Identification of New Dioxygenases Able to Recognize Polycyclic Aromatic Hydrocarbons with High Aromaticity

Abstract

Polycyclic aromatic hydrocarbons (PAHs), products from the incomplete combustion of crude oil, are pollutants present in nature. Ring hydroxylating dioxygenase enzymes are able to catalyze polycyclic aromatic hydrocarbons in the biodegradation process with a high degree of stereo-, regio-, and enantiospecificity. In this work, we present the first approximation of the binding modes of 9 PAHs with high aromaticity in the catalytic sites of biphenyl or naphthalene dioxygenases from four microorganisms usually used in bio-remediation processes: Sphingobium yanoikuyae, Rhodococcus jostii RHA1, Pseudomonas sp. C18, and Paraburkholderia xenovorans. Molecular modeling studies of two biphenyl dioxygenases from Sphingobium yanoikuyae and Paraburkholderia xenovorans showed good binding affinity for PAHs with 2–4 benzene rings (fluoranthene, pyrene, and chrysene), and both enzymes had a similar amount of substrate binding. Molecular docking studies using naphthalene dioxygenase from Pseudomonas sp. C18 showed that the enzyme is able to accommodate PAHs with high aromaticity (benzo(a)pyrene, indeno(1,2,3-cd)pyrene), with good docking scores. This study provides important insight into the utility of naphthalene dioxygenases in the degradation of HAPs with high aromaticity.

1. Introduction

Polycyclic aromatic hydrocarbons (PAHs), products from the incomplete combustion of crude oil, are pollutants present in nature [1,2]. There are more than 100 PAHs; their hydrophobicity increases and solubility decreases with an increased number of benzene rings. These compounds are of environmental concern because of their toxic, mutagenic, and carcinogenic properties [3]. Bioremediation is a highly efficient and cost-effective process that mainly uses microorganisms or enzymes (dioxygenases) to degrade contaminants in the environment (Figures S1 and S2) [4,5,6]. Communities of microorganisms with the capacity to degrade low-molecular-weight polycyclic aromatic hydrocarbons have been isolated and characterized, but those microorganisms were much less able to degrade high-molecular-weight PAHs (>4 benzene rings) [7,8,9,10,11,12]. Naphthalene is biodegraded by Sphingomonas (96%) [13], Sphingobiun, Actinobacteria, and Rhodococcus (100%). Anthracene is decomposed by Actinobacteria (93%), Sphingobium (28%), Mycobacterium (50–70%), Rhodococcus (90%), Nocardia, and Paracoccus [14]. Pyrene, fluoranthene, and chrysene are decomposed by Sphingomonas (80–90%), Rhodococcus (90%), Dyadobacter, Mycobacterium (50–70%), Sphingobium (20–60%), Pseudomonas aeroginos (60–70%), and Dyadobacter koreensis (80%) (Table S1) [8]. The concentration of PAHs with 2–3 benzene rings in nature decreases faster than the concentration of PAHs with 4–6 rings, which may be due to a recalcitrant effect or a lack of microorganisms capable of degrading these compounds [15].

PAH degradation by bacteria proceeds through dioxygenase attacks on a PAH to form a cis-dihydrodiol aromatic compound using two atoms of O₂ (Figure 1) [16,17] These proteins are also able to perform oxidation with a high degree of stereo-, regio-, and enantiospecificity at the catalytic mononuclear iron site [18]. Dioxygenases are metalloenzymes present in a wide variety of microorganisms that are able to oxidize PAHs using oxygen molecules and two electrons from a reductase to give cis-arene diols (Figure 1) [19,20]. A wide range of 3D dioxygenase structures have been determined, giving insight into substrate recognition and regioselectivity [19,21]. The structure of most dioxygenases is an α₃β₃ hexamer molecule, with each alpha subunit containing a Rieske [2Fe-2S] cluster and another alpha subunit containing a mononuclear iron center, where the catalytic reaction takes place [22,23]. The catalytic domain of these enzymes is antiparallel sheets, and the active site contains an Fe³⁺ ion usually coordinated by 2 His and 1 Asp amino acid [16]. In the Rieske center, Fe-1 is coordinated by 2 Cys and the second Fe by 2 His being conserved in this family of enzymes (Figure 1) [22].

Dioxygenases from different organisms have been studied for the deoxygenation of low PAHs (1–3 benzene rings). In particular, dioxygenase from Sphingomonas CHY-1 has been described as capable of recognizing different PAHs using molecular modeling [13,16,24]. Computational tools can be very useful for finding dioxygenases capable of degrading highly condensed PAHs, allowing these enzymes to be used in biocatalytic processes. In this paper, we carried out molecular docking studies of dioxygenases from different microorganisms involved in the biodegradation of highly condensed PAHs that are present in nature.

