Bacterial hydrolytic dehalogenases and related enzymes: occurrences, reaction mechanisms, and applications

Dehalogenases catalyze the cleavage of the carbon-halogen bond of organohalogen compounds. They have been attracting a great deal of attention partly because of their potential applications in the chemical industry and bioremediation. In this personal account, we describe occurrences, reaction mechanisms, and applications of bacterial hydrolytic dehalogenases and related enzymes, particularly L-2-haloacid dehalogenase, DL-2-haloacid dehalogenase, fluoroacetate dehalogenase, and 2-haloacrylate reductase. L-2-Haloacid dehalogenase is a representative enzyme of the haloacid dehalogenase (HAD) superfamily, which includes the P-type ATPases and other hydrolases. Structural and mechanistic analyses of this enzyme have yielded important insights into the mode of action of the HAD superfamily proteins. Fluoroacetate dehalogenase is unique in that it catalyzes the cleavage of the highly stable C--F bond of a fluorinated aliphatic compound. In the reactions of L-2-haloacid dehalogenase and fluoroacetate dehalogenase, the carboxylate group of Asp performs a nucleophilic attack on the alpha-carbon atom of the substrate, displacing the halogen atom. This mechanism is common to haloalkane dehalogenase and 4-chlorobenzoyl-CoA dehalogenase. DL-2-Haloacid dehalogenase is unique in that a water molecule directly attacks the substrate, displacing the halogen atom. The occurrence of 2-haloacrylate reductase was recently reported, revealing a new pathway for the degradation of unsaturated aliphatic organohalogen compounds.     

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.     

Multiple Reaction Pathways Operating in the Mechanism of Vinylogous Mannich‐Type Reaction Activated by a Water Molecule

A systematic search for reaction pathways for the vinylogous Mannich‐type reaction was performed by the artificial force induced reaction method. This reaction affords δ‐amino‐γ‐butenolide in one pot by mixing 2‐trimethylsiloxyfuran, imine, and water under solvent‐free conditions. Surprisingly, the search identified as many as five working pathways. Among them, two concertedly produce anti and syn isomers of the product. Another two give an intermediate, which is a regioisomer of the main product. This intermediate can undergo a retro‐Mannich reaction to give a pair of intermediates: an imine and 2‐furanol. The remaining pathway directly generates this intermediate pair. The imine and 2‐furanol easily react with each other to afford the product. Thus, all of these stepwise pathways finally converge to give the main product. The rate‐determining step of all five (two concerted and three stepwise) pathways have a common mechanism: concerted Si? O bond formation through the nucleophilic attack of a water molecule on the silicon atom followed by proton transfer from the water molecule to the imine. Therefore, these five pathways have comparable barriers and compete with each other.     

Hydrogen‐bonding interactions in the active site of a low molecular weight protein‐tyrosine phosphatase

Molecular dynamics simulation of the Michaelis complex, phospho‐enzyme intermediate, and the wild‐type and C12S mutant have been carried out to examine hydrogen‐bonding interactions in the active site of the bovine low molecular weight protein‐tyrosine phosphatase (BPTP). It was found that the S_γ atom of the nucleophilic residue Cys‐12 is ideally located at a position opposite from the phenylphosphate dianion for an inline nucleophilic substitution reaction. In addition, electrostatic and hydrogen‐bonding interactions from the backbone amide groups of the phosphate‐binding loop strongly stabilize the thiolate anion, making Cys‐12 ionized in the active site. In the phospho‐enzyme intermediate, three water molecules are found to form strong hydrogen bonds with the phosphate group. In addition, another water molecule can be identified to form bridging hydrogen bonds between the phosphate group and Asp‐129, which may act as the nucleophile in the subsequent phosphate hydrolysis reaction, with Asp‐129 serving as a general base. The structural difference at the active site between the wild‐type and C12S mutant has been examined. It was found that the alkoxide anion is significantly shifted toward one side of the phosphate binding loop, away from the optimal position enjoyed by the thiolate anion of the wild‐type enzyme in an S_N2 process. This, coupled with the high pK_a value of an alcoholic residue, makes the C12S mutant catalytically inactive. These molecular dynamics simulations provided details of hydrogen bonding interactions in the active site of BPTP, and a structural basis for further studies using combined quantum mechanical and molecular mechanical potential to model the entire dephosphorylation reaction by BPTP. © 2000 John Wiley & Sons, Inc. J Comput Chem 21: 1192–1203, 2000     

