The Catalytic Mechanism of Fluoroacetate Dehalogenase: A Computational Exploration of Biological Dehalogenation

The biological dehalogenation of fluoroacetate carried out by fluoroacetate dehalogenase is discussed by using quantum mechanical/molecular mechanical (QM/MM) calculations for a whole‐enzyme model of 10 800 atoms. Substrate fluoroacetate is anchored by a hydrogen‐bonding network with water molecules and the surrounding amino acid residues of Arg105, Arg108, His149, Trp150, and Tyr212 in the active site in a similar way to haloalkane dehalogenase. Asp104 is likely to act as a nucleophile to attack the α‐carbon of fluoroacetate, resulting in the formation of an ester intermediate, which is subsequently hydrolyzed by the nucleophilic attack of a water molecule to the carbonyl carbon atom. The cleavage of the strong C? F bond is greatly facilitated by the hydrogen‐bonding interactions between the leaving fluorine atom and the three amino acid residues of His149, Trp150, and Tyr212. The hydrolysis of the ester intermediate is initiated by a proton transfer from the water molecule to His271 and by the simultaneous nucleophilic attack of the water molecule. The transition state and produced tetrahedral intermediate are stabilized by Asp128 and the oxyanion hole composed of Phe34 and Arg105.     

Substrate specificity of fluoroacetate dehalogenase: an insight from crystallographic analysis, fluorescence spectroscopy, and theoretical computations

The high substrate specificity of fluoroacetate dehalogenase was explored by using crystallographic analysis, fluorescence spectroscopy, and theoretical computations. A crystal structure for the Asp104Ala mutant of the enzyme from Burkholderia sp. FA1 complexed with fluoroacetate was determined at 1.2 ? resolution. The orientation and conformation of bound fluoroacetate is different from those in the crystal structure of the corresponding Asp110Asn mutant of the enzyme from Rhodopseudomonas palustris CGA009 reported recently (J. Am. Chem. Soc. 2011, 133, 7461). The fluorescence of the tryptophan residues of the wild-type and Trp150Phe mutant enzymes from Burkholderia sp. FA1 incubated with fluoroacetate and chloroacetate was measured to gain information on the environment of the tryptophan residues. The environments of the tryptophan residues were found to be different between the fluoroacetate- and chloroacetate-bound enzymes; this would come from different binding modes of these two substrates in the active site. Docking simulations and QM/MM optimizations were performed to predict favorable conformations and orientations of the substrates. The F atom of the substrate is oriented toward Arg108 in the most stable enzyme-fluoroacetate complex. This is a stable but unreactive conformation, in which the small O-C-F angle is not suitable for the S(N)2 displacement of the F(-) ion. The cleavage of the C-F bond is initiated by the conformational change of the substrate to a near attack conformation (NAC) in the active site. The second lowest energy conformation is appropriate for NAC; the C-O distance and the O-C-F angle are reasonable for the S(N) 2 reaction. The activation energy is greatly reduced in this conformation because of three hydrogen bonds between the leaving F atom and surrounding amino acid residues. Chloroacetate cannot reach the reactive conformation, due to the longer C-Cl bond; this results in an increase of the activation energy despite the weaker C-Cl bond.     

Mapping the reaction coordinates of enzymatic defluorination

The carbon-fluorine bond is the strongest covalent bond in organic chemistry, yet fluoroacetate dehalogenases can readily hydrolyze this bond under mild physiological conditions. Elucidating the molecular basis of this rare biocatalytic activity will provide the fundamental chemical insights into how this formidable feat is achieved. Here, we present a series of high-resolution (1.15-1.80 ?) crystal structures of a fluoroacetate dehalogenase, capturing snapshots along the defluorination reaction: the free enzyme, enzyme-fluoroacetate Michaelis complex, glycolyl-enzyme covalent intermediate, and enzyme-product complex. We demonstrate that enzymatic defluorination requires a halide pocket that not only supplies three hydrogen bonds to stabilize the fluoride ion but also is finely tailored for the smaller fluorine halogen atom to establish selectivity toward fluorinated substrates. We have further uncovered dynamics near the active site which may play pivotal roles in enzymatic defluorination. These findings may ultimately lead to the development of novel defluorinases that will enable the biotransformation of more complex fluorinated organic compounds, which in turn will assist the synthesis, detoxification, biodegradation, disposal, recycling, and regulatory strategies for the growing markets of organofluorines across major industrial sectors.     