2. Results and Discussion

Genes responsible for the degradation of PAHs were studied for the first time in strains of Pseudomonas putida isolated in culture media with naphthalene or phenanthrene as the only carbon source (Figure S2b). Bacteria generally present high metabolic versatility and are capable of utilizing a broad spectrum of PAHs. To carry out the molecular docking studies, we selected naphthalene and biphenyl dioxygenases with 3D structure solved (PDB ID 2gbx, 1ulj, 4hjl, and 2xsh) from Sphingomonas, Rhodococcus, Pseudomonas, and Paraburkholderia (

Table 1. Protein Data Bank ID codes of dioxygenases from different microorganisms.

Code (PDB)	Organism	Dioxygenase/ Uniprot No.	Ions/Ligand
2ckf 2gbw 2gbx 2i7f	Sphingomonas sp. CHY-1 (Gram+) Sphingobium yanoikuyae (Gram+)	Naphthalene 1,2- Q65AT1 Biphenyl 2,3- /A2TC87	Fe2S2, Fe3+ ----   Biphenyl
2b1x 2b24	Rhodococcus sp. NCIMB 12038 (Gram-)	Naphthalene Q9 × 3r9	Fe2S2, Fe3+ Indol
1ulj 1uli	Rhodococcus jostii RHA1 (Gram-)	Biphenyl 2,3	Fe2S2, Fe3+ Biphenyl
1eg9 1ndo 1o7n 1o7p 3en1 3eqq	Pseudomonas putida (Gram-)	Naphthalene 1,2 P0A110 Toluene 2,3-/A5W4F2	Fe2S2, Fe3+, SO4− Indole Toluene ----
4hjl + 4hkv	Pseudomonas sp. C18 (Gram-)	Naphthalene 1,2-/P0A111	Fe2S2, Fe3+, SO4− Naphthalene Benzamide
2xsh 2xrx 2xso 2xsh	Paraburkholderia xenovorans LB400 (Gram-)	Biphenyl/ P37,334	Fe2S2, Fe3+, SO4− Biphenyl ---

⁺ To be published. Underlined pdb numbers correspond to proteins that have been used to study binding modes with PAHs by molecular docking.

).

The PAHs used in this study were anthracene, naphthalene, pyrene, chrysene, fluoranthene, benzo(a)pyrene, dibenzo(a,h)anthracene, indeno(1,2,3-cd)pyrene, and benzo(ghi)perylene (Figure 2). We chose these PAHs because they are listed by the United States Environmental Protection Agency (EPA) as priority pollutants.

The oxidation of these aromatic compounds by dioxygenases from different microorganisms is described in Table S1. Most dioxygenases are able to recognize up to three benzene rings (biphenyl, naphthalene, and anthracene); few enzymes are able to degrade compounds with more benzene rings [25].

Sphingobium yanoikuyae strain B1 was able to oxidize low-molecular-weight PAHs with four benzene rings (chrysene, pyrene, and fluorenthene) in in vitro assays [25]. Molecular modeling studies of PAHs into biphenyl 2,3-dioxygenase from Sphingobium yanoikuyae (PDB ID 2gbx) have shown that naphthalene, anthracene, fluoranthene, pyrene, and chrysene interact with nearby hydrophobic amino acid (F350, F201, L305, F404, V208, and T308) (Figure 3a). The experimental data support this enzyme as a good degrader of 2–4 benzene rings. PAHs were bound in the same hydrophobic internal pocket that the biphenyl substrate presents in the 3D structure of the protein. The chrysen–dioxygenase complex presents three Pi-Pi stacking interactions with two His (H207 and H293) of the enzyme in the binding site, giving a good docking score (−7.6 kcal/mol) (Table S3, Figure 3b). The surface area of the catalytic pocket of this enzyme is 361.44 Ų, so it is not surprising that it would not be able to accommodate larger PAHs (Table S2).

The same study was carried out with another biphenyl dioxygenase from Paraburkholderia xenovorans (also known as Burholderia xenovorans PDB ID 2xsh), which has a similar molecular surface area in the binding pocket of biphenyl dioxygenase from Sphingobium yanoikuyae (421.550 Ų) (see Supporting Materials). In addition, the amino acids that form the catalytic pocket are very similar in both enzymes (phenylalanines and histidines). The enzyme co-metabolizes many polychlorinated biphenyl compounds and has become a model system for the bacterial breakdown of very persistent environmental contaminants [26]. Figure 4 shows the binding of naphthalene, anthracene, fluoranthene, pyrene, and chrysene in the catalytic pocket, where their positions and conformations lie in very similar positions. The docking score results and structural features were very similar between these two biphenyl 2,3-enzymes. These studies demonstrate that the PAH binds in the enzyme with the atoms that would be oxidized closest to the Fe ion. Then, the enzymes can control the substrate orientation, as in product stereoselectivity/regioselectivity.

