Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase
ABSTRACT
Biphenyl dioxygenase, the first enzyme of the biphenyl catabolic pathway, is a major determinant of which polychlorinated biphenyl (PCB) congeners are metabolized by a given bacterial strain. Ongoing efforts aim to engineer BphAE, the oxygenase component of the enzyme, to efficiently transform a wider range of congeners. BphAEII9, a variant of BphAELB400 in which a seven-residue segment, 335TFNNIRI341, has been replaced by the corresponding segment of BphAEB356, 333GINTIRT339, transforms a broader range of PCB congeners than does either BphAELB400 or BphAEB356, including 2,6-dichlorobiphenyl, 3,3′-dichlorobiphenyl, 4,4′-dichlorobiphenyl, and 2,3,4′-trichlorobiphenyl. To understand the structural basis of the enhanced activity of BphAEII9, we have determined the three-dimensional structure of this variant in substrate-free and biphenyl-bound forms. Structural comparison with BphAELB400 reveals a flexible active-site mouth and a relaxed substrate binding pocket in BphAEII9 that allow it to bind different congeners and which could be responsible for the enzyme's altered specificity. Biochemical experiments revealed that BphAEII9 transformed 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl more efficiently than did BphAELB400 and BphAEB356. BphAEII9 also transformed the insecticide dichlorodiphenyltrichloroethane (DDT) more efficiently than did either parental enzyme (apparent kcat/Km of 2.2 ± 0.5 mM−1 s−1, versus 0.9 ± 0.5 mM−1 s−1 for BphAEB356). Studies of docking of the enzymes with these three substrates provide insight into the structural basis of the different substrate selectivities and regiospecificities of the enzymes.
IMPORTANCE Biphenyl dioxygenase is the first enzyme of the biphenyl degradation pathway that is involved in the degradation of polychlorinated biphenyls. Attempts have been made to identify the residues that influence the enzyme activity for the range of substrates among various species. In this study, we have done a structural study of one variant of this enzyme that was produced by family shuffling of genes from two different species. Comparison of the structure of this variant with those of the parent enzymes provided an important insight into the molecular basis for the broader substrate preference of this enzyme. The structural and functional details gained in this study can be utilized to further engineer desired enzymatic activity, producing more potent enzymes.
INTRODUCTION
The microbial degradation of environmental pollutants has been extensively studied over the past few decades (1–3). Some pollutants, including polychlorinated biphenyls (PCBs), pose a threat to human health and to the biosphere due to their toxicity and persistence. Apprehension about PCBs led to prohibition of their production and use as well as regulation of their disposal and/or remediation (3–7). Bacterial degradation plays a pivotal role in the bioremediation of PCB-contaminated soil and water (8, 9; K. Furukawa, presented at Bioremediation: the Tokyo ‘94 Workshop, 27 to 30 November 1994). Aerobic degradation involves the biphenyl (Bph) catabolic pathway, which includes four enzyme-catalyzed reactions. Bacteria can utilize the Bph pathway to cometabolize a variety of PCB congeners with differential effectiveness, and different PCB congeners are transformed in a strain-dependent fashion (10). Until now, structural studies have been done for all four enzymes to understand the detailed mechanism that underlies this pathway (11–16).
Biphenyl dioxygenase (BPDO), the first enzyme of the Bph pathway, is a three-component ring-hydroxylating Rieske-type oxygenase (RO) that utilizes NADH and O2 to transform biphenyl to cis-(2R,3S)-dihydro-dihydroxybiphenyl (Fig. 1A). The three components are a two-subunit oxygenase (BphAE), a ferredoxin (BphF), and a ferredoxin reductase (BphG) (17, 18). BphF and BphG deliver electrons from NADH to the oxygenase. As is typical of ring-hydroxylating ROs, the oxygenase is a 3-fold symmetric heterohexamer assembled from three larger BphA (α) subunits and three smaller BphE (β) subunits such that the overall shape resembles that of a mushroom, with the three α-subunits forming the cap and the three β-subunits forming the stem. Each α-subunit includes a [2Fe-2S] Rieske-type cluster (His2Cys2 ligation) involved in electron transfer from external reductants to an active site containing a mononuclear Fe2+ ion coordinated by two His residues, an Asp residue (His233, His239, and Asp388 in BphAELB400 [11]), and a conserved water molecule. The 3-fold symmetric arrangement of αβ-dimers brings the [2Fe-2S] cluster of one dimer close to the active site of another, such that the Rieske center of one α-subunit is 12 Å from the Fe2+ atom of the neighboring subunit. It is generally accepted that electron transfer from the Rieske cluster to Fe2+ occurs through a network of hydrogen bonds and that Asp230 plays a major role in connecting the sites (19, 20).
FIG 1
The enzymatic capabilities of BPDO have been studied extensively because its substrate preference and the nature of the reaction products are major determinants of the transformations of different PCBs by the Bph pathway. For the purpose of bioremediation, it is desirable to expand the range of PCB congeners transformed by the Bph pathway. Accordingly, an extensive effort in enzyme engineering has been directed toward broadening the substrate range of BPDO. For example, relatively unbiased directed evolution has been used to alter the substrate profiles of variants derived from the closely related BPDOs of Burkholderia xenovorans LB400 and Pseudomonas pseudoalcaligenes KF707, two bacterial strains that degrade PCBs relatively well (21, 22). An alternative, more targeted strategy reported by Mondello et al. took into account comparisons of amino acid sequences and functional properties, which identified four regions (regions I to IV) in the C-terminal domain of BphA that modulate the regioselectivity and regiospecificity of the enzyme (23). By shuffling the segment of bphA encoding the C-terminal portions of the B. xenovorans LB400 and Pandoraea pnomenusa B356 enzymes, Barriault et al. obtained several variants that exhibited an expanded substrate range (24).
