Three-Dimensional Structure of an Extradiol-Type Catechol Ring Cleavage Dioxygenase BphC derived from Pseudomonas sp. strain KKS102: Structural Features Pertinent to Substrate Specificity and Reaction Mechanisms

The three-dimensional structure of the BphC enzyme, which is a member of the extradiol-type catechol ring cleavage dioxygenases, was determined using a crystallographic technique at 1.8 Angstrom resolution. The crystal structure of the BphC enzyme complexed with its substrate, 2,3-dihydroxybiphenyl (2,3-DHBP), was also determined. It was revealed that (1) the active site is located inside the barrel-like structure of domain 2 of each subunit, and (2) the Fe ion in the active site coordinates His-145, His-209, and Glu-260. To gain further insight into the reaction mechanism of this enzyme, we have prepared more than 30 mutant proteins and carried out their biochemical and crystallographic analyses. These studies have revealed that (1) the shape of the substrate-binding pocket and its hydrophobic character are important for determining substrate specificity, (2) the shape and location of the putative "oxygen-binding cavity" are consistent with the reaction mechanism previously proposed based on spectroscopic studies on closely related enzymes, and (3) His-194 is indispensable for the enzymatic reaction, most probably playing the role of a proton acceptor whose existence has been suggested by previous spectroscopic studies.