Total Enzymatic Synthesis of the Cholecystokinin Octapeptide (CCK‐8)

The enzymatic synthesis of the cholecystokinin octapeptide (CCK‐8) is reported. The target octapeptide CCK‐8 is the minimum active sequence with the same biological activity as naturally occurring cholecystokinin and is a potential therapeutic agent in the control of gastrointestinal function as well as a drug candidate for the treatment of epilepsy and obesity. The protected CCK‐8 was obtained by incubation of Bz‐Arg‐Asp(OEt)‐Tyr‐Met‐OAl and Gly‐Trp‐Met‐Asp(OMe)‐Phe‐NH₂ with immobilized α‐chymotrypsin. The Bz‐Arg group was used as an N‐terminal protecting group in the synthesis of the tripeptide fragment. The protected CCK‐8 was treated with trypsin to remove the Bz‐Arg group successfully. Free or immobilized enzymes were used as catalysts. The effect of the acyl donor ester structure, the C(α) protecting group of the nucleophile, reaction media, enzyme, and the carrier of the enzymes on the outcome of the coupling reaction was studied.     

Mechanism of the Tyrosine Ammonia Lyase Reaction—Tandem Nucleophilic and Electrophilic Enhancement by a Proton Transfer

Quantum mechanics/molecular mechanics calculations in tyrosine ammonia lyase (TAL) ruled out the hypothetical Friedel–Crafts (FC) route for ammonia elimination from L ‐tyrosine due to the high energy of FC intermediates. The calculated pathway from the zwitterionic L ‐tyrosine‐binding state (0.0 kcal mol^?1) to the product‐binding state ((E)‐coumarate+H₂N? MIO; ?24.0 kcal mol^?1; MIO=3,5‐dihydro‐5‐methylidene‐4H‐imidazol‐4‐one) involves an intermediate (IS, ?19.9 kcal mol^?1), which has a covalent bond between the N atom of the substrate and MIO, as well as two transition states (TS1 and TS2). TS1 (14.4 kcal mol^?1) corresponds to a proton transfer from the substrate to the N1 atom of MIO by Tyr300? OH. Thus, a tandem nucleophilic activation of the substrate and electrophilic activation of MIO happens. TS2 (5.2 kcal mol^?1) indicates a concerted C? N bond breaking of the N‐MIO intermediate and deprotonation of the pro‐S β position by Tyr60. Calculations elucidate the role of enzymic bases (Tyr60 and Tyr300) and other catalytically relevant residues (Asn203, Arg303, and Asn333, Asn435), which are fully conserved in the amino acid sequences and in 3D structures of all known MIO‐containing ammonia lyases and 2,3‐aminomutases.     

Exploring nucleoside hydrolase catalysis in silico: molecular dynamics study of enzyme-bound substrate and transition state

The mechanism of action of inosine-uridine nucleoside hydrolase has been investigated by long-term molecular dynamics (MD) simulation in TIP3P water using stochastic boundary conditions. Five MD studies have been performed with enzyme substrate complex (E.S), enzyme substrate complex with protonated His241 (EH.S), enzyme transition state complex (E.TS), enzyme transition state complex with protonated His241 (EH.TS), and His241Ala transition state complex E(H241A).TS. Special attention has been given to the role of His241, which has been considered as the general acid catalyst to assist departure of the leaving nucleobase on the basis of its location in the active site in the X-ray crystal structure (). Yet on the basis of the location in the active site, Tyr229 is closer to the aniline ring of pAPIR as compared to His241. On initiation of MD simulations, His241 does not approach the nucleobase in the structures of EH.S, E.S, EH.TS, and E.TS. In the solvated enzyme, Tyr229, which is a member of the hydrogen bonding network inosine O2'.Asp14.His241.Tyr229.inosine N7, serves as a proton source to the leaving nucleobase. The loss of significant activity of His241Ala mutant is shown to be related to the disruption of the above hydrogen bonded network and the distancing of Tyr229 from inosine N7. The structures of the enzyme complexes with substrate or TS are not visibly altered on protonation of His241, a most unusual outcome. The bell-shaped pH dependence upon pK(app)'s of 7.1 and 9.1 may be attributed to the necessity of the dissociation of Asp10 or Asp15 and the acid form of Tyr229, respectively. In TS, the residue Ile81 migrated closer, whereas Arg233 moved away from the nucleobase. The probability of ribooxocarbenium ion stabilization by Asn168 and Asp14 is discussed. The Asp14-CO(2)(-) is hydrogen bonded to the ribose 2'-OH for 96% of the MD simulation time. Nucleophilic addition of water138 to ribooxocarbenium ion is suggested to be assisted by the proton shuttle from water138 --> Asp10 --> Asp15 --> water pool. An anticorrelation motion between Tyr229-OH and Asn168-OD1 in EH.S and E.S is observed. The relationship of this anticorrelated motion to mechanism, if any, deserves further exploration, perhaps the formation of a near attack conformation.     

