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 p4, 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.
Barriault
Diane
D
Institut National de la Recherche Scientifique, INRS-Institut Armand-Frappier, Laval, Québec H7V 1B7, Canada.
Lépine
François
F
Mohammadi
Mahmood
M
Milot
Sylvain
S
Leberre
Nicolas
N
Sylvestre
Michel
M
D001426:Bacterial Proteins / Q000378:metabolism*; D019117:Burkholderia / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000378:metabolism*; D009682:Magnetic Resonance Spectroscopy; D008968:Molecular Conformation; D015394:Molecular Structure; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism*; D011078:Polychlorinated Biphenyls / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
D001426:Bacterial Proteins; D007506:Iron-Sulfur Proteins; C012280:2,2'-dichlorobiphenyl; C068319:2,2',3,3'-tetrachlorobiphenyl; D011078:Polychlorinated Biphenyls; D010105:Oxygenases; C074161:biphenyl-2,3-dioxygenase; D010100:Oxygen