Several of the most potent PCB-transforming BphAE variants have been derived from BphAELB400 (25–27). BphAEII9 is a variant of BphAELB400 in which the seven residues of region III, 335TFNNIRI341, are replaced by the corresponding residues of BphAEB356, 333GINTIRT339. Among other characteristics, BphAEII9 is able to transform 2,3,4′-trichlorobiphenyl more efficiently than is either parental enzyme (28). BphAEII10 adds a single-residue substitution to the BphAEII9 background: Ala267 is replaced by Ser, as occurs in BphAEB356. Interestingly, the substrate range of BphAEII10 is significantly narrower than that of BphAEII9. Finally, BphAEP4 was created by substitution of two residues in region III of BphAELB400: T335A and F336M (25). In liquid culture depletion assays, this variant eliminates several congeners better than does BphAELB400, including the very persistent 2,6-dichlorobiphenyl (25). Structural analysis revealed that the altered substrate specificity of BphAEP4 is associated with both changes in direct side-chain–substrate interactions and changes in residue-residue interactions near the active site; the latter appear to relieve constraints on the induced fit between the enzyme and substrates (11).
In this study, we analyzed the crystal structure of BphAEII9 in the absence and presence of biphenyl to elucidate the molecular basis for the broader substrate preference of this enzyme. We focused on region III residues to better understand how their interactions with other residues modulate substrate competence and reaction regiospecificity. Furthermore, we report the results of biochemical assays and docking experiments with selected chlorinated biphenyl analogs and the insecticide 1,1,1-trichloro-2,2-bis(4-chlorophenyl)ethane (also known as dichlorodiphenyltrichloroethane [DDT]) directed toward the identification of the structural features of BphAEII9, BphAELB400, and BphAEB356 responsible for the differential abilities of the enzymes to transform these compounds.
MATERIALS AND METHODS
Manipulation of DNA and preparation of BphAEII9 protein.
The DNA for BphAEII9, a variant of BphAELB400, was obtained in a previous study by shuffling targeted regions of the bphALB400 and bphAB356 genes (24). bphAEII9 gene DNA was cloned into pET14b and transformed into Escherichia coli C41(DE3). BphAEII9 was produced in this strain as a His-tagged recombinant protein and purified by affinity chromatography according to protocols reported previously for BphAELB400 (26).
Crystallographic procedures.
Crystallization experiments were performed at 21°C in a glove box (Innovative Technologies, Newburyport, MA) with a circulating N2 atmosphere (<5 ppm oxygen). Good-quality, diffracting crystals were obtained from BphAEII9 by sitting-drop vapor diffusion using a well solution of 20 to 25% polyethylene glycol 800 (PEG 8000), 6% glycerol, 50 mM NaCl, and 50 mM 2-(N-morpholino)ethanesulfonic acid (MES) (pH 6.0). Crystals of the biphenyl-bound form of BphAEII9 were obtained by adding a small amount of powdered biphenyl (purchased from Sigma-Aldrich) directly to crystallization drops containing BphAEII9 crystals and incubating the mixture for 30 min before the crystals were harvested. Diffraction data from crystals flash-frozen by immersion in liquid N2 were collected at the BioCARS 14BM-C beamline at the Advanced Photon Source (Argonne National Laboratories). Data were acquired for both substrate-free and biphenyl-bound crystals. The diffraction data were indexed, integrated, and scaled by using the HKL2000 program suite.
Initial phases were obtained by molecular replacement using MOLREP from the CCP4 v.6.3.0 software suite (29, 30). The crystal structure of a single αβ-heterodimer of BphAELB400 (PDB accession no. 2XRX) was used as a search model. REFMAC 5.2 (31) was used for rigid-body refinement of the molecular replacement model and for subsequent rounds of restrained atomic parameter refinement. The program COOT was used for analysis of electron density maps and model building (32). Solvent molecules, Fe ions (for the Rieske clusters and mononuclear Fe2+), and biphenyl molecules were added where the Fo − Fc map had features of appropriate volume above 3 σ and the 2Fo − 2Fc map showed density at 1 σ. The stereochemical properties of the refined models were evaluated by using the program MOLPROBITY (33). The data collection and refinement statistics and model quality for the two structures are summarized in Table 1. All molecular figures were prepared by using the program PyMOL (34).
TABLE 1
| Parametera | Value(s) for BphAEII9 | |
|---|---|---|
| Biphenyl freeb | Biphenyl boundc | |
| Crystallographic data | ||
| Space group | H3 | P1 |
| Wavelength (Å) | 0.9 | 0.9 |
| Resolution range (Å) | 129.1–2.5 | 129.1–1.9 |
| Cell dimensions | ||
| a (Å) | 211.89 | 132.77 |
| b (Å) | 211.89 | 133.19 |
| c (Å) | 168.44 | 133.96 |
| α (°) | 90 | 102.31 |
| β (°) | 90 | 102.54 |
| γ (°) | 120 | 104.54 |
| No. of unique reflections | 97,519 | 634,861 |
| Completeness (%) (value for last shell) | 99.4 (99.9) | 94.0 (92.0) |
| Rsym (%) (value for last shell) | 10.0 (53.0) | 10.0 (40.0) |
| I/σ (value for last shell) | 9.3 (2.6) | 21 (3.3) |
| VM (Å3 Da−1) (% solvent) | 2.3 (46) | 2.3 (46) |
| Refinement statistics | ||
| No. of reflections (working/test) | 92,654/4,632 | 618,963/30,948 |
| No. of residues | 2,463 | 7,368 |
| No. of water molecules | 389 | 4,008 |
| Resolution range (Å) | 38.6–2.5 | 23.3–1.9 |
| Rwork (%) | 18.1 | 21.6 |
| Rfree (%) | 23.4 | 25.4 |
| Average B-factors (Å2) | ||
| Protein chains | ||
| AB | 33.9 | 33.9 |
| CD | 40.1 | 40.1 |
| EF | 49.3 | 49.3 |
| GH | 51.8 | 51.8 |
| IJ | 16.4 | |
| KL | 21.0 | |
| MN | 23.4 | |
| OP | 18.6 | |
| QR | 23.2 | |
| ST | 28.7 | |
| UV | 30.1 | |
| WX | 34.2 | |
| Water atoms | 44.0 | 36.4 |
| All atoms | 44.0 | 24.3 |
| RMSD for bond lengths (Å) | 0.008 | 0.005 |
| RMSD for bond angles (Å) | 1.25 | 1.07 |
| Ramachandran plot | ||
| % favored | 98.0 | 96.3 |
| % allowed | 99.8 | 99.9 |
| Outliers (no. of residues) | 4 | 11 |
Assays and kinetic studies of BPDO from the wild type and the variant.
