central Drug Research Institute, Lucknow* Department of Biophysics, All India Institute of Medical Sciences, New Delhi**
â—‹Parul Mishra* V. Bhakuni* R. Prem Kumar** Nagendra Singh** Sujata Sharma** P Kaur** Tej P. Singh**

Hyaluronate lyases (HylP2) are a class of endoglycosaminidase enzymes with considerable complexity and heterogeneity. Their main function is to degrade hyaluronan, the main polysaccharide component of the host connective tissues into unsaturated disaccharide units as the final product. HylP2 was cloned, overexpressed, and purified. The recombinant HylP2 exists as a homotrimer of molecular mass about 110 kDa under physiological conditions. 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. The cloned purified protein was crystallized using hanging drop vapour diffusion method with sodium formate as precipitating agent. The crystals belong to space group H32 with cell dimensions of a = b = 59.6Å, c = 588.5Å. The structure was determined and refined to an Rcryst factor of 22.3% (Rfree factor of 23.6%). The final model contains 2518 protein atoms. The structure shows elongated molecule having a middle wider region made up of antiparallel β-strands, whereas the N and C-termini contain two and one α-helices respectively. The active site is located in the middle part of the structure. There are three molecules of HylP2 intertwined together with a large number of intermolecular interactions between them to form a highly stable and biologically active homotrimer. The structure shows remarkable differences in the loop region A122 to S129, which lies near the active site residues, N135, D137 and Y149. This loop is probably involved in determining the substrate specificity of the enzyme.