Time‐Resolved Crystallography of the Reaction Intermediate of Nitrile Hydratase: Revealing a Role for the Cysteinesulfenic Acid Ligand as a Catalytic Nucleophile

The reaction mechanism of nitrile hydratase (NHase) was investigated using time‐resolved crystallography of the mutant NHase, in which βArg56, strictly conserved and hydrogen bonded to the two post‐translationally oxidized cysteine ligands, was replaced by lysine, and pivalonitrile was the substrate. The crystal structures of the reaction intermediates were determined at high resolution (1.2–1.3 Å). In combination with FTIR analyses of NHase following hydration in H₂¹⁸O, we propose that the metal‐coordinated substrate is nucleophilically attacked by the O(SO^?) atom of αCys114‐SO^?, followed by nucleophilic attack of the S(SO^?) atom by a βArg56‐activated water molecule to release the product amide and regenerate αCys114‐SO^?.     

Catalytic Mechanism of the Arylsulfatase Promiscuous Enzyme from Pseudomonas Aeruginosa

To elucidate the working mechanism of the “broad substrate specificity” by the Pseudomonas aeruginosa aryl sulfatase (PAS) enzyme, we present here a full quantum chemical study performed at the density functional level. This enzyme is able to catalyze the hydrolysis of the original p‐nitrophenyl‐sulfate (PNPS) substrate and the promiscuous p‐nitrophenyl‐phosphate (PNPP) one with comparable reaction kinetics. Based on the obtained results, a multistep mechanism including activation of the nucleophile, the nucleophilic attack, and the cleavage of the S? O (P? O) bond is proposed. Regarding the phosphate monoester, the results indicate that only some steps of the promiscuous reaction are identical to those in the native process. Differences concern mainly the last step in which the His115 residue acts as a general base to accept the proton by the O atom of the FGly51 in the PNPS, whereas in PNPP, the Asp317 protonated residue works as a general acid to deliver a proton by a water molecule to the oxygen atom of the C? O bond. The shapes of the relative potential‐ energy surface (PES) are similar in the two examined cases but the rate‐determining step is different (nucleophile attack vs. nucleophile activation). The influence of the dispersion contributions on the investigated reactions was also taken into account.     

Theoretical Studies on the Binding Mode and Reaction Mechanism of TLP Hydrolase kpHIUH

In this work, we have investigated the binding conformations of the substrate in the active site of 5-HIU hydrolase kpHIUH and its catalytic hydrolysis mechanism. Docking calculations revealed that the substrate adopts a conformation in the active site with its molecular plane laying parallel to the binding interface of the protein dimer of kpHIUH, in which His7 and His92 are located adjacent to the hydrolysis site C6 and have hydrogen bond interactions with the lytic water. Based on this binding conformation, density functional theory calculations indicated that the optimal catalytic mechanism consists of two stages: (1) the lytic water molecule is deprotonated by His92 and carries out nucleophilic attack on C6=O of 5-HIU, resulting in an oxyanion intermediate; (2) by accepting a proton transferred from His92, C6–N5 bond is cleaved to completes the catalytic cycle. The roles of His7, His92, Ser108 and Arg49 in the catalytic reaction were revealed and discussed in detail.     