DDT (99% pure) was obtained from Sigma-Aldrich, and 2,3,4′-trichlorobiphenyl and 2,2′,5,5′-tetrachlorobiphenyl were obtained from Ultra Scientific. The ability of BphAELB400, BphAEB356, and BphAEII9 to metabolize 2,3,4′-trichlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and DDT was assessed by using BPDO systems reconstituted from His-tagged components produced and purified as described previously (11). The enzyme assays were performed with 200 μl of a solution containing 100 to 800 nmol the substrate and buffered with 50 mM MES (pH 6.0) at 37°C as described previously (35). Metabolites were identified by gas chromatography-mass spectrometry (GC-MS) analysis after the reaction medium was incubated for 15 min. The metabolites were extracted at pH 6.0 with ethyl acetate and then treated with n-butylboronate (nBuB) prior to GC-MS analysis, as described previously (36), using a Hewlett Packard HP6980 series gas chromatograph interfaced with an HP5973 mass selective detector (Agilent Technologies). To obtain kinetic parameters with DDT as the substrate, metabolite production was monitored by high-performance liquid chromatography (HPLC) and UV-visible (UV-Vis) spectrometry according to a protocol described previously (37).
Docking studies.
Molecular docking of substrates was accomplished by using Schrodinger Maestro suite version 9.1 (Glide version 2.6; Schrodinger, Inc., New York, NY) (38). The crystal structures of BphAELB400 (PDB accession no. 2XRX), BphAEII9, and BphAEB356 (PDB accession no. 3GZX) with bound biphenyl were prepared for docking by using the Maestro protein preparation wizard: hydrogens were added, bond orders were assigned, and the structure was energy minimized by using the OPLS2001 force field until the root mean square deviation (RMSD) between the minimized structure and the starting structure reached 0.3 Å. For each of the prepared protein structures, a 12- by 12- by 12-Å receptor grid box was erected by using the centroid of the atoms in the bound biphenyl as the center of the box. The substrates DDT, 2,3,4′-trichlorobiphenyl, and 2,2′,5,5′-tetrachlorobiphenyl were prepared by using the Maestro Ligprep module (Schrodinger, Inc., New York, NY). Glide was then used for docking of the substrates using the Extra Precision (XP) mode (39, 40). The best conformation was selected on the basis of the Glide score and Emodel value. Visual inspection was used to confirm that the substrates were docked in a plausible orientation similar to that observed in the crystal structure of the BphAELB400:biphenyl complex.
Protein structure accession numbers.
RESULTS AND DISCUSSION
Crystal structures of substrate-free and biphenyl-bound forms of BphAEII9.
The crystal structures of the substrate-free and biphenyl-bound forms of BphAEII9 were determined to resolutions of 2.5 Å and 1.9 Å, respectively. Representative crystallographic data and refinement statistics are presented in Table 1.
The crystal structure of the substrate-free form was refined at a resolution of 2.5 Å to final Rcryst and Rfree values of 18.1% and 23.4%, respectively, with four αβ-heterodimers in the asymmetric unit of space group H3. Three dimers form one biological hexamer (α3β3) associated with the asymmetric unit, and the fourth forms a hexamer with symmetry-related heterodimers. The final model includes residues 18 to 143 and 153 to 459 for all α-subunits and residues 6 to 188 for all β-subunits. Interpretable electron density was not observed for residues 144 to 152 of the α-subunit. These residues were also absent in the wild-type BphAELB400 structure (11). Similar to other Rieske-type dioxygenases, each α-subunit binds a [2Fe-2S] Rieske-type cluster and a mononuclear Fe2+ ion at the active site, which is coordinated to two His residues (His233 and His239), an Asp residue (Asp388), and one or more water molecules.
The biphenyl-bound structure was refined to a final Rcryst of 21.6% and an Rfree of 25.4% at a resolution of 1.9 Å in space group P1. The structure includes 12 crystallographically independent αβ-heterodimers. α-subunits are labeled chains A, C, E, and G, etc., and β-subunits are labeled chains B, D, F, and H, etc. The final model includes residues 18 to 143 and 153 to 459 for all α-subunits except chain U (missing residues 143 and 459) and chain W (missing residue 143). For the β-subunits, all chains extend to residue 188, but the N-terminal content varies among the chains: chain F begins at residue 5, chain J begins at residue 14, and the remaining chains begin within the range of residues 6 to 13.
In eight of the α-subunits (chains C, E, I, K, M, O, Q, and W), initial unbiased (Fo − Fc) Fourier maps showed bulky electron density at the active site, consistent with bound biphenyl (Fig. 1B), whereas such density was not observed for the other four α-subunits (chains A, G, S, and U). Density consistent with a partially occupied biphenyl was observed for chain S at a later stage.
The absence of biphenyl in some active sites appears to be a consequence of crystal packing contacts associated with structural variation in the N-terminal segments of the β-subunits. For most β-subunits, the N-terminal segment interacts with residues of the same subunit or an adjacent β-subunit of the same α3β3 hexamer. However, for three β-subunits (chains D, F, and J), the N-terminal segment extends away from the rest of the β-subunit and interacts with a loop formed by α-subunit residues 247 to 263 at the mouth of one of the actives sites of a neighboring α3β3 hexamer. This contact apparently interferes with the binding of biphenyl. In fact, for chains D and F, the side chain of β-Phe9 extends just into the mouth of the active site of a neighboring α-subunit (chains A and G, respectively) and would prevent the entry of the substrate into the active site, as shown in Fig. S1 in the supplemental material. In the case of chain J, β-Phe9 was not modeled because interpretable electron density begins at residue 14. Nevertheless, the N-terminal segment in chain J extends toward chain U, and density for biphenyl was not observed in the active site of U.
Interactions, conformation, and orientation of biphenyl.
