人工设计逆醛缩酶RA95.5-8F催化β-羟基酮C—C裂解的理论研究

采用量子力学与分子力学组合(QM/MM)方法对人工设计逆醛缩酶RA95.5-8F催化β-羟基酮化合物裂解反应的机理进行了研究.结果表明,裂解反应主要包括赖氨酸Lys1083对底物的亲核进攻、Schiff碱形成、烯胺水解及C—N断裂等过程, C—N键裂解生成丙酮为整个反应的决速步骤,能垒为106.27 kJ/mol;活性中心的赖氨酸Lys1083、酪氨酸Tyr1051、天冬酰胺Asn1110和酪氨酸Tyr1180构成一个催化四联体, Lys1083通过与底物形成席夫碱对底物进行活化, Tyr1051作为催化酸碱参与质子转移过程,催化四联体的氢键网络有利于反应过渡态的稳定并使R-构型的底物更容易结合在活性位点,导致RA95.5-8F对R构型底物具有高的选择性和催化活性.     

How does the cAMP-dependent protein kinase catalyze the phosphorylation reaction: an ab initio QM/MM study

We have carried out density functional theory QM/MM calculations on the catalytic subunit of cAMP-dependent protein kinase (PKA). The QM/MM calculations indicate that the phosphorylation reaction catalyzed by PKA is mainly dissociative, and Asp166 serves as the catalytic base to accept the proton delivered by the substrate peptide. Among the key interactions in the active site, the Mg(2+) ions, glycine rich loop, and Lys72 are found to stabilize the transition state through electrostatic interactions. On the other hand, Lys168, Asn171, Asp184, and the conserved waters bound to Mg(2+) ions do not directly contribute to lower the energy barrier of the phosphorylation reaction, and possible roles for these residues are proposed. The QM/MM calculations with different QM/MM partition schemes or different initial structures yield consistent results. In addition, we have carried out 12 ns molecular dynamics simulations on both wild type and K168A mutated PKA, respectively, to demonstrate that the catalytic role of Lys168 is to keep ATP and substrate peptide in the near-attack reactive conformation.     

New insights into the mechanism of the Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase from S. enterica: a theoretical study

The reaction pathway of Schiff base hydrolysis catalyzed by type I dehydroquinate dehydratase (DHQD) from S. enterica has been studied by performing molecular dynamics (MD) simulations and density functional theory (DFT) calculations and the corresponding potential energy profile has also been identified. On the basis of the results, the catalytic hydrolysis process for the wild-type enzyme consists of three major reaction steps, including nucleophilic attack on the carbon atom involved in the carbon-nitrogen double bond of the Schiff base intermediate by a water molecule, deprotonation of the His143 residue, and dissociation between the product and the Lys170 residue of the enzyme. The remarkable difference between this and the previously proposed reaction mechanism is that the second step here, absent in the previously proposed reaction mechanism, plays an important role in facilitating the reaction through a key proton transfer by the His143 residue, resulting in a lower energy barrier. Comparison with our recently reported results on the Schiff base formation and dehydration processes clearly shows that the Schiff base hydrolysis is rate-determining in the overall reaction catalyzed by type I DHQD, consistent with the experimental prediction, and the calculated energy barrier of ～16.0 kcal mol(-1) is in good agreement with the experimentally derived activation free energy of ～14.3 kcal mol(-1). When the imidazole group of His143 residue is missing, the Schiff base hydrolysis is initiated by a hydroxide ion in the solution, rather than a water molecule, and both the reaction mechanism and the kinetics of Schiff base hydrolysis have been remarkably changed, clearly elucidating the catalytic role of the His143 residue in the reaction. The new mechanistic insights obtained here will be valuable for the rational design of high-activity inhibitors of type I DHQD as non-toxic antimicrobials, anti-fungals, and herbicides.     

Substrate‐Guided Front‐Face Reaction Revealed by Combined Structural Snapshots and Metadynamics for the Polypeptide N‐Acetylgalactosaminyltransferase 2

The retaining glycosyltransferase GalNAc‐T2 is a member of a large family of human polypeptide GalNAc‐transferases that is responsible for the post‐translational modification of many cell‐surface proteins. By the use of combined structural and computational approaches, we provide the first set of structural snapshots of the enzyme during the catalytic cycle and combine these with quantum‐mechanics/molecular‐mechanics (QM/MM) metadynamics to unravel the catalytic mechanism of this retaining enzyme at the atomic‐electronic level of detail. Our study provides a detailed structural rationale for an ordered bi–bi kinetic mechanism and reveals critical aspects of substrate recognition, which dictate the specificity for acceptor Thr versus Ser residues and enforce a front‐face S_Ni‐type reaction in which the substrate N‐acetyl sugar substituent coordinates efficient glycosyl transfer.     