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     

Amino acids of ricin and its polypeptides

Ricin and its corresponding polypeptides (A & B chain) were purified from castor seed. The molecular weight of ricin subunits were 29,000 and 28,000 daltons. The amino acids in ricin determined were Asp45 The22 Ser40 Glu53 Cys4 Gly96 His5 Ile21 Leu33 Lys20 Met4 Phe13 Pro37 Tyr11 Ala45 Val23 Arg20 indicating that ricin contains approximately 516 amino acid residues. The amino acids of the two subunits of ricin A and B chains were Asp23 The12 Ser21 Glu29 Cys2 Gly48 His3 Ile12, Leu17 Lys10 Met2 Phe6 Pro17 Tyr7 Ala35 Val13 Arg13 while in B chain the amino acids were Asp22 The10 Ser19 Glu25 Cys2 Gly47 His1 Ile10, Leu15 Lys11 Met1 Phe7 Pro6 Tyr5 Ala32Val11 Arg10. The total helical content of ricin came around 53.6% which is a new observation.     

A New Mechanism for β‐Lactamases: Class D Enzymes Degrade 1β‐Methyl Carbapenems through Lactone Formation

β‐Lactamases threaten the clinical use of carbapenems, which are considered antibiotics of last resort. The classical mechanism of serine carbapenemase catalysis proceeds through hydrolysis of an acyl‐enzyme intermediate. We show that class D β‐lactamases also degrade clinically used 1β‐methyl‐substituted carbapenems through the unprecedented formation of a carbapenem‐derived β‐lactone. β‐Lactone formation results from nucleophilic attack of the carbapenem hydroxyethyl side chain on the ester carbonyl of the acyl‐enzyme intermediate. The carbapenem‐derived lactone products inhibit both serine β‐lactamases (particularly class D) and metallo‐β‐lactamases. These results define a new mechanism for the class D carbapenemases, in which a hydrolytic water molecule is not required.     

MC1R Gene Polymorphism Affects Skin Color and Phenotypic Features Related to Sun Sensitivity in a Population of French Adult Women

The melanocortin-1 receptor ( MC1R ) gene is known to play a major role in skin and hair pigmentation and to be highly polymorphic in Caucasians. This study was performed to investigate the relationships between MC1R gene polymorphisms and skin color in a large sample of French middle-aged Caucasian women. The codons 60 to 265 and the codon 294 of the MC1R gene were sequenced in 488 women. The skin color was measured on the inner side of the forearm using a spectrophotometric instrument. Fifteen variants were identified: Arg151Cys, Arg160Trp, Arg142His, Asp294His, Ile155Thr, Asp84Glu, Val60Leu, Val92Met, Arg163Gln, Ser83Pro, Thr95Met, Pro256Ser, Val265Ile, Ala166Ala and Gln233Gln. Women carrying Arg151Cys, Asp294His, Arg160Trp and Asp84Glu variants had a significantly higher reflectance in the red region, which indicates a lower level of functional melanin. This association was the most pronounced for women carrying Asp84Glu. In contrast, no significant difference was observed for other variants. Moreover, associations between MC1R polymorphisms and the risks of experiencing sunburn and of having freckles were found independently of skin color. Our findings support the hypothesis that MC1R polymorphisms do not necessarily alter the skin color but should sensitize the skin to UV-induced DNA damage.     

Theoretical investigation of the reaction mechanism of the dinuclear zinc enzyme dihydroorotase

The reaction mechanism of the dinuclear zinc enzyme dihydroorotase was investigated by using hybrid density functional theory. This enzyme catalyzes the reversible interconversion of dihydroorotate and carbamoyl aspartate. Two reaction mechanisms in which the important active site residue Asp250 was either protonated or unprotonated were considered. The calculations establish that Asp250 must be unprotonated for the reaction to take place. The bridging hydroxide is shown to be capable of performing nucleophilic attack on the substrate from its bridging position and the role of Zn(beta) is argued to be the stabilization of the tetrahedral intermediate and the transition state leading to it, thereby lowering the barrier for the nucleophilic attack. It is furthermore concluded that the rate-limiting step is the protonation of the amide nitrogen by Asp250 coupled with C-N bond cleavage, which is consistent with previous experimental findings from isotope labeling studies.     