As observed in prior structures of biphenyl dioxygenases, the substrate binding pocket of BphAEII9 is sandwiched between the core β-strands and α-helices of the α-subunit. Biphenyl binds at the active site in a nonplanar conformation with the ring closer to the Fe2+ ion surrounded by residues Gln226, Phe227, Asp230, Met231, His233, and His323. The distal ring is surrounded by Ala234, His239, Ser283, Val287, Gly321, Gln322, Leu333, Ile336, Phe378, and Phe384 (Fig. 1B). Nonplanarity manifests through torsion about the C-1—C-1P bond, which averages −52° and ranges from −37° to −67°.
Consistent with a 2,3-dioxygenation of the substrate, the C-2 and C-3 atoms of biphenyl are generally closer to the Fe2+ ion than is C-1: the average distances are 4.5 Å for C-2 and C-3 and 5.1 Å for C-1. Despite variations in the C-2–Fe2+ and C-3–Fe2+ distances in the range of 4.2 Å to 4.8 Å, in no case is either C-2 or C-3 significantly closer to Fe2+. Furthermore, superposition of the α-subunits on the basis of C-α atoms (as described below) places the proximal rings in a tight cluster, as shown in Fig. 2B. The same superposition shows greater variability in the relative positions of the distal rings. This variance in the position of the distal ring is also seen in the structure of biphenyl-bound BphAELB400. However, the proximal ring binds in a similar fashion with respect to the Fe2+ ion in all chains.
FIG 2
Water molecules in contact with biphenyl and bound to Fe2+ were modeled in three different locations among the several active sites, and either one or two water molecules were included. Two of the locations correspond to the site of side-on binding of dioxygen observed in crystal structures of naphthalene dioxygenase (41), whereas the third site lies on the opposite side of a plane defined by C-2, C-3, and Fe2+. Based on the quality of the active-site density and the reliability of the atomic coordinates (judged by B-factors), the active-site models for chains C and O are the most reliable.
Comparison among α-subunits of the substrate-free and biphenyl-bound structures of BphAEII9.
The C-α atoms of the α-subunits from the substrate-free and biphenyl-bound BphAEII9 structures, 16 chains in total, can be superposed with a C-α RMSD of 0.5 Å or less, demonstrating close agreement. In some regions, however, variations in the local structure are observed.
Examination of superposed αβ-dimers reveals that the BphAEII9:biphenyl complex shows greater variability in the position of key active-site elements, as shown in Fig. 2B, than either the substrate-free BphAEII9 structure (Fig. 2C) or the biphenyl-bound form of its progenitor, BphAELB400 (Fig. 2A). For example, in the latter two crystal structures, the superposed positions of the Fe2+ ions lie in single, tight clusters. In contrast, in the BphAEII9:biphenyl complex, five Fe2+ ions (chains A, C, E, G, and U) are in one cluster, and six (chains I, K, M, O, Q, and W) are found in a second cluster ∼1.3 Å distant (average of all intercluster Fe2+-Fe2+ distances). Although the first cluster includes all chains for which biphenyl is not modeled (chains A, G, and U), two active sites with biphenyl present (chains C and E) are also members. The Fe2+ ion from chain S lies approximately between the two clusters and nearly equidistant (0.8 Å) from both.
Comparatively greater positional variability is also observed in the BphAEII9:biphenyl complex for two of the Fe2+-coordinating residues, Asp388 and His239, as well as Ser283, Val287, Gly321, and Gln322. The case of Asp388 is highlighted in Fig. 2A to C. Although the carboxylate groups of Asp338 for all chains lie close to a common plane, the C-δ atoms are as much as 1.5 Å apart. The variations for Asp388 extend to the backbone atoms: the average (0.8 Å) and maximum (1.5 Å) distances among C-α atoms are indistinguishable from the average and maximum distances for C-γ and Oδ1, which binds to the Fe2+ ion. Ligands would be expected to track with the Fe2+ ion, and thus, clusters are again observed for the Asp388 carboxylates, although they are not as distinct or as tightly clustered.
To understand the variations in Fe2+ and Asp388 locations, it is useful to consider them with reference to a vector from the C-α atom of Gly321 to the C-α atom of Asp388, which passes close to the center of biphenyl and close to the Fe2+ ion. The length of the vector is significantly shorter for chains without density for biphenyl (ŕ = 15.0 Å; range = 15.0 Å to 15.1 Å) than for chains with biphenyl (ŕ = 16.5 Å; range = 15.9 Å to 17.3 Å). Qualitatively, expansion of the active site along this vector moves Fe2+ and Asp388 away from Gly321, opening space for biphenyl.
The orientation of the peptide plane between Gly321 and Gln322 and the interactions of the carbonyl of Gly321 also differ between the substrate-free and biphenyl complexes and vary among the chains of the latter (Fig. 3A). In all four chains of the BphAEII9 substrate-free structure, ψ is in the range of 12° to 37° such that the carbonyl of Gly321 points into the active site and interacts with C-ε of Met231 through a distance of 3.0 Å to 3.2 Å. For the biphenyl-soaked structure, the carbonyl of Gly321 is similarly placed in six of the chains, including all three without density for biphenyl (chains A, G, and U) and three with biphenyl modeled (chains M, Q, and W). In the other six chains, all with biphenyl modeled (chains C, E, I, K, O, and S), ψ is in the range of 95° to 115°, such that the direction of the carbonyl of Gly321 differs by ∼90°. This allows the carbonyl to form a hydrogen bond with the amide NH of Tyr277 through a distance of 3.0 Å to 3.1 Å. As in the case of the Fe2+ location, the local structures of chains without biphenyl modeled are consistent, but the chains with biphenyl bound disperse between two groups, one that is consistent with the substrate-free structure and one that is distinct.
FIG 3
A difference in the extent of variability between the biphenyl complex and the substrate-free enzyme is also seen for the short α-helix (residues Pro281 to Met288) that forms one side of the active-site portal and one wall of the biphenyl binding site, where the side chains of Ser283 and Val287 are in contact with the distal ring of biphenyl. In the BphAEII9 biphenyl complex, the maximum difference in the positions of Ser283 C-α atoms for any pair of chains is 1.9 Å, but among the chains in the BphAEII9 substrate-free structure, the maximum value of the same measure is 0.6 Å.