Catalytic mechanism of 6-phosphogluconate dehydrogenase: a theoretical investigation

Density functional calculations are employed to theoretically explore the mechanism of all elementary reaction steps involved in the catalytic reaction of 6-phosphogluconate dehydrogenase (6PGDH). The model systems we choose for the enzyme contain the essential parts of the cofactor (NADP+), the substrate 6-phosphogluconate (6PG), and some key residues (Lys183 and Glu190) in the active site of sheep liver 6PGDH. The effect of the apoenzyme electrostatic environment on the studied reaction is treated by the self-consistent reaction-field method. Our calculations demonstrate that the first step of the catalytic reaction is the formation of a 3-keto 6PG intermediate, which proceeds through a concerted transition state involving a hydride transfer from 6PG to NADP+, and a proton transfer from 6PG to Lys183. The second step is the elimination of a CO2 molecule from 6-PG, concomitant with a proton transfer from Lys183 to 6-PG. In the final step, a concerted double proton transfer (one from Glu190 to the substrate, another from the substrate to Lys183) results in the final product, the keto form of ribulose 5-phosphate (Ru5P). The rate-limiting step is the formation of a 3-keto 6PG intermediate, with a free energy barrier of 22.7 kcal/mol at room temperature in the protein environment, and all three steps are calculated to be thermodynamically favorable. These results are in good agreement with the general acid/general base mechanism suggested from previous experiments for the 6PGDH reaction.     

Revisiting the Origin of Bacterial Bioluminescence: QM/MM Study on Oxygenation Reaction of Reduced Flavin in Protein

Bacterial bioluminescence is initiated by the oxygenation reaction of reduced flavin mononucleotide in luciferase. This enzymatic oxygenation occurs in a wide range of biological processes including cellular redox metabolism, biocatalysis, biosynthesis and homeostasis. However, little is known about the mechanism of the enzymatic reaction between singlet reduced flavin and triplet oxygen. To explore the enigmatic oxygenation, for the first time, the reaction of reduced flavin anion with oxygen was studied in bacterial luciferase by a combined quantum mechanics and molecular mechanics method as well as molecular dynamics simulation. The calculated results demonstrate that the reaction proceeds via a proton-coupled electron transfer (PCET) pathway, and the essential αHis44 acts as a catalytic acid to provide the proton. The currently proposed PCET mechanism clearly describes the initial steps of bacterial bioluminescence, and could be suitable for the other flavin oxygenation reactions in enzymes.     

Exploring the influence of conserved lysine69 on the catalytic activity of the helicobacter pylori shikimate dehydrogenase: A combined QM/MM and MD simulations

Shikimate dehydrogenase (SDH) catalyzes the reversible, NADPH-dependent reduction of 3-dehydroshikimate to shikimate, involved in the shikimate pathway. This pathway has emerged as an important target for the development of antimicrobial agent. Structural and functional analyses suggest that the conserved Lys69 plays an important role in the catalytic activity of Helicobacter pylori (H. pylori) SDH. However, the detailed mechanism how mutation of Lys69 affects the catalytic activity of H. pylori SDH remains unclear. Here, two-layered ONIOM-based quantum mechanics/molecular mechanics (QM/MM) calculation and molecular dynamics (MD) simulations were performed to explore the role of Lys69 in the H. pylori SDH. Our results showed that in addition to act as a catalytic base, the conserved Lys69 plays an additional, important role in the maintenance of the substrate shikimate in the active site, facilitating the catalytic reaction between the cofactor NADP⁺ and shikimate. Mutation of Lys69 triggers the movement of shikimate away from the active site of SDH, thereby disrupting the catalytic activity. This result can advance our understanding the catalytic mechanism of SDH family, which may benefit of the rational design of SDH inhibitors.     

The reaction mechanism for dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica

The fundamental reaction mechanism for the dehydration process catalyzed by type I dehydroquinate dehydratase from Gram-negative Salmonella enterica has been studied by density functional theory calculations. The results indicate that the dehydration process undergoes a two-step cis-elimination mechanism, which is different from the previously proposed one. The catalytic roles of both the highly conserved residue His143 and the Schiff base formed between the substrate and Lys170 have also been elucidated. The structural and mechanistic insight presented here may direct the design of type I dehydroquinate dehydratase enzyme inhibitors as non-toxic antimicrobials, anti-fungals, and herbicides.     

