Abstract
The use of halogen bond is widespread in drug discovery, design, and clinical trials, but is overlooked in drug biosynthesis. Here, the role of halogen bond in the nitrilase-catalyzed synthesis of ortho-, meta-, and para-chlorophenylacetic acid was investigated. Different distributions of halogen bond induced changes of substrate binding conformation and affected substrate selectivity. By engineering the halogen interaction, the substrate selectivity of the enzyme changed, with the implication that halogen bond plays an important role in biosynthesis and should be used as an efficient and reliable tool in enzymatic drug synthesis.
Similar content being viewed by others
Introduction
Halogens (X = F, Cl, Br, and I), as pharmaceutically active ligand substituents, are widely used in pharmacology1,2. Approximately 50% molecules in high-throughput screening are halogenated1 and around 40% drugs currently on the market or in clinical trials are halogenated3. Furthermore, an estimated 25% medicinal chemistry papers and patents involve the addition of halogen atoms at a late stage of the synthesis1. Halogens, treated primarily as electron-rich atoms that do not participate in specific interactions4, form a halogen bond (X-bond) with a proximal halogen-bond acceptor (such as O, N, S, and aromatic ring)5,6,7,8. The halogen bond, analogous to the hydrogen bond, is a highly directional and specific non-covalent interaction9. This bond has attracted great attention in pharmacology because halogen bonds, as orthogonal molecular interactions to hydrogen bonds, can be introduced to improve ligand affinities without disrupting other structurally important interactions10, and thus can be exploited for the rational design of halogenated ligands as inhibitors and drugs11.
The halogen bond, which has a wide application in the pharmaceutical sector, including drug discovery, design, and clinical trials, has been nonetheless overlooked in enzymatic catalysis, generally regarded as a practical and environmentally-friendly alternative to the traditional metallo- and organocatalysis in drug synthesis12. Nevertheless, the halogen bond is also popular in protein-ligand complexes, with >1000 structures in 2010 and >2000 in recent years13. Regardless, the importance or prevalence of the halogen bond in the biosynthesis of drugs or drug precursors remains unclear.
Nitrilase (EC 3.5.5.1), catalyzing the hydrolysis of nitriles to the corresponding acids in a single step reaction14, plays an important role in the manufacture of key building blocks for drugs, such as clopidogrel15, atorvastatin (Lipitor)16 and pregabalin17. This not only on account of the mild reaction conditions but also because of the regioselectivity and enantioselectivity of the nitrilase18. Each isomer of ortho-, meta-, and para-halogenated precursors or drugs should be used individually because of the specific pharmaceutical activity. For example, ortho-chlorophenylacetic acid can be used to synthesize diclofenac19 and clopidogrel20, an anti-inflammatory drug and anti-platelet aggregation drug, respectively; para-chlorophenylacetic acid can be used to synthesize indoxacarb21 and baclofen22, an insecticide and a muscle relaxer for treating muscle symptoms caused by multiple sclerosis, respectively. However, naturally occurring nitrilase is characterized mainly by meta-activity, seldom by para-activity, but not ortho-activity23. Therefore, it is crucial to engineer nitrilase substrate selectivity for each isomer of the ortho-, meta-, and para-halogenated compounds.
In this study, we undertook the design of nitrilase enzymes with altered specificities for substrate isomers. We used mutagenesis to specify potential halogen bonding interactions with the chloro-substituents at ortho-, meta-, or para-positions (Fig. 1A). We started by analyzing the active site of the wild type enzyme and, after performing molecular dynamics (MD) simulations, we designed mutants in the substrate binding pocket to engineer X-bonds between the substrate and protein side-chains. Thus, enzyme substrate specificity was directed towards one or more of the isomeric forms. The results of this study demonstrate the potential for exploiting X-bonds as a recognition element in protein engineering, particularly in helping to define and alter the specificity of enzymes in their catalytic site. Our study shed light on the role of halogen bonds in drug biosynthesis and suggests that more attention should be paid to the application of the halogen bond in enzymatic synthesis of drugs in the future.