Cloning of Haloacid Dehalogenase Gene from Bacillus Cereus Local Strain with the Addition of Restriction Sites

Previous studies had successfully isolated and characterized the haloacid dehalogenase gene from Bacillus cereus local strain, namely bcfd1 gene. In the further research, this gene would be sub cloned into expression vector in order to analyse its expression. However, this gene could not be directly sub cloned because it doesn’t have suitable restriction sites that facilitate correct orientation of cloning. Therefore, the addition of suitable restriction sites at both end of the gene was necessary. The research is started by designing specific pair of primers to amplify the bcfd1 gene from Bacillus cereus chromosome by adding EcoRI on forward primer and HindIII on reverse primer. The 870 bp of bcfd1 gene code for haloacid dehalogenase with suitable restiction sites has been successfully cloned into the pGEM-T Easy cloning vector. The recombinant clone that was obtained from screened by ampicillin resistant and ß-galactosidase activity was confirmed by size screening, restriction analysis, and re-PCR. This clone already to be performed for the further sub cloning process in order to get the right cloning direction.     

Molecular mechanical study of halogen bonding in drug discovery

A halogen bond is a noncovalent bond between a halogen atom (X) and a Lewis base (Y). This type of bond is attributed to the anisotropic distribution of the charge density on the halogen atom, resulting in the formation of a positive cap (called the σ-hole) centered on the A-X axis. The current research is the first reported molecular mechanical study of halogen bonding, the positive region centered on the halogen atom was represented by an extra-point (EP) of charge. The correlation between the X-EP distance and the X…Y bond length was explored to determine the optimal position of the EP. A test set of 27 halogen-containing molecules complexed to various Lewis bases was studied using molecular mechanical potentials. The molecular mechanical minimized halogen bond lengths and binding energies were in good agreement with the corresponding quantum mechanical values. The EP inclusion on the halogen atom resulted in an improvement in the accuracy of the electrostatic-potential derived charges. The solvation free energies of halobenzene molecules relative to benzene were calculated with and without EP inclusion to assess the accuracy of the developed approach. Molecular mechanical study of halo derivatives of benzotriazole complexed to cyclin-dependent protein kinase 2 (CDK2) was performed, and MM-PB(GB)SA binding energies were calculated as a case study in finding potent halogenated inhibitors that can serve as antitumor drugs.     

Whole-cell biodetection of halogenated organic acids

A whole-cell bacterial sensor system for short-chain halo-organic acids was constructed, using 2-chloropropionic acid (2-CPA) as a model pollutant. An Escherichia coli host was transformed with a moderate-copy plasmid containing a fusion of two foreign genetic elements: (a) a promoter-containing segment of the Pseudomonas DL-DEX (DL-2-haloacid dehalogenase) encoding gene and (b) bioluminescence (luxCDABE) genes of Photorhabdus luminescens. The resulting construct, named MT1, responded to the presence of 2-CPA by dose-dependent light emission, in a highly specific albeit a very insensitive manner. Thus, while the desired concept was successfully demonstrated, further genetic work is needed in order to make such a construct practical for environmental monitoring purposes.     

NLIP and HAD-like Domains of Pah1 and Lipin 1 Phosphatidate Phosphatases Are Essential for Their Catalytic Activities

Saccharomyces cerevisiae Pah1 phosphatidate phosphatase (PAP) catalyzes the dephosphorylation of phosphatidate to yield diacylglycerol, controlling phospholipids and triacylglycerol metabolisms. Pah1 and human Lipin 1 are intrinsically disordered proteins with 56% and 43% unfolded regions, respectively. Truncation analysis of the conserved and non-conserved regions showed that N- and C-conserved regions are essential for the catalytic activity of Pah1. PAP activities can be detected in the conserved N-terminal Lipin (NLIP) domain and C-terminal Lipin (CLIP)/haloacid dehalogenase (HAD)-like domain of Pah1 and Lipin 1, suggesting that the evolutionarily conserved domains are essential for the catalytic activity. The removal of disordered hydrophilic regions drastically reduced the protein solubility of Pah1. Thioredoxin is an efficient fusion protein for production of soluble NLIP–HAD recombinant proteins in Escherichia coli.     