Rhodococcus wratislaviensis IFP 2016 and Rhodococcus aetherivorans IFP 2017 were able to degrade 15 petroleum compounds (low-molecular-weight PAHs or aliphatic compounds: xylene, toluene, octane, and so on) (Table S2) [27]. The molecular docking of 9 PAHs into biphenyl dioxygenase from Rhodococcus jostii RHA1 (PDB ID 1ulj) revealed that only naphthalene and anthracene could be accommodated in the binding site, with good docking scores of −6.7 and −7.3 kcal/mol, respectively (Table S3) (Figure 5), although the enzyme presents a large catalytic pocket (surface area of 1360.404 Ų). Naphthalene and anthracene share similar special occupancies with two aromatic staking interactions in the catalytic pocket (F368 residue for naphthalene and H224 for anthracene) (Figure 5). This result may be due to the ability of the enzyme’s catalytic pocket to recognize low-molecular-weight PAHs.

The biodegradation of PAHs in a culture made up of a consortium of five microorganisms, including Pseudomonas aeruginosa, was able to completely eliminate benzo(k)fluoranthene, indeno(1,2,3-cd)pyrene, and dibenzo(a,h)anthracene [28]. Docking studies with PAHs inside the substrate pocket of naphthalene dioxygenase from Pseudomonas sp. C18 (ID 4hjl) (Figure 6 Table S3) reveal that seven PAHs interacted with hydrophobic amino acids (F202, A206, V260, V209, L207, F224, I408, and L253) (Figure 6) by Pi-Pi staking interactions. The docking scores with benzo(a)pyrene (five benzene rings) and indeno(1,2,3-cd)pyrene (six rings) into the naphthalene enzyme (−9.9 and −9.0 kcal/mol, respectively) were analyzed (Figure 6).

Residues F202, F224, and H208 are expected to affect the topology of the binding pocket and consequently select the shape of allowed PAHs (Figure 7). So, the protein could oxidize a wide range of PAHs because its binding site is large enough to accommodate up to six rings.

Dibenzo(a,h)anthracene and benzo(ghi)perylene (five or six fused aromatic rings) did not interact within any dioxygenases used in this study. Molecular modeling studies provide important insights into the utility of naphthalene or biphenyl dioxygenases in the degradation of HAPs with high aromaticity.

3. Materials and Methods

The docking evaluation was carried out for four proteins (Table 1). The crystal structure of the dioxygenases used in this evaluation was retrieved from the Protein Data Bank (PDB). The catalytic α-subunit was selected, and all water molecules were removed. An oxygen molecule was placed near the Fe ion [29], and a 3D structure model of the proteins was optimized using the Protein Preparation Wizard tool integrated in Maestro (Schrödinger, New York, NY, USA) [30]. Missing hydrogen atoms were added, assuming the standard protonation state of titratable residues. The grid of the enzymes was using the receptor grid generation tools in Glide. The size of the docked molecules was set to within 20 Å. The molecular surface of the proteins and ligands was carried out using the binding surface area analysis tool integrated in Maestro.

Three-dimensional structures of the PAHs were retrieved from PubChem with extension. sdf and CID: anthracene 8418, naphthalene 931, fluoranthene 9154, pyrene 31423, chrysene 9171, benzo(a)pyrene 2336, dibenzo(a,h)anthracene 5889, indeno(1,2,3-cd)pyrene 9131, and benzo(ghi)perylene 115240. These compounds were prepared using the LigPrep module in the Schrödinger package. All polycyclic aromatic hydrocarbons were minimized using the OPLS3e force field implemented in Maestro. Molecular dynamic simulations of protein–PAH complexes were carried out using the Schrödinger bioinformatics suite. To achieve this, molecular docking was performed using the Glide-dock XP module [31,32]. Ligand poses generated in this way were run through a series of hierarchical filters to evaluate ligand interactions with the receptor. Docking score, glide e-model, ionization penalty, glide gscore, and TPSA were used to select the docking poses.

4. Conclusions

Molecular modeling was performed in an attempt to identify novel dioxygenases from microorganisms able to degrade PAHs with high aromaticity. Biphenyl dioxygenase from Rodococcus jostii RHA1 only showed binding of naphthalene and anthracene (smallest PAHs). The other two biphenyl dioxygenases, from Sphingobium yanoikuyae and Paraburkholderia xenovorans, showed good binding affinity for 5 of the 9 PAHs studied (particularly fluoranthene, pyrene, and chrysene), and both enzymes had very similar volumes of substrate binding (421.550 and 361.44 A², respectively). Molecular docking of 9 PAHs into naphthalene dioxygenase from Pseudomonas sp. C18 showed the good accommodation of seven PAHs (5–6 fused aromatic rings) that presented large, accessible surface areas (1500.585 Ų). This study shows that structural features are essential for determining the binding mode of PAHs. This study provides important insight into the utility of naphthalene dioxygenases in the degradation of PAHs with high aromaticity.