HIGH RESOLUTION CRYSTAL STRUCTURES OF 2,3-DIHYDROXYBIPHENYL 1,2-DIOXYGENASE FROM A PCB-DEGRADING BACTERIUM COMPLEXED WITH SUBSTRATES Jeffrey T. Bolin, Seungil Han, Lindsay D. Eltisa, Biological Sciences/LILY, Purdue University, W. Lafayette, IN 47907, U.S.A; aBiochemistry Dept., Université Laval, Ste-Foy, Quebec, Canada
2,3-dihydroxybiphenyl 1,2-dioxygenase (DHBD) catalyses the ring cleavage step in the aerobic degradation of biphenyl and PCBs using an Fe(II)-dependent, extradiol cleavage mechanism. Crystal structures of DHBD in complexes with 2,3-dihydroxybiphenyl, 3-methylcatechol, and catechol have been determined at 2.3 Å, 1.9 Å, and 1.9 Å resolution, respectively. The structure of the free enzyme has also been determined at 1.9 Å resolution1.
These studies target the DHBD from Pseudomonas LB400, an organism that transforms a broad range of PCBs. The enzyme is a D4 symmetric homooctomer of 298 residue subunits and contains one Fe per subunit. Crystallization and diffraction studies were performed under anaerobic conditions so as to maintain the active, Fe(II) form of the enzyme.
These are the first structures of any extradiol dioxygenase in the Fe(II) form. As such they provide a structural framework for discussion of the mechanism of extradiol ring cleavage reactions as well as the potential adaptation of this enzyme and the biphenyl degradation pathway to achieve bioremediation of PCBs.
The structures of substrate complexes show bidentate binding via the vicinal hydroxyl oxygens. However, the two Fe-O bond lengths are markedly different, suggesting monoanionic binding and deprotonation of the 2-hydroxyl in the case of 2,3-dihydroxybiphenyl. The active site structure in the complexes is similar to that of the free enzyme, but there are several important differences including a > 0.5 Å shift of the Fe and its ligands, and subtle changes in the hydrogen bonding pattern that may be important for the mechanism.
1. S. Han et al., Science 270, 976-980, 1995.