Roles of K151 and D180 in L‐2‐haloacid dehalogenase from Pseudomonas sp. YL: Analysis by molecular dynamics and ab initio fragment molecular orbital calculations

L ‐2‐haloacid dehalogenase (L ‐DEX) catalyzes the hydrolytic dehalogenation of L ‐2‐haloalkanoic acids to produce the corresponding D ‐2‐hydroxyalkanoic acids. This enzyme is expected to be applicable to the bioremediation of environments contaminated with halogenated organic compounds. We analyzed the reaction mechanism of L ‐DEX from Pseudomonas sp. YL (L ‐DEX YL) by using molecular modeling. The complexes of wild‐type L ‐DEX YL and its K151A and D180A mutants with its typical substrate, L ‐2‐chloropropionate, were constructed by docking simulation. Subsequently, molecular dynamics (MD) and ab initio fragment molecular orbital (FMO) calculations of the complexes were performed. The ab initio FMO method was applied at the MP2/6‐31G level to estimate interfragment interaction energies. K151 and D180, which are experimentally shown to be important for enzyme activity, interact particularly strongly with L ‐2‐chloropropionate, catalytic water, nucleophile (D10), and with each other. Our calculations suggest that K151 stabilizes substrate orientation and balances the charge around the active site, while D180 stabilizes the rotation of the nucleophile D10, fixes catalytic water around D10, and prevents K151 from approaching D10. Further, D180 may activate catalytic water on its own or with K151, S175, and N177. These roles are consistent with the previous results. Thus, MD and ab initio FMO calculations are powerful tools for the elucidation of the mechanism of enzymatic reaction at the molecular level and can be applied to other catalytically important residues. The results obtained here will play an important role in elucidating the reaction mechanism and rational design of L ‐DEX YL with improved enzymatic activity or substrate specificity. © 2009 Wiley Periodicals, Inc. J Comput Chem, 2009     

Organic fluorines as halogen bond donors: Theoretical study and crystallographic evidence

Organic fluorines usually cannot act as halogen bond donors, as a result of the extreme electronegativity and least polarizability of the fluorine atom. However, when the electronegative ability of the substituents bound to the carbon atom is very strong, organic fluorines do show positive electrostatic potentials (ESPs) along the C? F bond and thus can form halogen bonds with electron donors. In this work, the effects of six different substituents, i.e., NO₂, CF₃, CN, COOH, CHO, and CCH, on the ESPs of the F atom were studied using the M06‐2x method. When two or three substituents with very strong electron‐withdrawing ability, such as NO₂ and CN, are linked to the C atom, positive ESPs take place on the outermost portion of the F atom. However, the ESPs remain negative along the C? F bond with the introduction of relatively weak electron‐withdrawing substituents, irrespective of the number of the subsituents. In addition, some complexes between fluorinated molecules with positive ESPs on the F atom and NH₃ were calculated, to characterize the structural and energetic features of these specific halogen bonds. Finally, some crystal structures extracted from the Cambridge Structural Database were selected to provide experimental evidence of these interactions involving the C? F bond. The results presented in this work are very important for the modern definition of halogen bonding as well as for the development of pharmaceuticals and a wide range of functional material. © 2015 Wiley Periodicals, Inc.     

Theoretical Studies on the Halogen Bond between Propargyl Radical and Dihalogen Molecules

MP2/aug‐cc‐pVDZ calculations are carried out on the geometries, vibrational frequencies, interaction energies and topological properties for the π‐type halogen‐bonded complexes between propargyl radical and dihalogen molecules ClF, BrF and BrCl. There are two kinds of geometries: complex ( a ) involves the interaction between the X (X=Cl, Br) atom and the midpoint of C(1) –C(2) bond, complex ( b ) involves the interaction between the X atom and C(3) atom. The lengths of the halogen bond, the frequencies of the halogen bond, the elongation extent of the X–Y (XY=ClF, BrF, BrCl) bond, topological parameters at the BCPs of the halogen bond and X–Y bond are all well consistent with the interaction energies. The interaction of complex ( a ) is stronger than that of complex ( b ); the interaction of propargyl···BrF is stronger than that of propargyl···ClF and propargyl···BrCl. For the complexes ( a ) and ( b ), the charge transfer is observed from propargyl radical to XY, the atomic energy, the dipolar polarization, and the volume of the halogen atom X decrease upon complex formation.     