Finally, a comparable extent of variability is seen in both structures for the backbone and side-chain atoms from residues Pro249 to Thr260. Here, maximum C-α displacements of up to 0.8 Å and 2 Å in the BphAEII9 biphenyl-bound structure and of up to 3.8 Å and 1.1 Å in the BphAEII9 biphenyl-free structure are seen at residues Ser254 and Ile258, respectively. A comparable variation is observed for side-chain atom positions and conformations. This segment contains a large fraction of solvent-exposed residues and does not contribute directly to biphenyl binding.
Observed structural changes due to shuffled region III.
Previous studies suggested that each residue in region III can influence the functional properties of the enzyme by direct or indirect interactions with active-site residues (11, 26). We have compared the structure of BphAEII9 with those of the parental enzymes, BphAELB400 and BphAEB356, to consider the influence of specific substitutions on the functional properties of the oxygenase. BphAEII9 has four substitutions in region III with respect to BphAELB400: Thr335Gly, Phe336Ile, Asn338Thr, and Ile341Thr. In a previous report (28), we showed that the kcat values of BphAEII9 and BphAELB400 for biphenyl are similar. This indicates that the structural changes brought about by these substitutions did not alter the electron transfer system in BphAEII9. However, as we discuss below, the structural analysis shows that each of these substitutions may contribute to the increased flexibility of the active site.
The Thr335Gly substitution.
In BphAELB400, a hydrogen bond between Thr335 Oγ and Gly321 N presumably restricts the conformation of the Val320-Gly321 dipeptide. The Thr335Gly substitution in BphAEII9 abolishes the hydrogen bond, and the Val320-Gly321 segment, which lines the active-site pocket, is more relaxed (Fig. 3B). As noted above, the Gly321-Gln322 peptide plane exists in two orientations among the chains of the biphenyl complex. One of these, as illustrated by chain C, would accommodate bulkier substrates. For example, for chain C of the BphAEII9:biphenyl complex, a ψ angle of 107° at Gly321 increases the distance between the carbonyl oxygen of Gly321 and ortho and meta carbon atoms of biphenyl's distal ring to 5.4 Å and 5.0 Å, respectively. These distances are typically 4 Å or less in the BphAELB400:biphenyl complex.
As was the case for the previously studied BphAELB400 variant BphAEp4, the substitution of Thr335 by a smaller amino acid such as Ala in BphAEp4 or Gly in BphAEII9 eliminates a hydrogen bond between the Thr335 Oγ and Gly321 N, relieving a conformational constraint on the Val320-Gly321 dipeptide, residues that line the active site of the protein (11). Due to the absence of this constraint on Gly321, the carbonyl of the peptide bond between Gly321 and Gln322, which points toward the active site in the BphAEII9 substrate-free structure, points away from the substrate binding site in the BphAEII9 biphenyl-bound structure. This makes more space in the active site for the binding of bulkier substrates. Also, in the presence of biphenyl, Val320 in BphAEII9 is displaced from its original position by 1.0 Å and makes polar contact with Ser283. At its new position, Ser283 interacts with the main chain of residues Ala286 and Val287. This is responsible for the variability in the position of the short α-helix (Pro281-Met288).
The Phe336Ile substitution.
The Phe336Ile substitution places a smaller side chain at the surface of the active site, directly increasing the volume available for substrate binding. The minimum distance between residue 336 and meta and/or para carbons of biphenyl's distal ring increases from 3.6 Å (median) for BphAELB400 (Phe336LB400 C-ε and/or C-ζ in a −60,−40 rotamer) to 4.1 Å (median) for BphAEII9 (Ile366II9 C-δ1 in a −60,−60 rotamer). Moreover, the minimum distance increases to 5.1 Å in chain Q, wherein Ile366II9 assumes a readily accessible −60,−60 rotamer. It should be noted that mutation to Ile336 affects the conformation of a neighboring residue, Phe378, pushing it closer to biphenyl (Fig. 3C), such that Phe378II9 C-ζ approaches the ortho and/or meta atoms of the distal ring. The typical closest-contact distances remain longer than 4 Å, but distances as short as 3.7 Å were observed. These contact distances can be readily altered by a change in the torsion angle between biphenyl's rings, whereas contacts between the side chain of residue 336 and the para carbon are unaffected by the conformation of biphenyl.
The Asn338Thr substitution.
In the case of the Asn338Thr substitution, an effect mediated by the difference in chain lengths of Thr and Asn is conceivable. For both variants, the side-chain oxygen atom accepts a hydrogen bond from the Nε of a neighboring residue, Arg340. It is clearly seen that due to the difference in the side-chain lengths of Asn and Thr, the side chain of Arg340 is placed at different positions in BphAELB400 and BphAEII9, respectively (Fig. 3D). While a hydrogen bond between Arg340 Nη2 and the carbonyl of Phe378 is clearly visible in the BphAELB400 structure, it is not observed in BphAEII9. This impedes the movement of the side chain of Phe378 in BphAELB400 but not in BphAEII9. As mentioned above, Phe378 lies close to the active site, and the freedom associated with its side chain can easily affect the orientation of the bound substrate. In BphAEII9, the phenyl ring of Phe378 is 4.9 Å away from the vicinal ring and 4.4 Å away from the distal ring of the biphenyl. These distances in BphAELB400 are 5.5 Å from the vicinal ring and 4.9 Å from the distal ring of the substrate. Thus, the type of residue at position 338 indirectly affects the orientation and the position of oxygenation of the substrate at the active site.
The Ile341Thr substitution.
The influence of the Ile341Thr substitution on substrate range or other aspects of enzyme activity remains enigmatic from the perspective of the crystal structures. Relative to the biphenyl binding site, the side chain for position 341 lies on the opposite surface of the central β-sheet, and its C-β atom is >14 Å distant from the closest atom of biphenyl. Significant changes in local backbone conformation are not observed, and minor changes in the placement or conformation of neighboring side chains directed toward the active site (Arg340 and Trp342) cannot be attributed uniquely to the Ile341Thr substitution.