Results
Nitrilase from Syechocystis sp. PCC6803, whose structure has been reported in our previous work (PDBID: 3WUY)24, exhibited high selectivity for meta-chlorobenzyl cyanide (1a) but not para-chlorobenzyl cyanide (1b) (Table 1). The difference between 1a and 1b concerns just the location of the halogen atom, Cl, which can form halogen bond with the proximal halogen-bond acceptor. The halogen bonds in the two complexes were carefully analyzed by a 10-ns MD simulation (Supporting Information Figure S1). In the nitrilase WT–1a complex, a halogen bond was formed between 1a and Gly195, while it was formed with Tyr173 for WT–1b complex. The different halogen atom location caused a distinct distribution of the halogen bond. Because the interactions between the substrates and the enzyme contribute to the conformation of the substrate-enzyme conformations25,26, this changing distribution of the halogen bond may result in a distinct substrate binding mode, greatly contributing to the substrate selectivity27.
Careful analysis of the binding states of the two substrates was performed. First, no steric clashes were apparent for 1b because of the large binding pocket of this nitrilase (Fig. 2A). According to the catalytic mechanism28 of the nitrilase superfamily (Fig. 1B), the nitrilase utilizes the Glu53–Lys135–Cys169 catalytic triad to hydrolyze non-peptide carbon-nitrogen bonds. The first and decisive step is a nucleophilic attack on a cyano or carbonyl carbon atom by the active site cysteine residue. DC8-SG describes the distance from the C8 carbonyl carbon atom of the substrate to the mercapto sulfur SG of the catalytic cysteine, which determines whether the nucleophilic attack can occur (Fig. 1B). Meanwhile, the lysine formed hydrogen bonds with the substrate to provide stabilization for the intermediate. DN1-HZ is the distance between the N1 cyano nitrogen of the substrate and HZ of the catalytic lysine, which affects the stabilization of the substrates (Fig. 1B). Therefore, shorter DC8-SG and DN1-HZ both indicate a greater probability of the substrate being catalyzed. A 10-ns MD simulation of the two complexes (WT-1a and WT-1b) indicated that for this nitrilase, the average DC8-SG and DN1-HZ values with 1a were 3.76 Å and 2.83 Å, respectively; and those with 1b were 6.54 Å and 9.49 Å, respectively. The binding conformation of 1a facilitated the nucleophilic attack and the formation of transition state. This was not the case for the binding conformation of 1b. Therefore, for WT enzyme, the orientation of the cyano group of 1a toward the catalytic triad was stabilized by the halogen bond between 1a and Gly195 and the hydrophobic stacking interaction between 1a and Pro193 (Fig. 2B). Meanwhile, in the WT–1b complex, the main halogen bond was formed between 1b and Tyr173. Because the triple-bond between the carbon and nitrogen atoms and the phenyl ring are structurally rigid, the direction change on one side of the phenyl ring, such as para-Cl, would cause a corresponding direction change on the other side, such as a triple-bond of carbon and nitrogen. Thus, the direction change of para-Cl caused the cyano group to position away from the catalytic triad, which was further retained by an perpendicular aromatic interactions formed between Phe202 and 1b (Fig. 2C).
Hence, the halogen bonds, together with aromatic interactions, affected substrate selectivity of WT for 1a and 1b, with the halogen bonds serving as a crowbar and the aromatic interactions controlling the rotation of substrates. These interactions may be maintaining the cyano group in the proximity of the catalytic triad, and positioning it in an orientation favorable for the enzymatic reaction to occur. It seems to be feasible that the modulation of the halogen bonds would affect enzyme substrate selectivity. However, a direct and precise modulation is rather difficult since the halogen bond, like the hydrogen bond, is a complex network of interactions. Here, a simple protocol was designed to model modulation of the halogen bond. First, residues in the substrate binding pocket24 were selected to be mutated to aromatic amino acids (Phe, Tyr, Trp), since the binding pocket is “aromatic rich” and aromatic amino acids are involved in halogen bond7,8. The mutants were constructed by in silico mutation. Second, enzyme-substrate complex conformations, reflecting both interactions25,26 and catalytic property27, were used to screen the shifted selectivity mutants. The screening criteria were that both DC8-SG and DN1-HZ should be shorter than the corresponding distances in WT. MD Simulation (10-ns MD) was performed to analyze the enzyme-substrate complex conformations. Finally, mutagenesis and catalytic experiments with the screened mutants were performed in vitro.
