Revisiting the Regiospecificity of Burkholderia xenovorans LB400 Biphenyl Dioxygenase toward 2,2′-Dichlorobiphenyl and 2,3,2′,3′-Tetrachlorobiphenyl

2,2′-Dichlorobiphenyl (CB) is transformed by the biphenyl dioxygenase of Burkholderia xenovorans LB400 (LB400 BPDO) into two metabolites ( 1 and 2 ). The most abundant metabolite, 1 , was previously identified as 2,3-dihydroxy-2′-chlorobiphenyl and was presumed to originate from the initial attack by the oxygenase on the chlorine-bearing ortho carbon and on its adjacent meta carbon of one phenyl ring. 2,3,2′,3′-Tetrachlorobiphenyl is transformed by LB400 BPDO into two metabolites that had never been fully characterized structurally. We determined the precise identity of the metabolites produced by LB400 BPDO from 2,2′-CB and 2,3,2′,3′-CB, thus providing new insights on the mechanism by which 2,2′-CB is dehalogenated to generate 2,3-dihydroxy-2′-chlorobiphenyl. We reacted 2,2′-CB with the BPDO variant p 4 , which produces a larger proportion of metabolite 2 . The structure of this compound was determined as cis -3,4-dihydro-3,4-dihydroxy-2,2′-dichlorobiphenyl by NMR. Metabolite 1 obtained from 2,2′-CB- d 8 was determined to be a dihydroxychlorobiphenyl- d 7 by gas chromatographic-mass spectrometric analysis, and the observed loss of only one deuterium clearly shows that the oxygenase attack occurs on carbons 2 and 3. An alternative attack at the 5 and 6 carbons followed by a rearrangement leading to the loss of the ortho chlorine would have caused the loss of more than one deuterium. The major metabolite produced from catalytic oxygenation of 2,3,2′,3′-CB by LB400 BPDO was identified by NMR as cis -4,5-dihydro-4,5-dihydroxy-2,3,2′,3′-tetrachlorobiphenyl. These findings show that LB400 BPDO oxygenates 2,2′-CB principally on carbons 2 and 3 and that BPDO regiospecificity toward 2,2′-CB and 2,3,2,′,3′-CB disfavors the dioxygenation of the chlorine-free ortho - meta carbons 5 and 6 for both congeners.