Therefore, except for the Ile341Thr substitution, structural analysis clearly shows that the other three substitutions in BphAEII9 may contribute to the expanded substrate range of the enzyme. The indirect effect of Phe336Ile, Asn338Thr, and Ile341Thr substitutions on the active site influences the constraints placed on Phe378, a residue that lines the active site and is particularly close to the bound biphenyl (Fig. 4). The Asn338Thr and Ile341Thr substitutions appear to act together, pulling the Arg340 side chain away from the backbone carbonyl of Phe378. As a consequence, the hydrogen bond involving Arg340 and Phe378 of BphAELB400 is eliminated in BphAEII9, freeing at least one major constraint on the position of Phe378. In contrast, the Phe336Ile substitution acts more directly on the position of Phe378, by displacing the ring of Phe378 in the direction of the Fe2+ ion. Interestingly, comparison of the position of Phe378LB400 with the corresponding positions of Phe376B356 and Phe376B356 exhibits a shift toward the Fe2+ ion as well (Fig. 4).
FIG 4
Additionally, variation was seen in the binding positions and orientations of biphenyl in various αβ-heterodimers of the BphAEII9 biphenyl-bound structure. First, the variation in the distance from the Fe2+ ion to the vicinal ring of biphenyl demonstrates a structure with a high tolerance for a variety of substrate binding. Next, the variation seen in the position of the distal ring of biphenyl demonstrates a binding pocket that has a volume larger than is necessary to bind biphenyl and is capable of binding larger substrates. These aspects of substrate binding again point to a relaxed active site capable of accommodating a variety of substrate analogs, which explains the ability of BphAEII9 to metabolize an expanded range of PCB congeners.
Thus, although the chains modeled in the structures of the BphAEII9 substrate-free and biphenyl-bound forms show a high degree of similarity with each other and with the chains of BphAELB400, there are a number of conformational variations within localized regions. The regions of high variability (specifically the iron ligands Asp388 and His239, residues Gly321 and Gln322, the α-helix Pro281-Met288, and the region of Pro249 through Thr260) demonstrate a high degree of flexibility in and around the active site (Fig. 4). Some of this flexibility, for example, the region spanning Pro249 to Thr260, is observed in the parent enzyme, but much of it appears unique to BphAEII9.
Transformation of 2,3,4′-trichlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and DDT and docking studies.
To provide more insight into the structural features of BphAEII9, BphAELB400, and BphAEB356 responsible for their differential abilities to metabolize biphenyl analogs, we examined the biochemistry and the structural interaction of these enzymes toward 2,2′,5,5′-tetrachlorobiphenyl, 2,3,4′-trichlorobiphenyl, and DDT.
Biochemical studies were performed to assess the ability of BphAEII9 to transform DDT and two PCB congeners, 2,3,4′-trichlorobiphenyl, 2,2′,5,5′-tetrachlorobiphenyl, and the regiospecificity of the reactions.
BphAELB400 generated one major metabolite from 2,3,4′-trichlorobiphenyl as determined by examination of the GC-MS profile of the assay solution (Fig. 5A). The mass spectral features of this metabolite's nBuB derivative were consistent with those of a dihydro-dihydroxy-trichlorobiphenyl, namely, a fragmentation pattern exhibiting a molecular ion at m/z 356 and diagnostically important ions at m/z 340 (M+ − O), m/z 321 (M+ − Cl), m/z 286 (M+ − 2Cl), and m/z 256 (M+ − nBuBO2). A second metabolite exhibiting mass spectral features of a dihydro-dihydroxy-trichlorobiphenyl was also detected, but these features are not observable in Fig. 5A, as this metabolite was present in only trace amounts. BphAEII9 transformed 2,3,4′-trichlorobiphenyl into the same major metabolite, as shown in Fig. 5A; however, the amount produced was significantly larger. BphAEB356 produced the same metabolite from 2,3,4′-trichlorobiphenyl, and the amount produced was similar to that produced by BphAELB400 (not shown). Therefore, our assay demonstrated that the major product produced by three enzymes is the same metabolite and that BphAEII9 has a greater ability to degrade 2,3,4′-trichlorobiphenyl, as demonstrated by the much larger peak for the metabolite observed in the chromatogram. The substrate was docked into the active site of the three enzymes.
FIG 5
For BphAELB400, 2,3,4′-trichlorobiphenyl docked with the C-3 and C-3–chlorine atoms in approximately the same location as the C-2 and C-4 atoms of biphenyl in its complex with BphAELB400, and the proximal ring is in approximately the same plane as found in the biphenyl complex. This places the dichlorinated ring in a location and orientation consistent with dioxygenation across the C-4—C-5 bond. In this pose, as shown in Fig. S2B in the supplemental material, C-4 and C-5 are 4.3 Å and 4.4 Å from the Fe2+ ion, respectively, and 2.8 Å and 3.0 Å from a water molecule that occupies the presumed binding site for dioxygen, respectively.
For BphAEII9, docking of 2,3,4′-trichlorobiphenyl produced a pose consistent with the same regio- and stereospecificity as those for BphAELB400, but the binding mode is remarkably different (Fig. 6A and B; see also Fig. S2B in the supplemental material). The dichlorinated ring does not penetrate as deeply into the active site, such that its location is comparable to that of the distal ring in the BphAEII9:biphenyl complex. In addition, relative to the biphenyl complex, the orientation of the dichlorinated ring is shifted by ∼70°, and the torsion angle between the rings is shifted by +110°. Nevertheless, the distances from C-4 and C-5 to the water molecule are 3.3 Å and 3.0 Å, respectively, and the distances to the Fe2+ ion are 5.1 Å and 4.5 Å, respectively. For BphAEB356, 2,3,4′-trichlorobiphenyl docked in a manner similar to that of BphAEII9, as shown in Fig. S2A in the supplemental material, but with reduced docking scores.
