Effect of F, Cl, Br and I substitution on the BphB enzyme for the degradation of halogenated biphenyls, revealed by quantum and molecular mechanics
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Halogenated biphenyls are worldwide persistent pollutants of great environmental concern. In particular, polychlorinated biphenyls and polybrominated biphenyls have been globally used for industrial purposes until they were found highly toxic, mutagenic and carcinogenic to humans. Therefore, ecological strategies to remove halogenated biphenyls, such as enzyme-catalyzed degradation, are needed. Here, we studied the effect of substitution of F, Cl, Br or I at the 4,4′-positions of 2,3-dihydro-2,3-dihydroxybiphenyl-2,3-dehydrogenase (BphB) on the degradation of halogenated biphenyls by quantum and molecular mechanics. Results show that Boltzmann-weighted average degradation barriers of substituted BphB are all lower than the unsubstituted biphenyl, except for chlorinated biphenyl. The roles of residues nearby the active site, e.g., isoleucine89, asparagine115, serine142, asparagine143, proline184, methionine187 and threonine189, were also investigated.
KeywordsQuantum mechanics/molecular mechanics Polyhalogenated biphenyls Metabolites Dehydrogenation Electrostatic influence
2-Hydroxyl-6-oxo-6-phenylhexa-2,4-dienoic acid hydrolase
Nicotinamide adenine dinucleotide (coenzyme)
Isoleucine and its sequence number in BphB enzyme
Quantum mechanics/molecular mechanics
The degradation pathway of polyfluorobiphenyls degradation
The degradation pathway of polybrominated biphenyls degradation
The degradation pathway of polyiodobiphenyls degradation
Polyhalogenated biphenyls include polyfluorobiphenyls (PFBs), polychlorinated biphenyls (PCBs), polybrominated biphenyls (PBBs) and polyiodobiphenyls (PIBs). They are widely used as hydraulic fluids, plasticizers and flame retardants. PCBs and PBBs have been discontinued in many countries as they were found highly mutagenic and carcinogenic to humans (Srogi 2008; Dsikowitzky and Schwarzbauer 2014; Nidheesh 2018). Due to their persistence and bioaccumulation, PCBs and PBBs have become ubiquitous pollutants in mobile environmental reservoirs and food chain, which have caused serious threats to human health (Kuswandi 2018; Sharma and Roy 2018). Environmental biotransformation is an excellent method that can provide solutions with the lowest input and has received worldwide acceptance to treat biphenyl and polyhalogenated biphenyls (Dannan et al. 1982). Previous structural, molecular docking and biochemical studies reported that dehydrogenase BphB can efficiently degrade biphenyl and polyhalogenated biphenyls (Vezina et al. 2007; Zhuang et al. 2016; Jain et al. 2018; Kulshreshtha 2018).
Four enzymes, BphA (biphenyl dioxygenase), BphB, BphC (dioxygenase) and BphD (hydrolase), are involved in the biphenyl and polyhalogenated biphenyls degradation processes (Field and Sierra-Alvarez 2008; Qu et al. 2013). BphA, the first enzyme in the degradation process, inserts two oxygen atoms into the vicinal ortho-meta carbons of the substrates. BphB, the second enzyme, catalyzes a dehydrogenation reaction. BphC and BphD sequentially metabolize 2,3-dihydroxybiphenyl and finally to 2-hydroxypenta-2,4-dienoic acid (Li et al. 2015). The dehydrogenation step by BphB is crucial in the polyhalogenated biphenyls degradation.
BphB is a dehydrogenase and belongs to a very large short-chain dehydrogenase/reductase family, and the sequence alignments of BphB are similar to its family (Jornvall et al. 1995; Vedadi et al. 2000). The X-ray structure indicates that active residues serine142, deprotonated tyrosine155 and coenzyme NAD (nicotinamide adenine dinucleotide) form the active site of BphB and accommodate substrate polyhalogenated biphenyls through hydrogen bonds (Borja et al. 2005; Piccoli et al. 2014). Structural studies are vital for understanding BphB-catalyzed processes. However, it is still obscure substitution effect that affects the degradation of halogenated biphenyls. Given the environmental importance of PFBs, PCBs, PBBs and PIBs, it is significant and urgent to understand the substitution effect of BphB-catalyzed degradation.
