ANALYSIS OF A NEW DIMERIC EXTRADIOL DIOXYGENASE FROM A NAPHTHALENESULFONATE-DEGRADING SPHINGOMONAD

A new extradiol dioxygenase was cloned by screening a gene bank from t he naphthalenesulfonate-degrading bacterial strain BN6 for colonies wi th 2,3-dihydroxybiphenyl dioxygenase (DHBPDO) activity. A 1.6 kb DNA f ragment was sequenced and an ORF of 954 bp identified. Comparison of t he deduced amino acid sequence of DHBPDO II from strain BN6 with previ ously published sequences showed the closest relationship to a metapyr ocatechase (MpcII) from Alcaligenes eutrophus JMP 222. Thus, the enzym e was only distantly related to the main groups of catechol 2,3-dioxyg enases or DHBPDOs. The dioxygenase was expressed using a T7 expression vector and the enzymic characteristics of the protein were examined. The enzyme oxidized 2,3-dihydroxybiphenyl, 3-isopropylcatechol, 3-meth ylcatechol, 4-fluorocatechol and 1,2-dihydroxynaphthalene. Comparison of the UV/visible spectrum of the product formed from 3,5-dichlorocate chol with previous reports suggested that this substrate is oxidized b y different extradiol dioxygenases either by proximal or distal ring c leavage. The enzyme required Fe2+ for maximal activity. In contrast to most other extradiol dioxygenases, the enzyme consisted of only two i dentical subunits.