STRUCTURAL STUDIES ON SOME METALLOPROTEINS: BIPHENYL DIOXYGENASE AND DAHP SYNTHASE

Abstract

Biphenyl dioxygenase (BPDO), the first enzyme of the biphenyl degradation pathway is responsible for the initial dioxygenation step during the metabolism of biphenyl. Variants II- 9 (BPDO119) and 111-52 (BPDOm52) generated by Barriault et al (2002) through family shuffling of a targeted region of bphA coding for the large subunit (BphA) of biphenyl dioxygenase from Burkholderia xenovorans LB400 (BPDOLB4oo) exhibited enhanced degrading capabilities towards a wide range of polychlorinated biphenyl (PCB) substrates (Barriault et al, 2002). In this work, the biphenyl dioxygenase from these variants, BPDO119 in native state, BPDO119 complexed with biphenyl and BPDO11152 were crystallized to carry out structural studies towards identifying the underlying reason for their better degrading potential as compared to other well studied strains. The crystals were all rhombohedral in shape. BPDO119 native crystals belonged to space group R3 and diffracted to 2.50 A resolution, BPDO119 crystals complexed with biphenyl belonged to PI space group and diffracted to 1.95 A resolution while the BPDO11152 crystals belonged to space group R3 and diffracted to 4.50A resolution. Crystal structures of BPDO119 and BPDO11152 were determined by using the coordinates of BPDOlb4oo (Kumar et al, unpublished) as the search model, and twelve molecules were placed in the asymmetric unit of biphenyl complex of BPDO119 and 4 molecules were placed in BPDO119 substrate free and BPDO11152. After rigid body refinement, strict NCS was applied for the first rounds of refinement, followed by restrained NCS refinement. BPDO119 is 013P3 hexamer is similar to Napthalene-l,2-Dioxygenases (NDOs) and other Biphenyl Dioxygenases (BPDOs). The a subunit can be divided into two distinct domains: a Rieske domain that contains the Rieske [2Fe-2S] center and the catalytic domain that contains the active site mononuclear non-heme ferrous iron. The iron atom at the active 49 Studies on (BipHenyC(Dioxygenase Variants site is pentacoordinated by His 233, His 239, Asp 388 and by two water molecules and the geometry changes when it binds substrate and becomes tetracoordinated as only one water molecule coordinates in this form. The ligation of the non heme iron (mononuclear centre) and Rieske (2Fe-2S) centres and the bridging between them in neighbouring catalytic subunits by hydrogen bonds through asingle amino acid (Asp230) are similar to BPDOs and NDOs (Naphthalene dioxygenases). The final models of BPDOn9 in substrate free form and BPDO119 complex with biphenyl obtained after refinement were subjected to threedimensional structure comparisons with the other related Rieske oxygenases. The entrance to the active site of BPDOn9 is different from the entrance to the site of other BPDOs and NDOs, as the loop forming the mouth ofBPDOn9 entrance block the mouth of the entrance of other related dioxygenases. It was found out that the catalytic pocket of the variants had larger volumes as compared to other known BPDOs and NDOs. The docking studies of various PCBs shows that the amino acids mutated in BPDO„9 were more likely to accommodate greater variety of PCB congeners which could be responsible for the observed enhanced substrate specificity. In addition, BPDOn9 structure supports the report that p subunit play important role in determining substrate specificity (Hurtubise et al, 1998). BPDO119 displays ahydrogen bonding network between Lys 95 of p- subunit and Asp 387 of a subunit, which is nearest neighbor ofAsp 388 that coordinates with mononuclear iron in the active site.