Department of biophysics, All india institute of medical sciences, New Delhi -110029
○Ishfaq Ahmed Sheikh A. S. Ethayathulla Amit Kumar Singh Nagendra Singh Sujata Sharma T. P. Singh
The crystal structure of the complex of buffalo lactoperoxidase with thiocyanate has been determined at 2.75 A resolution and refined to Rcryst and R free factors of 0.182 and 0.216 respectively. The structure contains 4698 protein atoms, 43 heme atoms, 3 thiocyanate atoms, 4 carbonate atoms. 8 iodide ions, 1 calcium ion, 145 atoms from three glycan chains and 183 water molecules. The heme is covalently attached to the protein via two ester linkages between carboxyl groups of Asp 94 and Glu 242 and modified methyl groups on pyrrole rings of the heme group. In addition to these linkages, residues Ala 100, Arg 333, His 337 and Arg 424 interact through hydrogen bonds. The Ca 2+ ion is held in a coordination sphere with pentagonal pyramidal geometry. A single carbonate ion is held in a positively charge environment created by Asn 81, Arg 488, Arg 490 and NAG 1. It is a unique structure of lactoperoxidase with 8 iodide ions that are held at distal cavities. The secondary structure is largely alpha-helical with a very little beta-sheet. The structure determination has also confirmed the presence of a calcium-binding site and three sites of aspargine linked glycosylation (Asn 189, Asn 225 and Asn 317) as well as the identities of the proximal His 336 and distal His 95. The structure determination has also revealed a number of halide-binding sites and their possible relevance to the catalytic mechanism of the enzyme. The complex formation with thiocyanate has been also analyzed in detail.