Computer simulation of the interaction of Cu(I) with cys residues at the binding site of the yeast metallochaperone Cu(I)-Atx1

The copper binding site and electronic structure of the metallochaperone protein Atx1 were investigated using the combination of quantum mechanics methods and molecular mechanics methods in the ONIOM(QM:MM) scheme at the density functional theory (DFT) B3LYP/ 6-31G(d):AMBER level. The residues in the binding site, -Met13-Thr14-Cys15-Cu(I)-Cys18-Gly17-Ser16-, were modeled with QM and the rest of the residues with MM. Our results indicate that the structure for Cu(I)-Atx1 has the copper atom coordinated to two sulfur atoms from Cys15 (2.110 A) and Cys18 (2.141 A) with an angle S-Cu(I) -S of 166 degrees . The potential energy surface of the copper atom is used to estimate its binding energy and the force field for the copper ligands. The potential surface is shallow for the bending mode S-Cu-S, which explains the origin of the disorder observed in crystallographic and nuclear magnetic resonance studies. Using molecular dynamics for Cu(I)-Atx1 in a box of water molecules and in vacuum, with the force field derived in this work, we observed a correlated motion between the side chains of Thr14 and of Lys65 which enhances distortions in the S-Cu-S geometry. The results are compared with recent experiments and the previous models. The vibrational spectra for the copper ligands and for the residues in the binding site were computed. The localized modes for the copper ligands and the amide bands were assigned. The presence of the copper atom affects the amide bands' frequencies of the residues Cys15 and Cys18, giving resolved bands that can be used to sense changes in the binding site upon translocation of copper atom or interaction with target proteins. Furthermore, the EXAFS (extended X-ray absorption fine structure) spectrum of the proposed structure for Cu(I)-Atx1 was calculated and reproduced the experiments fairly well.     

Computational Characterization of Reactive Intermediates of Carbon Monoxide Dehydrogenase

The catalytic oxidation of CO to CO2 by carbon monoxide dehydrogenases has been explored theoretically, and a large C-cluster model including the metal core [Ni-4Fe-4S] and surrounding residues and crystal water molecules was used in density functional calculations. The key species involved in the oxidation of CO at the C-cluster, Cred1, Cred2 and Cint, have been elucidated. On the basis of computational results, the plausible enzymatic mechanism for the CO oxidation was proposed. In the catalytic reaction, the first proton abstraction from the Fe（1）-bound water leads to a precursor to accommodate CO binding and the subsequently consecutive proton transfers from the metal-bound carboxylate to the amino acid residues facilitate the release of CO2. The hydrogen-bond network around the C-cluster formed by conserved residues His93, His96, Glu299, Lys563, and four water molecules in the active domain plays an important role in proton transfer and intermediate stabilization. Predicted geometries of key species show good agreement with the reported crystal structures.     

Promiscuity in alkaline phosphatase superfamily. Unraveling evolution through molecular simulations

We here present a theoretical study of the alkaline hydrolysis of a phosphodiester (methyl p-nitrophenyl phosphate or MpNPP) in the active site of Escherichia coli alkaline phosphatase (AP), a monoesterase that also presents promiscuous activity as a diesterase. The analysis of our simulations, carried out by means of molecular dynamics (MD) simulations with hybrid quantum mechanics/molecular mechanics (QM/MM) potentials, shows that the reaction takes place through a D(N)A(N) or dissociative mechanism, the same mechanism employed by AP in the hydrolysis of monoesters. The promiscuous activity observed in this superfamily can be then explained on the basis of a conserved reaction mechanism. According to our simulations the specialization in the hydrolysis of phosphomonoesters or phosphodiesters, developed in different members of the superfamily, is a consequence of the interactions established between the protein and the oxygen atoms of the phosphate group and, in particular, with the oxygen atom that bears the additional alkyl group when the substrate is a diester. A water molecule, belonging to the coordination shell of the Mg(2+) ion, and residue Lys328 seem to play decisive roles stabilizing a phosphomonoester substrate, but the latter contributes to increase the energy barrier for the hydrolysis of phosphodiesters. Then, mutations affecting the nature or positioning of Lys328 lead to an increased diesterase activity in AP. Finally, the capacity of this enzymatic family to catalyze the reaction of phosphoesters having different leaving groups, or substrate promiscuity, is explained by the ability of the enzyme to stabilize different charge distributions in the leaving group using different interactions involving either one of the zinc centers or residues placed on the outer side of the catalytic site.     

L1 β-Lactamase催化反应机理研究

用混合量子力学和分子力学(QM/MM)方法和密度泛函理论讨论了L1 β-Lactamase催化Nitrocefin水解的过程, 研究结果表明, 反应为多步反应: 第一步亲核进攻反应为反应的决速步骤, 并且伴随着酰胺键的断裂, 第二步反应为质子迁移反应. 同时讨论了金属锌在反应中的作用.     