Effect of electrostatic interaction on the mechanism of dehalogenation catalyzed by haloalkane dehalogenase

Theoretical calculation has been carried out for the nucleophilic displacement reaction of 1,2‐dichloroethane catalyzed by haloalkane dehalogenase. The results indicate that different hydrogen bond patterns of the oxyanion hole and the halide‐stabilizing residues play an important role in the dehalogenation reaction. They cause concertedly an earlier transition state (TS) with the activation barrier of 16.60 kcal/mol. The stabilization effect of Trp125 and Trp175 on chlorine atom in the TS is larger than that of the reactant complex by 15.67 kcal/mol so that, they make contribution to the stabilization of the TS. Moreover, the reaction shows the enzymatic action can be attributed to a combination of reactant‐state destabilization and transition‐state electrostatic stabilization. © 2011 Wiley Periodicals, Inc. Int J Quantum Chem, 2011     

A new unconventional halogen bond C?X···H?M between HCCX (X = Cl and Br) and HMH (M = Be and Mg): An ab initio study

In this article, a new type of halogen‐bonded complex YCCX···HMY (X = Cl, Br; M = Be, Mg; Y = H, F, CH₃) has been predicted and characterized at the MP2/aug‐cc‐pVTZ level. We named it as halogen‐hydride halogen bonding. In each YCCX···HMY complex, a halogen bond is formed between the positively charged X atom and the negatively charged H atom. This new kind of halogen bond has similar characteristics to the conventional halogen bond, such as the elongation of the C? X bond and the red shift of the C? X stretch frequency upon complexation. The interaction strength of this type of halogen bond is in a range of 3.34–10.52 kJ/mol, which is smaller than that of dihydrogen bond and conventional halogen bond. The nature of the electrostatic interaction in this type of halogen bond has also been unveiled by means of the natural bond orbital, atoms in molecules, and energy decomposition analyses. © 2009 Wiley Periodicals, Inc. J Comput Chem 2010     

Halogen‐ and Hydrogen‐Bonded Salts and Co‐crystals Formed from 4‐Halo‐2,3,5,6‐tetrafluorophenol and Cyclic Secondary and Tertiary Amines: Orthogonal and Non‐orthogonal Halogen and Hydrogen Bonding,and Synthetic Analogues of Halogen‐Bonded Biological Systems

Co‐crystallisation of, in particular, 4‐iodotetrafluorophenol with a series of secondary and tertiary cyclic amines results in deprotonation of the phenol and formation of the corresponding ammonium phenate. Careful examination of the X‐ray single‐crystal structures shows that the phenate anion develops a C?O double bond and that the C?C bond lengths in the ring suggest a Meissenheimer‐like delocalisation. This delocalisation is supported by the geometry of the phenate anion optimised at the MP2(Full) level of theory within the aug‐cc‐pVDZ basis (aug‐cc‐pVDZ‐PP on I) and by natural bond orbital (NBO) analyses. With sp² hybridisation at the phenate oxygen atom, there is strong preference for the formation of two non‐covalent interactions with the oxygen sp² lone pairs and, in the case of secondary amines, this occurs through hydrogen bonding to the ammonium hydrogen atoms. However, where tertiary amines are concerned, there are insufficient hydrogen atoms available and so an electrophilic iodine atom from a neighbouring 4‐iodotetrafluorophenate group forms an I???O halogen bond to give the second interaction. However, in some co‐crystals with secondary amines, it is also found that in addition to the two hydrogen bonds forming with the phenate oxygen sp² lone pairs, there is an additional intermolecular I???O halogen bond in which the electrophilic iodine atom interacts with the C?O π‐system. All attempts to reproduce this behaviour with 4‐bromotetrafluorophenol were unsuccessful. These structural motifs are significant as they reproduce extremely well, in low‐molar‐mass synthetic systems, motifs found by Ho and co‐workers when examining halogen‐bonding interactions in biological systems. The analogy is cemented through the structures of co‐crystals of 1,4‐diiodotetrafluorobenzene with acetamide and with N‐methylbenzamide, which, as designed models, demonstrate the orthogonality of hydrogen and halogen bonding proposed in Ho’s biological study.     

