Biochemical and genetic characterization comparison of four extradiol dioxygenases in Rhizorhabdus wittichii RW1.

Rhizorhabdus (previously Sphingomonas) wittichii RW1 uses a diverse array of aromatic organic compounds as energy and carbon sources, including some extremely recalcitrant compounds such as dibenzo-p-dioxin and dibenzofuran. Extradiol dioxygenases play a key role in the metabolism of dibenzofuran (DBF), dibenzo-p-dioxin (DBD), PCBs, and various other aromatic compounds. In this study, a detailed kinetic analysis of four extradiol dioxygenases identified in R. wittichii RW1 (DbfB, Edo2, Edo3, and Edo4) showed all of them to be typical 2,3dihydroxybiphenyl (DHB) dioxygenases with DHB as preferred substrate (k/K values of 0.13-188 (µM  s)) and only slightly lower activity against trihydroxybiphenyl (THB) whereas monocyclic substrates were, to different extents, poor substrates due to high k values. All extradiol dioxygenases analyzed were subject to mechanism-based inactivation by 2,2,3-trihydroxybiphenylether (THBE) the intermediate of DBD degradation. However, Edo4 was superior as reflected by the relatively high partition ratio and the comparably low efficiency of inactivation. Significant differences were observed with respect to their inactivation by 3-chlorocatechol. The absence of any significant mechanism-based inactivation makes Edo3 a perfect candidate for being recruited for chlorobiphenyl degradation where inactivation of extradiol dioxygenases by this intermediate creates significant metabolic problems. KEY POINTS: • Characterization of additional extradiol dioxygenases encoded by RW1 • Identification of differences in 2,2,3-trihydroxybiphenylether transformation • Identification of differences in inhibition by 3-chlorocatechol.