With 1b, T54Y mutant DC8-SG and DN1-HZ distances were shorter compared with WT (Supporting Information Table S1). Next, 10-ns MD simulation was performed to investigate the dynamic conformation of T54Y (Supporting Information Figure S2). The productive binding conformation involved a T54Y–1b complex with small and stable DC8-SG and DN1-HZ (average values of 4.31 Å and 7.90 Å, respectively), while those with 1a bound to T54Y were relatively large (average values of 5.74 Å and 8.54 Å, respectively). In addition, the distribution of halogen bond in T54Y-1a and T54Y-1b was changed compared with WT. In the T54Y–1b complex, the halogen bond was formed between 1b and Gln205. Additionally, when Thr54 was mutated to Tyr, the increased steric occupation pushed the loop of residues 139–146 back, away from the center of the binding pocket, making space for the side chain of Trp170. Further, 1b formed π-π stack interaction with Trp170 and Phe202, pulling the cyano group of 1b toward the catalytic triad (Fig. 2D). On the other hand, in the T54Y–1a complex, 1a formed halogen bond with Tyr173 and an aromatic interaction with Phe202, with both keeping the cyano group away from the catalytic triad. Hence, the modulation of the halogen bonds, together with the aromatic interactions, manipulated the substrate binding conformation. Binding conformations of T54Y with 1a and 1b suggested enzyme substrate selectivity for 1b. Hence, T54Y was selected for testing. As expected, KcatKM−1 analysis revealed that the catalytic efficiency of T54Y with 1b, 0.56 s−1 mM−1, was much higher than that with 1a, 0.01 s−1 mM−1 (Table 1). The activity of T54Y with ortho-chlorobenzyl cyanide (1c) was also tested, but it was below the detection limit. We thus verified that the halogen bonds formed between the substrates and the enzyme strongly affect enzymatic substrate specificity and can be employed to shift substrate selectivity.
Furthermore, since few known natural nitrilases display ortho-selective activity, it is more challenging and valuable to shift the substrate selectivity to ortho-selectivity. Here, the same protocol was applied to design mutants with substrate selectivity for ortho-chlorobenzyl cyanide (1c) as above. No steric clashes were apparent for 1c binding because of the large binding pocket of this nitrilase (Fig. 2A). The only difference between 1a and 1c was the halogen bond. In the WT–1c complex, the chlorine atom of 1c formed a halogen bond with Trp170. A 10-ns MD simulation of this complex yielded average DC8-SG and DN1-HZ values of 7.60 Å and 9.06 Å, respectively. They were both larger than those of 1a. Therefore, the binding conformation of 1c was not conducive to nucleophilic attack and transition state formation, because the halogen bond formed between ortho-chlorine atom and Trp170, and the perpendicular aromatic interactions between substrate and Trp173, cooperatively kept 1c cyano group away from the catalytic triad (Fig. 2E). We constructed and analyzed the mutants, as described above, and in silico screened their interaction with 1c.
With 1c, H141W mutant DC8-SG and DN1-HZ distances were short, indicating a potential productive conformation (Supporting Information Table S1). Additionally, the dynamic conformation of H141W complexed with 1c and 1a was analyzed by a 10-ns MD simulation. Distances DC8-SG and DN1-HZ of H141W–1c complex remained small and stable (average values of 3.79 Å and 3.46 Å, respectively), while those with 1a were not only large but also variable (average values of 10.40 Å and 14.76 Å, respectively; Supporting Information Figure S2). These results of conformational analyses demonstrated a potential substrate selectively of H141W toward 1c. This binding mode change may stem from the rearrangement of the halogen bond. As expected, in the H141W–1c complex, the halogen bond was mainly formed with Phe202 (80%), positioning the CN group to the active site, with the assistance of an aromatic interaction with Trp141 (Fig. 2F). On the other hand, for 1a, the main halogen bond formed with Ile201 (95.08%) and a sigma-hole…π interaction arose with Phe202, cooperatively placing the cyano group away from the catalytic triad. Therefore, H141W was selected to testing. As anticipated, the H141W mutant, which showed a productive binding conformation with 1c, was really highly selective for 1c, according to the KcatKM−1 analysis (0.33 s−1 mM−1, Table 1). The catalytic efficiency of the mutant was below the detection limit, for both 1a and 1b. Hence, the data once again confirmed that the halogen bonds, working as a crowbar for the cyano group, together with the aromatic interactions, strongly affect substrate selectivity in biosynthesis.
