Figure 1.
Scheme showing the initial reaction in the Bph pathway.
Table 1.
Refinement parameters and statistics.
Figure 2.
Ribbon diagram showing the overall structure of BPDOB356.
Two orthogonal views showing three αβ protomers arranged around the crystallographic three-fold axis to form the active hexamer. This arrangement allows the Rieske domain (green ribbons) from the tan α-subunit to interact with the catalytic Fe2+(rust sphere) in the adjacent subunit (purple ribbons). All structural graphics were created using Pymol (www.pymol.org).
Figure 3.
Unrooted phylogenetic tree obtained from a crystal structure-based sequence alignment of 25 α-subunits of related Rieske Oxygenases.
Bootstrap values out of 100 replicates are indicated. The proteins are abbreviated using the gene name and strain as follows: biphenyl dioxygenase from Pandoraea pnomenusa B-356 (BphAB356, GenPeptID: AAC44526), Burkholderia xenovorans LB400 (BphALB400, GenPeptID: YP_556409), Rhodococcus globerulus P6 (BphA1P6, GenPeptID: CAA56346), Rhodococcus jostii RHA1 (BphA1RHA1, GenPeptID: BAA06868), and Sphingobium yanoikuyae B1 (BphA1fB1, GenPeptID: ABM91740); benzene dioxygenase from Pseudomonas putida ML2 (BedC1ML2, GenPeptID: Q07944); benzoate dioxygenase from P. putida F1 (BnzAF1, GenPeptID: A5W4F2); cumene dioxygenase from Pseudomonas fluorescens IP01 (CumA1IP01, GenPeptID: BAA07074); dibenzofuran dioxygenase from Terrabacter sp. YK3 (DfdA1YK3, GenPeptID: BAC06602); diterpenoid dioxygenase from Pseudomonas abietaniphila BKME (DitABKME, GenPeptID: AAD21063); dinitrotoluene dioxygenase from Burkholderia cepacia R34 (DntAcR34, GenPeptID: AAL50021); dibenzo-p-dioxin dioxygenase from Sphignomonas (DxnA1RW1, GenPeptID: CAA51365); ethylbenzene dioxygenase from R jostii RHA1 (EbdA1RHA1, GenPeptID: BAC92718); 3-phenylpropionate dioxygenase from E. coli K-12 (HcaEK12, GenPeptID: ACB03690); naphthalene dioxygenase from Rhodococcus sp. NCIMB 12038 (NarA12038, GenPeptID: AAD28100) and Pseudomonas putida 9816-4 (NdoB9816, GenPeptID: P0A110); nitrobenzene dioxygenase from Comomonas sp. JS765 (NbzA JS765, GenPeptID: AAL76202); polyaromatic hydrocarbon dioxygenase from Mycobacterium vanbaalenii PYR-1(NidAPYR1, GenPeptID: AAF75991, NidA3PYR1, GenPeptID: AAY85176) and Burkholderia sp. RP007 (PhnAcRP07, GenPeptID: AAD09872); phthalate dioxygenase from R. jostii RHA1 (PadAaRHA1, GenPeptID: ABG99212) and Terrabacter sp. DBF63 (PhtADBF63, GenPeptID: BAC54156); phenanthrene dioxygenase from Nocardioides sp. KP7 (PhdAKP7, GenPeptID: BAA94708) and Sphingomonas sp. CHY1 (PhnA1CHY1, GenPeptID: CAG17576); and tetraline dioxygenase from Sphingomonas macrogoltabidus TFA (ThnA1TFA, GenPeptID: AAN26443). Proteins for which the crystal structure was used for alignment are indicated in bold text in the figure.
Figure 4.
2Fo-Fc electron density maps (contoured at 1σ above the mean) showing the coordination geometry of the mononuclear Fe2+.
a) The mononuclear Fe2+ geometry found in the crystal structure determined in the presence of MES buffer, and b) in the presence of MES buffer and biphenyl. The Fe atom is coordinated by two histidines, a bidentate aspartate, and one water molecule (W1).
Figure 5.
The positions and surface representations of the active site invaginations of BPDOB356, NDO9816-4, BPDORHA1, CumDOIP01, and NDO12038.
a) Shows the overall active site cavity of BPDOB356 as determined by the program VOIDOO. b) The overall active site cavity of NDO9816-4 determined similar to a). The solvent accessible surface representations calculated by the program Pymol for c) BPDOB356, d) NDO9816-4, e) BPDORHA1, f) CumDOIP01, and g) NDO12038. BPDOB356 has a much larger active site cavity relative to BPDORHA1. The distal pocket of CumDOIP01 is less pronounced than that of BPDOB356. The view of the active site cavity of d) NDO9816-4 and g) NDO12038 has been rotated slightly relative to that of BPDOB356 in order to provide an unobstructed view of the entrance passageway.
Figure 6.
The steady-state dihydroxylation of 2,2′-dichlorobiphenyl by BPDO variants; dependence of the initial velocity of O2-uptake on biphenyl concentration in air-saturated buffer.
BPDOB356 wild-type (▪); BPDOB356 M231A (•); BPDOB356 M231T (⧫).
Table 2.
Apparent steady-state kinetic parameters of BPDOB356 wild-type (wt) and variants (M231A and M231T) for biphenyl and 2,2′-dichlorobiphenyl.
Table 3.
Summary of crystallographic data.