Some measures for making halogen bonds stronger than hydrogen bonds in H2CS-HOX (X = F, Cl, and Br) complexes

The properties and applications of halogen bonds are dependent greatly on their strength. In this paper, we suggested some measures for enhancing the strength of the halogen bond relative to the hydrogen bond in the H(2)CS-HOX (X = F, Cl, and Br) system by means of quantum chemical calculations. It has been shown that with comparison to H(2)CO, the S electron donor in H(2)CS results in a smaller difference in strength for the Cl halogen bond and the corresponding hydrogen bond, and the Br halogen bond is even stronger than the hydrogen bond. The Li atom in LiHCS and methyl group in MeHCS cause an increase in the strength of halogen bonding and hydrogen bonding, but the former makes the halogen bond stronger and the latter makes the hydrogen bond stronger. In solvents, the halogen bond in the Br system is strong enough to compete with the hydrogen bond. The interaction nature and properties in these complexes have been analyzed with the natural bond orbital theory.     

Co-existing Intermolecular Halogen Bonding and Hydrogen Bonding in the Compound Trans-5,10-bis（1-bromodifluoro-acetyl-1-ethoxycarbonyl-methylidene）thianthrene

Trans-5,10-bis(1-bromodifluoroacetyl-l-ethoxycarbonyl-methylidene)thianthrene (1b) was prepared from the reaction of BrCF2COC(N2)CO2Et with thianthrene. X-ray single crystal diffraction analysis showed that the intermolecular halogen bonding and hydrogen bonding coexisted in this compound. The bromine atom acted as an electron acceptor in the halogen bond and an electron donor in the hydrogen bond. It is the first example that the bromine atom acted as such a dual role in the hydrogen and halogen bond.     

Theoretical study on the hydrolysis mechanism of N,N-dimethyl-N'-(2-oxo-1, 2-dihydro-pyrimidinyl)formamidine: water-assisted mechanism and cluster-continuum model

The hydrolysis reaction of N,N-dimethyl-N'-(2-oxo-1, 2-dihydro-pyrimidinyl)formamidine (DMPFA), a model compound of the antivirus drug amidine-3TC (3TC = 2', 3'-dideoxy-3'-thiacytidine), is investigated by the hybrid density functional theory B3LYP/6-31+G (d,p) method. The hydrolysis reaction of the title compound is predicted to undergo via two pathways, each of which is a stepwise process. Path A is the addition of H2O to the C=N double bond in the amidine group to form a tetrahedral structure in its first step, and then the transfer of the H atom of hydroxyl leads to the corresponding products via four possible channels. Path B simultaneously involves the nucleophilic attack of H2O to the C atom of the C=N bond and the proton transfer to the N atom of amino group leading to the cleavage of the C-N single bond in the amidine group. The results indicate that path A is more favorable than path B in the gas phase. Moreover, to simulate the title reaction in aqueous solution, water-assisted mechanism and the cluster-continuum model, based on the SCRF/CPCM model, are taken into account in our work. The results indicate that it is rational for two water molecules served as a bridge to assist in the first step of path A and that cytosine rather than the cytosine-substituted formamide should be released from the tetrahedral intermediate via s six-membered cycle transition state (channel 2). Our calculations exhibit that the process toward the tetrahedral intermediate is the rate-determining step both in the gas phase and in aqueous solution.     

Weakly coordinating anions: crystallographic and NQR studies of halogen-metal bonding in silver, thallium, sodium, and potassium halomethanesulfonates