Additionally, the putative X-bonding substituent Cl was varied to methyl group at each isomer position to futher evidence that the Cl in any of the isomeric forms participate in X-bonding in this enzyme system. From the experiment results (Supporting Information Table S2),wild-type showed activity to both meta-, and para-methylbenzyl cyanide (relative acitvity of 100% and 46.2%,respectively), and H141W and T54Y both showed selectivity to meta-methylbenzyl cyanide (relative acitvity of 27.18% and 20.54%, respectively). The H141W and T54Y showed similar selectivity with wild-type. The resulting isomer activity may come from steric or hydrophobic effects since the methyl substituent does not involve in halogen bond. Compared with methyl substituent, when the substituent is Cl, wild-type showed more rigid substrate selectivity to meta-isomer, indicating the halogen bond enhanced the substrate selectivity. Besides, wild-type, H141W and T54Y exhibited the different substrate selectivity to chlorobenzyl cyanide, but the same s pecificity to meta-methylbenzyl cyanide, indicating that the halogen bond participated in the substrate selectivity of the enzymes. Therefore, the engineered enzymes actually utilized X-bonding as the driver of substrate specificity in this study.
Discussion
In conclusion, the role of halogen bond in nitrilase substrate selectivity was herein investigated. Detailed conformational analysis revealed that the halogen bond induced a change of substrate binding mode, serving as a crowbar, and the aromatic interactions controlled the positioning of the benzene ring of the substrate. Together, the bonds held the substrate cyano group in proximity to the catalytic triad, and positioning it in a reaction-favoring orientation. Furthermore, by regulating the halogen bond, two types of artificial enzymes were obtained, with selective activity toward para-chlorobenzyl cyanide and ortho-chlorobenzyl cyanide, respectively. Therefore, halogen bond formed between the substrate and the nitrilase strongly affected substrate selectivity of the enzyme.
Besides, the current study assessed the performance of standard Amber ff99SB force field (Supporting Information Table S4 and Figures S4–S6) and the positive extra-point (PEP) modified one in describing the halogen bond. In the PEP approach29,30, the σ-hole on the halogen atom is represented by an extra-point of positive charge. From the MD results of PEP, the different distribution of halogen bond contributes majorly to the substrate selectivity of wild-type. Also H141W showed shorter DC8-SG and DN1-HZ to 1c and T54Y exhibited both shorter key distances to 1b, indicating H141W and T54Y were selectively active to 1c and 1b, respectively. Compared with the standard form Amber force field, the residues paticipating in X-bond were more focused in PEP, indicating the X-bonds were enhanced with EP integrated. Therefore, the modified Amber force field such as PEP is more accurate in describing the property of X-bonds.
Generally, halogen bonds formed between proteins and their ligands have been widely employed in drug design because of their role in improving ligand binding affinities. However, enzymatic catalysis, which is generally regarded as a practical and-environmentally friendly approach to drug synthesis, has ignored the halogen bond. This study demonstrated for the first time that halogen bond significantly affects substrate binding conformation and, further, plays an important role in substrate selectivity. Therefore, in a protein-ligand biosystem, the halogen bond can shift the enzyme’s substrate selectivity by adjusting the substrate binding conformation. It can be used as a powerful tool, similarly to hydrogen bond7,8,31,32 and electrostatic interactions33 in enzyme design. Consequently, the halogen bond in the biocatalysis cannot be ignored and more attention should be paid to it.
Methods
The expression and purification of nitrilase and the enzyme assay were conducted as described previously15,24,34, while an eluting solvent system was phosphoric acid (0.1%, v/v) and methanol (40:60, v/v).