Theoretical studies on the deacylation step of serine protease catalysis in the gas phase, in solution, and in elastase

The deacylation step of serine protease catalysis is studied using DFT and ab initio QM/MM calculations combined with MD/umbrella sampling calculations. Free energies of the entire reaction are calculated in the gas phase, in a continuum solvent, and in the enzyme elastase. The calculations show that a concerted mechanism in the gas phase is replaced by a stepwise mechanism when solvent effects or an acetate ion are added to the reference system, with the tetrahedral intermediate being a shallow minimum on the free energy surface. In the enzyme, the tetrahedral intermediate is a relatively stable species ( approximately 7 kcal/mol lower in energy than the transition state), mainly due to the electrostatic effects of the oxyanion hole and Asp102. It is formed in the first step of the reaction, as a result of a proton transfer from the nucleophilic water to His57 and of an attack of the remaining hydroxyl on the ester carbonyl. This is the rate-determining step of the reaction, which requires approximately 22 kcal/mol for activation, approximately 5 kcal/mol less than the reference reaction in water. In the second stage of the reaction, only small energy barriers are detected to facilitate the proton transfer from His57 to Ser195 and the breakdown of the tetrahedral intermediate. Those are attributed mainly to a movement of Ser195 and to a rotation of the His57 side chain. During the rotation, the imidazolium ion is stabilized by a strong H-bond with Asp102, and the C(epsilon)(1)-H...O H-bond with Ser214 is replaced by one with Thr213, suggesting that a "ring-flip mechanism" is not necessary as a driving force for the reaction. The movements of His57 and Ser195 are highly correlated with rearrangements of the binding site, suggesting that product release may be implicated in the deacylation process.     

A DFT study on the formation of a phosphohistidine intermediate in prostatic acid phosphatase

Histidine phosphatases are a class of enzymes that are characterized by the presence of a conserved RHGXRXP motif. This motif contains a catalytic histidine that is being phosphorylated in the course of a dephosphorylation reaction catalyzed by these enzymes. Prostatic acid phosphatase (PAP) is one such enzyme. The dephosphorylation of phosphotyrosine by PAP is a two-step process. The first step involves the transfer of a phosphate group from the substrate to the histidine (His12). The present study reports on the details of the first step of this reaction, which was investigated using a series of quantum chemistry calculations. A number of quantum models were constructed containing various residues that were thought to play a role in the mechanism. In all these models, the transition state displayed an associative character. The transition state is stabilized by three active site arginines (Arg11, Arg15, and Arg79), two of which belong to the aforementioned conserved motif. The work also demonstrated that His12 could act as a nucleophile. The enzyme is further characterized by a His257-Asp258 motif. The role of Asp258 has been elusive. In this work, we propose that Asp258 acts as a proton donor which becomes protonated when the substrate enters the binding pocket. Evidence is also obtained that the transfer of a proton from Asp258 to the leaving group is possibly mediated by a water molecule in the active site. The work also underlines the importance of His257 in lowering the energy barrier for the nucleophilic attack.     

Key role of active-site water molecules in bacteriorhodopsin proton-transfer reactions

The functional mechanism of the light-driven proton pump protein bacteriorhodopsin depends on the location of water molecules in the active site at various stages of the photocycle and on their roles in the proton-transfer steps. Here, free energy computations indicate that electrostatic interactions favor the presence of a cytoplasmic-side water molecule hydrogen bonding to the retinal Schiff base in the state preceding proton transfer from the retinal Schiff base to Asp85. However, the nonequilibrium nature of the pumping process means that the probability of occupancy of a water molecule in a given site depends both on the free energies of insertion of the water molecule in this and other sites during the preceding photocycle steps and on the kinetic accessibility of these sites on the time scale of the reaction steps. The presence of the cytoplasmic-side water molecule has a dramatic effect on the mechanism of proton transfer: the proton is channeled on the Thr89 side of the retinal, whereas the transfer on the Asp212 side is hindered. Reaction-path simulations and molecular dynamics simulations indicate that the presence of the cytoplasmic-side water molecule permits a low-energy bacteriorhodopsin conformer in which the water molecule bridges the twisted retinal Schiff base and the proton acceptor Asp85. From this low-energy conformer, proton transfer occurs via a concerted mechanism in which the water molecule participates as an intermediate proton carrier.