Theoretical Investigation On The Catalytic Mechanisms Of Hydroxynitrile Lyase And Two O-methyltransferases

Enzymes are biological catalysts, which catalyze most biochemical processes effectively and specially in living organisms. This is crucial for all forms of life, such as biosynthesis and metabolism. Detailed understanding enzyme action is therefore of widespread importance for exploring the rule of living action and promising routes to new drugs, analysis of the effects of genetic variation and mutation and design of new catalysts, and is also a problem of significance and challenge in biology. Experimentally, X-ray crystallography and NMR spectroscopy have revealed the atomic-level structure of many enzymes and provided suggestions of how reactions occur, and detailed kinetic and isotopic labeling study have defined many features of enzymatic reaction schemes. However, it is extremely difficult to study directly transient species such as transition states and reaction intermediates in enzymes using experimental techniques, and the ability to investigate these species is vital to understand how enzymes how work. Furthermore, the relation between the enzymatic structure and its catalytic functions remain controversial, and it has proved difficult to distinguish between alternative mechanisms and to analyse contributions to catalysis. Computer modeling methods that have emerged and is maturing rapidly can be used to interpret, complement and expand results obtained from experiment. In particular, modeling of enzyme reactions at the atomic level can offer uniquely detailed insights. Starting from experimentally obtained structure information, different reaction mechanism can be evaluated and transition states and reaction intermediates can be modeled. Quantum chemical methodology is today a very important tool in the elucidation of properties and reaction mechanisms of enzyme active sites. In this thesis, quantum chemistry, in particular the B3LYP density functional theory, is used to investigate catalytic mechanisms of Hydroxynitrile (HNL), Chalcone O-methyltransferase (ChOMT) and Isoflavone O-methyltransferase.1. Catalytic Mechanism of Hydroxynitrile Lyase from Hevea brasiliensis: A Theoretical InvestigationHydroxynitrile lyases, HNLs(EC 4.1.2.39), are important enzymes for the catabolism of cyanogenic glycosides during cyanogenesis and the metabolism of these compounds during seedling development in several species of cyanogenic plants. HNLs can catalyze the cleavage of cyanohydrins into hydrocyanic acid (HCN) and the corresponding aldehyde or ketone. The liberated HCN not only plays a significant defensive role in plant system against herbivores and microbial attack but also serves as a nitrogen source for biosynthesis of L-asparagine. In addition, by employing the reversible enzymatic reaction, HNLs are used as versatile biocatalysts for the enantioselective synthesis of chiral cyanohydrins, which are important synthetic intermediates in the fields of fine chemicals, pharmaceuticals, and agrochemicals. Therefore, as important enzymes and biocatalysts, HNLs have attracted considerable biologists′and chemists′attention over the past years.Density functional theory (DFT) calculations using the hybrid functional B3LYP have been performed to investigate the catalytic mechanism of hydroxynitrile lyase from Hevea brasiliensis (Hb-HNL). This enzyme catalyzes the cleavage of acetone cyanohydrin to hydrocyanic acid plus acetone. Two models (A and B) of the active site consisting of 105 and 155 atoms, respectively, were constructed on the basis of the crystal structure. Good consistency between the two models provides a verification of the proposed mechanism. Our calculations show that the catalytic reaction proceeds via three elementary steps: (1) deprotonation of the OH-Ser80 by His235 and concomitant abstraction of a proton from the substrate hydroxyl by Ser80; (2) the C-C bond cleavage of the acetone cyanohydrin; and (3) protonation of the cleaved cyanide by His235. The cleavage of the C-C bond is the rate-limiting step with the overall free energy barrier of 13.5 kcal/mol for relatively smaller model A (14.9 kcal/mol for a larger model B) in the protein environment, which is in good agreement with experimental rate. The present results give support to the previously proposed general acid/base catalytic mechanism, in which the catalytic triad acts as a general acid/base. Moreover, the calculated results for model C, with the positive charge of Lys236 removed from model A, show that Lys236 with the positive charge plays a vital role in lowering the reaction barrier of the rate-determining and helps in stabilizing the negatively charged CN- by forming a hydrogen bond with the substrate, consistent with the experimental analysis.2. Reaction Mechanism of IsoavoneO-methyltransferase:A Theoretical InvestigationMethylation of oxygen (O-methylation) is a universial process and plays a key role in