Computational Details
The initial structure of the nitrilase was crystal structure (PDBID: 3WUY) obtained in our previous work. All of the molecular docking experiments and virtual mutation utilized here were performed on Discovery Studio 4.035. Default parameters were used in our docking experiments. The substrate conformation with highest score calculated by Discovery Studio 4.0 was selected for MD simulation. The substrates halogenated by chlorine were constructed and their geometries were optimized at the HF/6-31G* level by Gaussian 03. The atomic partial charges were then evaluated with incorporating an extra point of charge on each isomer’s halogen atom using the restrained electrostatic potential (RESP) approach36. The distance between the halogen and the extra point is 1.90 Å37. The parameters used for the extra point were set as described by Ibrahim38. The Amber ff99SB force field39 was applied to treat the proteins, and the Generalized Amber Force Field (GAFF)40 was employed to deal with the substrates. The complex was solvated in a TIP3P water box, and sodium ions were added to ensure the electric neutrality. MD simulation was carefully performed with Amber1441,42 in 7 stages. The system was minimized with position restraints of 50.0 kcal mol−1 Å−2 and 20.0 kcal mol−1 Å−2 in the first and second stages, respectively, and with no restraint in the third stage. The minimization for each stage took 20,000 steps to reach convergence. Then, the system was heated from 0 K to 300 K in 0.05 ns with a restraint of 10.0 kcal mol−1 Å−2 using the Andersen temperature coupling scheme43. The next stage was the equilibration with a restraint of 2.0 kcal mol−1 Å−2 for 0.05 ns, followed by full system equilibration with no restraints for 0.5 ns. Finally, the MD simulation was performed at 300 K for 10 ns. Non-bonding interactions were calculated using a cutoff of 14 Å. The SHAKE algorithm44 was employed to restrain all bonds involving hydrogen atoms. Langevin dynamics45 was applied to regulate the temperature with a collision frequency of 2.0 ps−1. The pressure was controlled using the isotropic position scaling protocol. All MD simulations were performed using the AMBER14 program. The criteria of halogen bond was set according to Weiliang Zhu et al.3,13, as X···Y distances shorter than the sum of vdW radii, (d(Cl···O) < 3.27 Å, d(Cl···N) < 3.30 Å, d(Cl···S) < 3.55 Å, the C–X···Y angle β is larger than 140°. For C–X···π halogen bond, π systems from aromatic residues (Phe, Tyr, His, and Trp) are considered in this study with the following criteria: d(Cl···π) < 4.2 Å, α < 60°, and θ > 146° (Fig. 3).
Additional Information
How to cite this article: Jiang, S. et al. The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis. Sci. Rep. 6, 34750; doi: 10.1038/srep34750 (2016).
References
Hernandes, M. Z. et al. Halogen atoms in the modern medicinal chemistry: hints for the drug design. Current drug targets 11, 303–314 (2010).
Lu, Y. et al. Halogen bonding for rational drug design and new drug discovery. Expert opinion on drug discovery 7, 375–383 (2012).
Xu, Z. et al. Halogen bond: its role beyond drug–target binding affinity for drug discovery and development. Journal of chemical information and modeling 54, 69–78 (2014).
Metrangolo, P. & Resnati, G. Halogen bonding: a paradigm in supramolecular chemistry. Chemistry-a European Journal 7, 2511–2519 (2001).
Hardegger, L. A. et al. Systematic investigation of halogen bonding in protein–ligand interactions. Angewandte Chemie International Edition 50, 314–318 (2011).
Auffinger, P., Hays, F. A., Westhof, E. & Ho, P. S. Halogen bonds in biological molecules. Proceedings of the National Academy of Sciences of the United States of America 101, 16789–16794 (2004).
Matter, H. et al. Evidence for C-Cl/C-Br⋅⋅⋅ π Interactions as an Important Contribution to Protein–Ligand Binding Affinity. Angewandte Chemie 121, 2955–2960 (2009).
Andrea, V. R. The role of halogen bonding in inhibitor recognition and binding by protein kinases. Current topics in medicinal chemistry 7, 1336–1348 (2007).
Parisini, E., Metrangolo, P., Pilati, T., Resnati, G. & Terraneo, G. Halogen bonding in halocarbon–protein complexes: a structural survey. Chemical Society Reviews 40, 2267–2278 (2011).
Voth, A. R., Khuu, P., Oishi, K. & Ho, P. S. Halogen bonds as orthogonal molecular interactions to hydrogen bonds. Nature chemistry 1, 74–79 (2009).
