CRYSTAL STRUCTURE OF A PCB--DEGRADING DIOXYGENASE, THE BphC ENZYME FROM A PSEUDOMONAS SP. Y. Mitsui, T. Senda, M.Fukuda, Department of BioEngineering, Nagaoka University of Technology, Nagaoka, Niigata 94-21, Japan

Polychlorinated biphenyls (PCBs) are widely distributed environmental pollutants. Because of their chemical stability, they are hard to decompose. Pseudomonas sp. strain KKS102 is one of the PCB-degrading microorganisms and the "BphC" enzyme (2,3-dihydroxybiphenyl dioxygenase) is a key enzyme in its PCB degrading pathway. The BphC enzyme has been characterized as one of the extradiol type dioxygenases which, when active, have one ferrous iron in their active sites. It is an oligomeric enzyme made up of eight identical subunits each of 292 amino acid residues. The total molecular weight is ca. 250kDa.

The structure was solved by MIR method at 1.8 Å resolution(1). Crystal structures of the complexes with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT), have also been solved(2). In these crystals, the Fe ion was found to be in a ferric state explaining the existence of the intact substrates in the active sites.

The Fe ion is coordinated by the side chain atoms of His145, His209, Glu260 and two solvent molecules forming a square-pyramid. In the complexes with its substrates, two hydroxyl groups from the catechol ring moiety of the substrates (rather than the solvent molecules) were bound to the Fe ion forming a trigonal bipyramid. The van der Waals surface of the active site and that of the bound substrate are almost complimentary to each other. Thus the basic coordination geometry of the BphC enzyme in complex with its substrate must be mainly determined by the stereochemical effect and may be retained even if the Fe oxidation state were changed. Apparently in accord with this notion, the polyhedra formed by the Fe ion (in a ferrous state) and the three equivalent protein ligands found in the substrate--free crystal structure of a similar enzyme (having ca.66 % amino acid sequence homology), which was solved later under unaerobic condition(3), was superimposable on the present polyhedra (involving a ferric Fe ion) with a rms deviation of only 0.19 A(2). Details of the present structure and further studies on several mutated enzymes will be given in a poster presented by T.Senda et al.

(1) K. Sugiyama, T. Senda et al. Proc. Japan Acad. 71B, 32 -35 (1995).

(2) T. Senda, M. Fukoda et al. J.Mol. Biol. 255, 735 - 752 (1996).

(3) S. Han, L. D. Eltis, K. N. Timmis et al. Science 270, 976 - 980 (l995).