ANALYSIS OF THE SUBSTRATE SPECIFICITY AND THE ENZYMATIC ACTIVITY OF AN EXTRADIOL TYPE DIOXYGENASE. T.Senda, H.Narita, K.Taguchi, K.Sugimoto, E.Masai, M.Fukuda and Y.Mitsui, Department of BioEngineering, Nagaoka, University of Technology, Nagaoka, Niigata 940-21, JAPAN.
The substrate specificity and the enzymatic activity of the "BphC" enzyme have been analyzed applying crystallographic and biochemical techniques for several mutant proteins. The BphC enzyme is a member of the extradiol type dioxygenases and known as a key enzyme in the PCB degrading pathway of microorganisms. We have already solved the three-dimensional structure of the free form of the BphC enzyme at 1.8 Å resolution and the substrate-bound forms at 2.6 Å resolution1). The active site of the enzyme is situated inside the ß-barrel-like structure in the C-terminal domain. The amino acid residues in contact with the catechol ring moiety of the substrate, which is cleaved by the enzyme, are well conserved among the related enzymes indicating the almost identical catalytic mechanism within the extradiol type dioxygenase family. On the other hand, amino acids around the benzene ring moiety of the substrate are less conserved. Thus these residues seem to determine the substrate specificity of the enzyme. To analyze the role of these amino acid residues in detail, we have prepared the mutant proteins and determined the crystal structures of these mutants as well as the kinetic parameters, Km and kcat. These structural and biochemical analyses have revealed clearly that 1) Ilel74, Thr280 and Phe201 affect the substrate specificity, 2) the amino acid residues bound to the Fe ion in the active center are essential to the enzymatic activity, and 3) His194, His240 and Tyr249, which are located close to the catechol ring moiety of the substrate, are also essential to the activity of the enzyme.
1) Sugiyama et al. (1995) Proc Japan Acad. 71B, 33-35.; Senda et al
(1996) J.Mol. Biol. 255, 735-752