Voth, A. R., Hays, F. A. & Ho, P. S. Directing macromolecular conformation through halogen bonds. Proceedings of the National Academy of Sciences 104, 6188–6193 (2007).
Bornscheuer, U. et al. Engineering the third wave of biocatalysis. Nature 485, 185–194 (2012).
Lu, Y., Wang, Y. & Zhu, W. Nonbonding interactions of organic halogens in biological systems: implications for drug discovery and biomolecular design. Physical Chemistry Chemical Physics 12, 4543–4551 (2010).
Wang, M.-X. Enantioselective Biotransformations of Nitriles in Organic Synthesis. Accounts of chemical research 48, 602–611 (2015).
Wang, H., Sun, H., Gao, W. & Wei, D. Efficient production of (R)-o-chloromandelic acid by recombinant Escherichia coli cells harboring nitrilase from Burkholderia cenocepacia J2315. Organic Process Research & Development 18, 767–773 (2013).
DeSantis, G. et al. Creation of a productive, highly enantioselective nitrilase through gene site saturation mutagenesis (GSSM). Journal of the American Chemical Society 125, 11476–11477 (2003).
Winkler, M., Knall, A. C., Kulterer, M. R. & Klempier, N. Nitrilases catalyze key step to conformationally constrained GABA analogous γ-amino acids in high optical purity. The Journal of organic chemistry 72, 7423–7426 (2007).
Ramteke, P. W., Maurice, N. G., Joseph, B. & Wadher, B. J. Nitrile‐converting enzymes: An eco‐friendly tool for industrial biocatalysis. Biotechnology and applied biochemistry 60, 459–481 (2013).
Bandarage, U. K. et al. Nitrosothiol esters of diclofenac: Synthesis and pharmacological characterization as gastrointestinal-sparing prodrugs. Journal of medicinal chemistry 43, 4005–4016 (2000).
Bousquet, A. & Musolino, A. Hydroxyacetic ester derivatives, preparation method and use as synthesis intermediates. US Patent patent (2003).
Li, X. et al. Research and comparison of the synthetic routes of indoxacarb. Modern Agrochemicals 8, 23–26 (2009).
Duan, Y. et al. Biocatalytic desymmetrization of 3-substituted glutaronitriles by nitrilases. A convenient chemoenzymatic access to optically active (S)-Pregabalin and (R)-Baclofen. Science China Chemistry 57, 1164–1171 (2014).
Yeom, S.-J., Lee, J.-K. & Oh, D.-K. A positively charged amino acid at position 129 in nitrilase from Rhodococcus rhodochrous ATCC 33278 is an essential residue for the activity with meta-substituted benzonitriles. FEBS letters 584, 106–110 (2010).
Zhang, L. et al. Structural insights into enzymatic activity and substrate specificity determination by a single amino acid in nitrilase from Syechocystis sp. PCC6803. Journal of structural biology 188, 93–101 (2014).
Sborgi, L. et al. Interaction Networks in Protein Folding via Atomic-Resolution Experiments and Long-Timescale Molecular Dynamics Simulations. Journal of the American Chemical Society 137, 6506–6516 (2015).
Koga, N. et al. Principles for designing ideal protein structures. Nature 491, 222–227 (2012).
Rago, F., Saltzberg, D., Allen, K. N. & Tolan, D. R. Enzyme substrate specificity conferred by distinct conformational pathways. Journal of the American Chemical Society 137, 13876–13886 (2015).
Fernandes, B. et al. Nitrile hydratase activity of a recombinant nitrilase. Advanced Synthesis & Catalysis 348, 2597–2603 (2006).
Ibrahim, M. A. AMBER empirical potential describes the geometry and energy of noncovalent halogen interactions better than advanced semiempirical quantum mechanical method PM6-DH2X. The Journal of Physical Chemistry B 116, 3659–3669 (2012).
Kolář, M. H. & Hobza, P. Computer Modeling of Halogen Bonds and Other σ-Hole Interactions. Chemical reviews 116, 5155–5187 (2016).
Cui, D. et al. A computational strategy for altering an enzyme in its cofactor preference to NAD (H) and/or NADP (H). FEBS Journal 282, 2339–2351 (2015).