lignin biosynthesis, stress tolerance, and disease resistance in plants. Plant O-methyltransferases (OMTs) constitute a large family of enzymes, which methylate the oxygen atom of a variety of secondary metabolites including phenylpropanoids, flavonoids, and alkaloids in plants. Isoavone O-methyltransferase (IOMT) belongs to S-adenosyl-L-methionine (SAM) dependent plant natural product O-methyltransferase(OMT) involved in secondary metabolism and is a key enzyme for the biosynthesis of the antimicrobial pterocarpan phytoalexin medicarpin, which is regarded as an important component of disease resistance response in alfalfa (Medicago sativa L.). In vivo, IOMT catalyzes the transfer of a methyl group from SAM to the B-ring 4'-hydroxyl group of the isoflavone daidzein, leading to the formation of S-adenosy-L-homocysteine (SAH) and formononetin, and the latter is an essential intermediate for synthesizing medicarpin in the isoflavonoid branch of the phenylpropanoid pathway. However, in vitro, the A-ring 7-hydroxyl group of the isoflavone daidzein is preferentially methylated, yielding the isoformononetin, which is rarely found in plants and has no known physiological function.The methyl-transfer reaction mechanism catalyzed by Isoavone O-methyl -transferase (IOMT) and the roles of some residues around the active site are investigated by employing density functional method with the models of different size. The calculations conform that the proton transfer from the substrate daidzein to His257 occurs spontaneously with no barrier when the substrate daidzein enters into the active site. The proton transfer barrierless is also certified that it is possible to occur by calculating the pKa values of Nεof His257 with or without Glu318. Then, the phenolate ion (strong nucleophile) abstracts the cationic methyl group from S-adenosyl-L-methionine (SAM) via a single SN2 step. On inclusion of salvation effects, the activation barrier is calculated to be 17.0 kcal/mol based on Model F, being in good agreement with the experimental value, and the reaction was exothermic by 12.8 kcal/mol. The calculated results by using several models with different size suggest that Glu318 and Asp288 play important roles in lowering the reaction barrier, while residues Asn258, Asn310, Met168 and Met311 may be responsible for constraining the spatial orientation of the reactant. In addition, the catalytic role of Glu318 would proceed through the electrostatic interaction between carboxyl group of Glu318 and the incipient imidazolium cation of His257, rather than the charge-relay mechanism suggest by experiment.3. Elucidation of the Methyl Transfer Mechanism Catalyzed by Chalcone O-methyltransferase: A Density Functional StudyChalcone O-methyltransferases (ChOMT) found in Medicago sativa L (alfalfa), is one of capable of S-adenosyl-L-methionine (SAM) dependent OMTs using isoliquiritigenin (4,2',4'-trihydroxychalcone) as substrate, yielding S-adenosyl-L-homocysteine (SAH) and 4,4'-dihydroxy-2'-methoxychalcone product. The latter product serves as a potent nodulation (nod) gene inducer of soil rhizobia and is the most efficient transcriptional activator of nod genes among the diverse compounds released from alfalfa roots. Also, through ChOMT regulating methylation of isoliquiritigenin can prevent the cyclization of isoliquiritigenin catalyzed by chalcone isomerase (CHI) to the flavanone liquiritigenin (7,4'-dihydroxyflavanone) , thus inhibiting the subsequent conversion of flavanones into a variety of structurally diverse natural products such as anthocyanins, flavonols, pterocarpans and isoflavones, etc..The mechanism of the methyl transfer catalyzed by chalcone O-methyltransferase (ChOMT) has been computationally investigated by employing the hybrid functional B3LYP. Two models (modelsⅠandⅡ) are devised based on the two conformations adopted by the substrate isoliquiritigenin within the active site in the X-ray structure, and the detailed reaction processes were obtained. The calculations show for both models the O-methylation reactions catalyzed by ChOMT take place via a two-step mechanism, in which the first step is a water-mediated deprotonation process, i.e., the proton is transferred from the 2'-hydroxyl group of the substrate to His278 through a water bridge, and the second step is the methyl transfer from SAM to the newly generated phenolate anion of the substrate via an SN2 mechanism. The rate-limiting step in the whole catalytic reaction is the methyl transfer, and the caluculated rate-determining barriers are 19.6 and 21.0 kcal/mol with solvent effects for conformations A and B, respectively, in very good agreement with the experimental value of 19.8 kcal/mol deduced from Isoavone O-methyl -transferase (IOMT). Conformation A is preferred for the reaction due to the lower rate-limiting barrier, while the contribution of conformation B to the catalytic reaction should not be ignored owing to the larger exothermicity.