Here, we carried out quantum mechanics/molecular mechanics calculations to investigate the substitution effect of BphB-catalyzed degradation. Quantum mechanics/molecular mechanics method can provide the atomistic details of catalyst mechanisms and has become an increasingly important tool to supplement experimental enzyme chemistry (Muthusaravanan et al. 2018). The degradation details of fluorined, chlorined, bromined and iodined substituents (at 4,4′-positions) of cis-2,3-dihydro-2,3-dihydroxybiphenyl were revealed.
The quantum mechanics (QM) region of the degradation system contains substrates (polyhalogenated biphenyls), deprotonated tyrosine155 and coenzyme nicotinamide adenine dinucleotide (NAD). The entire degradation system was placed in a water sphere with a radius of 41 Å. The employed quantum mechanics/molecular mechanics (QM/MM) method is similar to our previous study (Li et al. 2014; Zhang et al. 2018), so only important details will be briefly summarized here. The QM/MM calculations were performed with the aid of ChemShell platform (Metz et al. 2014), which integrates TURBOMOLE and DL-POLY programs (Smith and Forester 1996). The QM region was treated with B3LYP/cc-pVDZ//CHARMM22 level (Billeter et al. 2000). Single point energy was calculated at the M06-2X/6-311G(d,p)//CHARMM22 level (Qu et al. 2012). The M06-2X/6-311G(d,p)//B3LYP/cc-pVDZ level is an appropriate method for calculations due to the high accuracy of calculation with less computational source. Five different snapshots were extracted every 0.5 ns from 4 to 6 ns of the stochastic boundary molecular dynamic simulations for each degradation systems, which are named bf-4.0, bf-4.5, bf-5.0, bf-5.5, bf-6.0 for the polyfluorobiphenyls degradation process and bb-4.0, bb-4.5, bb-5.0, bb-5.5, bb-6.0 for the polybrominated biphenyls degradation process and bi-4.0, bi-4.5, bi-5.0, bi-5.5, bi-6.0 for the polyiodobiphenyls degradation process. These extracted structures will be treated as the initial configurations in geometry optimization and transition state search.
Results and discussion
According to room-temperature single-molecule experiments, the rate constant of an enzyme-catalyzed reaction shows large fluctuations (Min et al. 2005). Here, the Boltzmann-weighted averaging method was employed to obtain average barrier (Billeter et al. 2000; Lonsdale et al. 2012). In the following paragraphs, the “average barrier” mentioned refers to the Boltzmann-weighted average barrier from five different snapshots.
Reaction mechanism and potential energetic results
Catalytic itinerary and structural details
Individual residue influence
A positive ΔEi−0 value means that neglecting the ith residue will increase the potential barrier and suppress the enzyme reaction (Li et al. 2013). The calculation method of ΔEi−0 value is presented in supplementary materials.
This work investigated the substitution effect of dehydrogenase BphB-catalyzed degradation of halogenated biphenyls by using the quantum mechanics/molecular mechanics method (QM/MM). The degradation details of fluorined, chlorined, bromined and iodined substituents (at 4,4′-positions) of cis-2,3-dihydro-2,3-dihydroxybiphenyl were revealed. Five snapshots were investigated for each of the halogenated substrates, which consistently revealed that the corresponding Boltzmann-weighted average degradation barriers are all lower than the unsubstituted cis-2,3-dihydro-2,3-dihydroxybiphenyl, except for chlorined biphenyl. Our results deepen the understanding of the BphB-catalyzed degradation processes and can serve as the model for studying other polyhalogenated biphenyls. The electrostatic influence analysis reveals that serine142 facilitates the degradation reaction and residue asparagine143 suppresses the degradation reaction, which may assist searching for new experimental mutation targets for future enzyme modification.
The work was financially supported by National Natural Science Foundation of China (Project Nos. 21337001, 21577082, 21876102) and Taishan Scholars (No. ts201712003) and National Major Science and Technology Program for Water Pollution Control and Treatment (No. 2017ZX07202-002).
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