Cui, D. et al. Computational design of short-chain dehydrogenase Gox2181 for altered coenzyme specificity. Journal of biotechnology 167, 386–392 (2013).
Zhang, L. et al. Structure, mechanism, and enantioselectivity shifting of lipase LipK107 with a simple way. Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics 1844, 1183–1192 (2014).
Qiu, J. et al. Cloning, overexpression, and characterization of a high enantioselective nitrilase from Sphingomonas wittichii RW1 for asymmetric synthesis of (R)-Phenylglycine. Applied biochemistry and biotechnology 173, 365–377 (2014).
Inc, A. S. Discovery Studio Modeling Environment, Release 4.0, http://accelrys.com/ (2007).
Bayly, C. I., Cieplak, P., Cornell, W. & Kollman, P. A. A well-behaved electrostatic potential based method using charge restraints for deriving atomic charges: the RESP model. The Journal of Physical Chemistry 97, 10269–10280 (1993).
Kolar, M. & Hobza, P. On Extension of the Current Biomolecular Empirical Force Field for the Description of Halogen Bonds. Journal of chemical theory and computation 8, 1325–1333, doi: 10.1021/ct2008389 (2012).
Ibrahim, M. A. Molecular mechanical study of halogen bonding in drug discovery. Journal of computational chemistry 32, 2564–2574 (2011).
Cornell, W. D. et al. A second generation force field for the simulation of proteins, nucleic acids, and organic molecules. Journal of the American Chemical Society 117, 5179–5197 (1995).
Wang, J., Wolf, R. M., Caldwell, J. W., Kollman, P. A. & Case, D. A. Development and testing of a general amber force field. Journal of computational chemistry 25, 1157–1174 (2004).
Case, D. A. et al. AMBER 14, University of California, San Francisco, http://ambermd.org/ (2014).
Case, D. A. et al. The Amber biomolecular simulation programs. Journal of computational chemistry 26, 1668–1688 (2005).
Andrea, T. A., Swope, W. C. & Andersen, H. C. The role of long ranged forces in determining the structure and properties of liquid water. The Journal of chemical physics 79, 4576–4584 (1983).
Ryckaert, J.-P., Ciccotti, G. & Berendsen, H. J. Numerical integration of the cartesian equations of motion of a system with constraints: molecular dynamics of n -alkanes. Journal of Computational Physics 23, 327–341 (1977).
Pastor, R. W., Brooks, B. R. & Szabo, A. An analysis of the accuracy of Langevin and molecular dynamics algorithms. Molecular Physics 65, 1409–1419 (1988).
Acknowledgements
This work was supported by National Natural Science Foundation of China (No. 31571786), National Basic Research Program of China (973) (No. 2012CB721003), Natural Science Foundation of Shanghai (No. 16ZR1449500), National Key Research Program of China (No. 2016YFA0501701), Special Program for Applied Research on Super Computation of the NSFC-Guangdong Joint Fund (the second phase), Open Funding Project of the State Key Laboratory of Bioreactor Engineering.
Author information
Authors and Affiliations
Contributions
D.W., L.Z. and S.J. conceived the experiments. S.J., D.C. and Z.Y. did the simulations. S.J. and J.L. conducted the experiments, B.G. analysed the results. All authors reviewed the manuscript.
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Electronic supplementary material
Rights and permissions
This work is licensed under a Creative Commons Attribution 4.0 International License. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in the credit line; if the material is not included under the Creative Commons license, users will need to obtain permission from the license holder to reproduce the material. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/
About this article
Cite this article
Jiang, S., Zhang, L., Cui, D. et al. The Important Role of Halogen Bond in Substrate Selectivity of Enzymatic Catalysis. Sci Rep 6, 34750 (2016). https://doi.org/10.1038/srep34750
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/srep34750
This article is cited by
-
Novel fluorinated quaternary ammonium salts and their in vitro activity as trypanocidal agents
Medicinal Chemistry Research (2019)
-
Synthesis and in vitro evaluation of novel N-cycloalkylcarbamates as potential cholinesterase inhibitors
Monatshefte für Chemie - Chemical Monthly (2017)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.