Structure determination of reaction intermediates for 2,3-dihydroxybiphenyl 1,2-dioxygenase (the BphC enzyme) derived from Pseudomonas sp strain KKS102

2,3-Dihydroxybiphenyl 1,2-dioxygenase (the BphC enzyme) derived from Pseudomonas sp. strain KKS102, an extradiol type catecholic dioxygenase, is a non-heme iron-containing enzyme playing a key role in the degradation of biphenyl/polychlorinated biphenyl (PCB) in the microbe. In order to elucidate the catalytic mechanism of the enzyme, crystal structures of BphC (substrate-free form), the BphC-2,3-hydroxylbiphenyl complex (binary complex), the BphC-2,3-hydroxybiphenyl-NO complex (ternary complex) and the BphC-3-chlorocatechol (3Cl-catechol) complex were determined using the active-form enzyme. The coordination geometries of these structures are consistent with those derived from the X-ray absorption spectroscopy (XAS). A comparison of these structures shows that small conformational shifts occur around the active site upon substrate binding. As a result of the conformational shifts, His194, which has been suggested to be the catalytic base, hydrogen bonds with a hydroxyl group of the substrate. This hydrogen bond seems to be required for the proton transfer from the substrate to His194, and the hydrogen bond network, including Asp170, His193 and His194, seems to facilitate this proton transfer. (C) 2002 Elsevier Science B.V. All rights reserved.