FIG 6
Our docking study showed that the preferred docking pose for all three enzymes was one that positioned the substrate to be converted to a 3,4-dihydro-3,4-dihydroxy-2,3,4′-trichlorobiphenyl. This proposed product would not be further degraded into a chlorobenzoate, which may explain why Seeger et al. did not detect any chlorobenzoate produced from 2,3,4′-trichlorobiphenyl in their experiment with BphAELB400, since BphAELB400 metabolizes this substrate through 3,4-dioxygenation (42). Furthermore, the docking study showed that the preferred pose for BphAELB400 oriented the substrate much farther into the active site than the preferred pose for BphAEII9 (Fig. 6B; see also Fig. S2B in the supplemental material). This deeper-binding mode may explain why BphAELB400 exhibits lower activity toward 2,3,4′-trichlorobiphenyl than does BphAEII9.
BphAEII9 and BphAELB400 produced dihydro-dihydroxy-tetrachlorinated as the only metabolite of 2,2′,5,5′-tetrachlorobiphenyl, and on the basis of the peak area, the amounts produced were approximately the same for both enzymes (Fig. 5B). Consistent with data from a previous report (43), the dihydro-dihydroxy-tetrachlorobiphenyl produced from 2,2′,5,5′-tetrachlorobiphenyl by BphAEII9 and BphAELB400 must have been 3,4-dihydro-3,4-dihydroxy-2,2′,5,5′-tetrachlorobiphenyl, and GC-MS analysis showed that the metabolite produced by BphAEII9 was the same as the one produced by BphAELB400.
The bulkier 2,2′,5,5′-tetrachlorobiphenyl docked at the active sites of BphAELB400, BphAEII9, and BphAEB356 in distinct locations and orientations (Fig. 7B). Once again, the deepest penetration was found for BphAELB400, where docking of 2,2′,5,5′-tetrachlorobiphenyl gave a single pose competent for dioxygenation at positions C-3 and C-4, consistent with available biochemical data (Table 2). C-3 and C-4 are almost equidistant from the key water molecule at 3.3 Å and from the Fe2+ ion at 4.2 Å (see Fig. S3B in the supplemental material). The C-5–chlorine faces toward His323, the C-2–chlorine points toward Phe384, and the proximal ring of docked 2,2′,5,5′-tetrachlorobiphenyl is nearly in the same orientation as that observed for the proximal ring of biphenyl in its complex with BphAELB400.
FIG 7
TABLE 2
a
m, p, meta-para dioxygenation; o, m, ortho-meta dioxygenation.
In the active site of BphAEII9, 2,2′,5,5′-tetrachlorobiphenyl docked in multiple poses that would allow ortho-meta dioxygenation across the C-5—C-6 or C-2—C-3 bonds as well as meta-para dioxygenation across the C-3—C-4 bond. Since dechlorinated products were not observed in the biochemical analysis, poses consistent with ortho-meta dioxygenation were ruled out for the purposes of this study. The orientation most competent for meta-para dioxygenation, consistent with the biochemical data, was chosen for further analysis, although its docking metrics were lower. In this pose, as shown in Fig. 7A, carbons 3 and 4 are 3.0 Å and 4.2 Å away from the water molecule and 4.1 Å and 5.4 Å away from the Fe2+ ion, respectively. Unlike BphAELB400, the chlorine at the 2-C position of 2,2′,5,5′-tetrachlorobiphenyl is positioned toward His239 in BphAEII9. The chlorine at the 5-C position faces toward His323 in BphAEII9 but in a different orientation than that in BphAELB400.
Interestingly, the docking experiments with the two enzymes showed very different preferred poses for the binding of this substrate. The binding pose of 2,2′,5,5′-tetrachlorobiphenyl in BphAEII9 would likely generate a steric conflict between the substrate and Phe336 if adopted in BphAELB400. The pose adopted in BphAELB400 likewise appears to be inaccessible to BphAEII9, as it would create a steric conflict with Phe378. However, as discussed above, Phe378 is less constrained in BphAEII9 than in BphAELB400. Therefore, Phe378II9 may be more flexible in reality than was allowed in our docking experiments, which may explain the differences between the results of the biochemical and docking experiments.
For BphAEB356, the best-scoring pose places 2,2′,5,5′-tetrachlorobiphenyl much farther from the water molecule (at distances of 4.1 Å and 4.3 Å for C-3 and C-4, respectively) and Fe2+ (at a distance of 5.6 Å for both carbons) than in BphAELB400 and BphAEII9 (Table 2; see also Fig. S3A in the supplemental material). C-3 and C-4 are closest to the Fe2+ ion, but the increased distances reflect a binding mode that is likely incompetent for the reaction, consistent with biochemical data that show that BphAEB356 has poor activity toward 2,2′,5,5′-tetrachlorobiphenyl.
BphAEII9 retains activity toward 2,2′,5,5′-tetrachlorobiphenyl, while BphAEB356 shows very little activity toward this particular congener. This apparent contradiction is resolved when one considers the role of an adjacent residue, Thr375B356, in the position of Phe376B356. Thr375B356 Oγ1 makes a hydrogen bond to the backbone carbonyl of Gln371B356. This hydrogen bond forces Phe376B356 into a position closer to the Fe2+ ion (Fig. 8). The residue corresponding to Thr375B356 is Asn377LB400/II9. Asn377LB400/II9 also forms a hydrogen bond with the backbone carbonyl of His373LB400/II9 (corresponding to Gln371B356) and with the backbone carbonyl of Val287LB400/II9 (Fig. 8). The second interaction (between residues Thr375B356 and Gln371B356) is missing in BphAEB356, appears to anchor Phe378II9, restricts how far toward the Fe2+ ion the residue can move, and thus seems responsible for preserving BphAEII9's activity toward 2,2′,5,5′-tetrachlorobiphenyl.
