Department of Biophysics, All India Institute of Medical Sciences* Nutritional Science Laboratory, Morinaga Milk Industry Co. Ltd, Japan**
○Sujata Sharma* Amit Singh* Kouichirou Shin** Mitsunori Takase** Nagendra Singh* Tej Singh*
Lactoperoxidase is a heme containing enzyme that catalyzes the inactivation of a wide range of micro-organisms. It is a prominent member of a family of homologous mammalian peroxidases that includes thyroid peroxidase, eosinophil peroxidase and myeloperoxidase. This is the first crystal structure from the sub-family of lactoperoxidases. The protein was purified from bovine milk and crystallized using hanging drop method. The crystals belong to monoclinic space group P21 with cell dimensions, a=54.5 Å, b=80.6 Å, c=77.6 Å, beta=102.6 with two molecules in the unit cell. The structure was refined to Rcryst and Rfree factors of 0.173 and 0.218 respectively. The final structure contains 4697 protein atoms, 1 calcium ion bound firmly in the calcium-binding site, 1 heme prosthetic group, 2 nitrate ions, 1 carbonate ion, 1 molecule of hypothiocyanate and 318 water molecules. Bovine lactoperoxidase is a single polypeptide chain consisting of 583 amino acid residues with 4 potential N-glycosylation sites having N-X-S/T sequence motifs which are at N81, N189, N225 and N317. The electron densities of glycan chains have been observed at all the four sites. The heme group is covalently linked to the side chains of D94 and E244. It forms two strong ionic interaction with R334 and R426. The calcium ion is coordinated by the three backbone oxygen atoms of D96, T170 and F172, two carboxyl Od1 atoms of D96 and D174 and two Og atoms of T170 and S176 resulting in the formation of a coordination sphere of the calcium ion with pentagonal bipyramidal geometry. The nitrate anion is hydrogen bonded to W32, L33, V328, R383 and T544 while carbonate anion interacts with N81, R82 and R490. Finally, hypothiocyanate is observed in contact with Q91, H95, and the heme Fe3+ ion.