35Cl, (79,81)Br, and (127)I NQR (nuclear quadrupole resonance) spectroscopy in conjunction with X-ray crystallography is potentially one of the best ways of characterizing secondary bonding of metal cations such as Ag(+) to halogen donor atoms on the surfaces of very weakly coordinating anions. We have determined the X-ray crystal structure of Ag(O(3)SCH(2)Cl) (a = 13.241(3) A; b = 7.544(2) A; c = 4.925(2) A; orthorhombic; space group Pnma; Z = 4) and compared it with the known structure of Ag(O(3)SCH(2)Br) (Charbonnier, F.; Faure, R.; Loiseleur, H. Acta Crystallogr., Sect. B 1978, 34, 3598-3601). The halogen atom in each is apical (three-coordinate), being weakly coordinated to two silver ions. (127)I NQR studies on Ag(O(3)SCH(2)I) show the expected NQR consequences of three-coordination of iodine: substantially reduced NQR frequencies nu(1) and nu(2) and a fairly small NQR asymmetry parameter eta. The reduction of the halogen NQR frequency of the coordinating halogen atom in Ag(O(3)SCH(2)X) becomes more substantial in the series X = Cl < Br < I, indicating that the coordination to Ag(+) strengthens in this series, as expected from hard-soft acid-base principles. The numbers of electrons donated by the organic iodine atom to Ag(+) have been estimated; these indicate that the bonding to the cation is weak but not insignificant. We have not found any evidence for the bonding of these organohalogen atoms to another soft-acid metal ion, thallium. A scheme for recycling of thallium halide wastes is included.     

A density functional theory study on the catalytic mechanism of hydroxycinnamoyl‐CoA hydratase‐lyase

Hydroxycinnamoyl‐CoA hydratase‐lyase (HCHL), a particular member of the crotonase superfamily, catalyzes the bioconversion of feruloyl‐CoA to the important flavor and fragrance compound vanillin. In this article, the catalytic mechanism of HCHL has been studied by using hybrid density functional theory method with simplified models. The calculated results reveal that the mechanism involves the hydration of the C?C double bond of feruloyl‐CoA and thence the cleavage of C? C single bond of β‐hydroxythioester. The hydration step is a typical concerted process, whereas C? C bond cleavage follows a concerted but asynchronous mechanism. The calculated energy barrier of hydration reaction is only slightly lower than that of cleavage process, implying both of two processes are rate limiting. By using three substrate analogs, the substrate specificity of HCHL was further examined. It is found that the p‐hydroxyl group of aromatic ring is necessary for the catalytic reaction. © 2013 Wiley Periodicals, Inc.     

The competition of Y⋯o and X⋯n halogen bonds to enhance the group V σ‐hole interaction in the NCY⋯oPH3⋯NCX and OPH3⋯NCX⋯NCY (X,YF,Cl, and Br) complexes

The positive electrostatic potentials (ESP) outside the σ‐hole along the extension of O? P bond in O?PH₃ and the negative ESP outside the nitrogen atom along the extension of the C? N bond in NCX could form the Group V σ‐hole interaction O?PH₃?NCX. In this work, the complexes NCY?O?PH₃?NCX and O?PH₃?NCX?NCY (X, Y?F, Cl, Br) were designed to investigate the enhancing effects of Y?O and X?N halogen bonds on the P?N Group V σ‐hole interaction. With the addition of Y?O halogen bond, the V _{S, max} values outside the σ‐hole region of O?PH₃ becomes increasingly positive resulting in a stronger and more polarizable P?N interaction. With the addition of X?N halogen bond, the V _{S, min} values outside the nitrogen atom of NCX becomes increasingly negative, also resulting in a stronger and more polarizable P?N interaction. The Y?O halogen bonds affect the σ‐hole region (decreased density region) outside the phosphorus atom more than the P?N internuclear region (increased density region outside the nitrogen atom), while it is contrary for the X?N halogen bonds. © 2015 Wiley Periodicals, Inc.     

Competition between halogen bond and hydrogen bond in complexes of superalkali Li3S and halogenated acetylene XCCH (X = F,Cl, Br,and I)

Complexes of superalkali Li₃S and XCCH (X = F, Cl, Br, and I) have been studied with theoretical calculations at the MP2/aug‐cc‐pVTZ level. Three types of structures are found: (A) the X atom combines with the S atom through a halogen bond; (B) the X atom interacts with the π electron of Li₃S by a π halogen bond; (C) the H atom combines with the S atom through a hydrogen bond. For A and B, a heavier halogen atom makes the interaction stronger, while for C, the change of interaction energy is not obvious, showing a small dependence on the nature of the X atom in HCCX. A is more stable than B and their difference in stability decreases as X varies from Cl to I. For the F and Cl complexes, A is weaker than C, however, the former is stronger than the latter in the Br and I complexes. The above three types of interactions have been analyzed by means of electron localization function, electron density difference, and energy decomposition, and the results show that they have similar nature and features with conventional interactions. © 2014 Wiley Periodicals, Inc.