FIG 8
Based on this analysis, one would expect that if Thr375B356 was mutated to an Asn residue, BphAEB356 could potentially gain the ability to oxygenate 2,2′,5,5′-tetrachlorobiphenyl. Indeed, BphAEKF707, from a strain that exhibits no activity toward 2,2′,5,5′-tetrachlorobiphenyl, gains the ability to oxygenate this particular congener by a Thr376Asn substitution (corresponding to Thr375B356 and Asn377LB400/II9) (44, 45). Likewise, the reverse is true: when region III of BphAELB400 is replaced by region III of BphAEKF707 along with mutation of Asn377LB400 to Thr (as in BphAEKF707/B356), the enzyme loses its activity against ortho-substituted congeners (46). However, it was also reported previously by Mondello et al. that the Asn377LB400Thr substitution alone (i.e., without swapping of region III) did not affect the substrate specificity of the mutated enzyme (23). Thus, neither the single Asn377Thr substitution nor the swapping of region III alone appears to restructure BphAELB400 enough to disrupt its ability to act against ortho-substituted congeners.
As reported previously (37), on the basis of product formation, the Km and kcat values of BphAEB356 for DDT were 174 ± 5 μM and 0.15 ± 0.08 s−1, respectively. During the current work, we found that the activity of BphAELB400 toward DDT was too poor to calculate accurate steady-state kinetic values. On the other hand, BphAEII9 was able to metabolize DDT, exhibiting Km and kcat values of 82 ± 2 μM and 0.18 ± 0.04 s−1, respectively. The GC-MS chromatogram produced from BphAEB356 against DDT shows two metabolites identified as stereoisomers of 1,1,1-trichloro-2,(4-chlorophenyl-2,3-dihydroxy-4,6-cyclohexadiene)-2-(4′-chlorophenyl)ethane, as shown in Fig. 5C. BphAEII9 produced the same two stereoisomers but in an inverse ratio to that of BphAEB356 (Fig. 5C).
The active sites of BphAEII9, BphAELB400, and BphAEB356 all accommodated DDT with corresponding 4-Cl-phenyl rings in similar locations. For the proximal ring, the distances between C-4 atoms are 1.1 Å to 1.4 Å, and the orientations of the rings are also similar (Fig. 9B). Nevertheless, the placement of the trichloromethyl group distinguishes the BphAELB400 complex from the others, and this complex had the poorest docking metrics (Table 2).
FIG 9
For BphAEII9, the top-ranked pose is consistent with dioxygenation across the C-2—C-3 bond, and the trichloromethyl group is directed away from the Fe2+ ion toward Gly321 (Fig. 9A). The distances from C-2 and C-3 of the proximal ring to the water molecule that marks the dioxygen binding site are 2.6 Å and 2.3 Å, respectively, and the distances to Fe2+ are 4.5 Å and 4.1 Å, respectively. The best pose for BphAEB356 is similar (see Fig. S4A in the supplemental material), but the C-2 and C-3 atoms are more distant from the water atom, 3.3 Å and 3.1 Å, respectively, and from the Fe2+ ion, 4.9 Å and 4.3 Å, respectively. The trichloromethyl group points in a similar direction toward Gly319 (aligns with Gly321 of BphAEII9).
In the best pose for the BphAELB400 complex, the C-2 and C-3 atoms lie 3.4 Å from the water molecule and 4.0 Å and 4.1 Å from the Fe2+ ion, respectively; however, the trichloromethyl group lies near Phe378 and the Fe2+ ion, as shown in Fig. S4B in the supplemental material, such that the shortest Cl-Fe2+ distance is 4.1 Å. Moreover, although the C-water and C-Fe2+ distances are similar in the three docked complexes, the geometric relationships between the ring, the water molecule, and the Fe2+ ion in BphAELB400 differ markedly. In the BphAEII9 and BphAEB356 complexes, the plane defined by C-2, C-3, and the Fe2+ ion is nearly orthogonal to the plane of the ring, and the water molecule is only ∼0.8 Å out of the C-2–C-3–Fe2+ plane but >2.0 Å out of the plane of the ring. In contrast, in the BphAELB400 complex, the angle between the planes is ∼45°, and the water molecule is ∼1.5 Å away from the C-2–C-3–Fe2+ plane and within 1.0 Å of the plane of the ring.
Therefore, our study demonstrates that BphAEII9 and BphAEB356 have similar levels of activity toward DDT. This finding is further supported by docking studies that showed that BphAEB356 and BphAEII9 have preferred binding poses for DDT that are quite similar. Unlike the other two enzymes, BphAELB400 metabolized DDT very poorly, and the docking experiments generated a binding pose that was completely different from that of the other two enzymes. On the basis of biochemical data, the preferred binding pose modeled in BphAELB400 is not a productive orientation. However, if DDT was oriented in BphAELB400 in a way similar to what was observed for the docking with BphAEII9, the substrate would interfere with Gly321-Gln322 and Phe336. This interference is not present in BphAEII9 due to the substitution of Phe336 with Ile and the elimination of a hydrogen bond that constrains the position of Gly321-Gln322.
In conclusion, our study of the structure of BphAEII9 suggests an enzyme with increased flexibility in and around its active site compared to its two parent enzymes. This increased flexibility would allow it to better accommodate a wide variety of potential substrates, and this is reflected in its enhanced substrate profile compared to its parent enzymes.
ACKNOWLEDGMENTS
This work was supported by the DRDO, India. This research used resources of the Advanced Photon Source, a U.S. Department of Energy (DOE) Office of Science User Facility operated for the DOE Office of Science by the Argonne National Laboratory under contract no. DE-AC02-06CH11357. Use of BioCARS was also supported by the National Institute of General Medical Sciences (NIGMS) of the National Institutes of Health under grant number R24GM111072. D.B.N. is supported by grant number GM103403 from the NIGMS.
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Published In
Journal of Bacteriology
Volume 198 • Number 10 • 15 May 2016
Pages: 1499 - 1512
Editor: W. W. Metcalf
PubMed: 26953337
Copyright
© 2016 American Society for Microbiology.
History
Received: 30 November 2015
Accepted: 25 February 2016
Published online: 28 April 2016
Contributors
Editor
W. W. Metcalf
Editor
Metrics & Citations
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Citations
Citation
2016.
Structural Basis of the Enhanced Pollutant-Degrading Capabilities of an Engineered Biphenyl Dioxygenase. J Bacteriol
198:.
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