3-DIMENSIONAL STRUCTURES OF FREE-FORM AND 2 SUBSTRATE COMPLEXES OF ANEXTRADIOL RING-CLEAVAGE TYPE DIOXYGENASE, THE BPHC ENZYME FROM PSEUDOMONAS SP STRAIN KKS102

The crystal structure of an enzyme having polychlorinated-biphenyl deg rading activity, the BphC enzyme from Pseudomonas sp, strain KKS102 ha s been solved as a free form at 1.8 Angstrom resolution. This is the f irst three-dimensional structure among the extradiol-type dioxygenases . Based on 34,387 reflections (10.0 to 1.8 Angstrom, completeness 87.8 %), a current R-factor of 20.4% (with a free R-factor of 24.3%) was ob tained with a model obeying standard geometry within 0.011 Angstrom in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a ho mo-octamer and each subunit is composed of two domains: Domain 1 (N-te rminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the ''beta alpha beta beta beta' ' motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid re sidues (His145, His209 and Glu260) and two solvent molecules. The coor dination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structu res of the BphC enzyme complexed with its substrates, 2,3-dihydroxybip henyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were fo und intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present cry stals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligand s, were found to form a penta-coordinated system around the Fe ion rou ghly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mech anism proposed so far. (C) 1996 Academic Press Limited.