Organocatalytic asymmetric tandem Michael-Henry reactions: a highly stereoselective synthesis of multifunctionalized cyclohexanes with two quaternary stereocenters

A novel organocatalytic asymmetric tandem Michael-Henry reaction catalyzed by 9-amino-9-deoxyepiquinine (VI) has been developed. The reaction was efficiently catalyzed by catalyst VI to give highly functionalized cyclohexanes with four stereogenic carbons including two quaternary stereocenters in excellent enantioselectivities (97 to >99% ee) and high diastereoselectivities (93:7-99:1 dr). Thus, the first organocatalytic asymmetric Henry reaction of common ketones as acceptors is shown.     

Artificial ribonucleases. 5. Synthesis and ribonuclease activity of tripeptides composed of amino acids involved in catalytic centers of natural ribonucleases

The characteristic features of the spatial arrangement of the main functional groups involved in catalytic centers of ribonucleases and nucleases were revealed by computer analysis of the catalytic centers of these enzymes. Based on the results of computer simulation, tripeptides containing Lys, Arg, His or Hia, Thr, and Asn in different combinations were synthesized. In these tripeptides, the distances between the corresponding functional groups are equal to those observed in natural enzymes. The efficacy of RNA cleavage with Arg- and His-containing tripeptides depends on their structure and correlates with the overall positive charge of these compounds. Of all the tripeptides under consideration, compounds bearing the overall charge of +4 exhibit the highest ribonuclease activity.     

Retaining glycosyltransferase mechanism studied by QM/MM methods: lipopolysaccharyl-α-1,4-galactosyltransferase C transfers α-galactose via an oxocarbenium ion-like transition state

Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.     

The α-amino group of the threonine substrate as the general base during tRNA aminoacylation: a new version of substrate-assisted catalysis predicted by hybrid DFT

Density functional theory-based methods in combination with large chemical models have been used to investigate the mechanism of the second half-reaction catalyzed by Thr-tRNA synthetase: aminoacyl transfer from Thr-AMP onto the (A76)3'OH of the cognate tRNA. In particular, we have examined pathways in which an active site His309 residue is either protonated or neutral (i.e., potentially able to act as a base). In the protonated His309-assisted mechanism, the rate-limiting step is formation of the tetrahedral intermediate. The barrier for this step is 155.0 kJ mol(-1), and thus, such a pathway is concluded to not be enzymatically feasible. For the neutral His309-assisted mechanism, two models were used with the difference being whether Lys465 was included. For either model, the barrier of the rate-limiting step is below the upper thermodynamic enzymatic limit of ~125 kJ mol(-1). Specifically, without Lys465, the rate-limiting barrier is 122.1 kJ mol(-1) and corresponds to a rotation about the tetrahedral intermediate C(carb)-OH bond. For the model with Lys465, the rate-limiting barrier is slightly lower and corresponds to the formation of the tetrahedral intermediate. Importantly, for both "neutral His309" models, the neutral amino group of the threonyl substrate directly acts as the proton acceptor; in the formation of the tetrahedral intermediate, the (A76)3'OH proton is directly transferred onto the Thr-NH(2). Therefore, the overall mechanism follows a general substrate-assisted catalytic mechanism.     

Neutral loss of water from the b ions with histidine at the C-terminus and formation of the c ions involving lysine side chains

Neutral loss of water from the amide bond induced by the His side chain has been reported. The proposed fragmentation pathway is a retro-Ritter reaction catalyzed by the imidazole nitrogen. In our MS/MS study of the neuropeptide GAHKNYLRFamide, we observed that the neutral loss of water from the b(3) ion is abundant. The b(3) ion has a His residue at the C-terminus. As reported previously, in the b ions with His at the C-terminus, the imidazole residue is connected to the carbonyl carbon to form a five-membered ring. Therefore, it is unlikely that the neutral loss of water from the b(3) ion is catalyzed by the imidazole nitrogen. Through MS2 and MS3 studies of a synthetic peptide standard AGHKLL and its chemically labeled and isotope-encoded forms, we discovered that the water loss from the b(3) ion involves the carbonyl group of His, the hydrogen connected to the alpha-carbon of Gly, and the amide hydrogen of His. We also discovered the formation of an unusual c(x) ion in peptides with a Lys or Arg residue at the (x + 1) position of the peptide.     

Catalytic specificity exhibited by p-sulfonatocalix[n]arenes in the methanolysis of N-acetyl-l-amino acids

Specific acid catalysis of p-sulfonatocalix[n]arenes (n = 4, Calix-S4; n = 6, Calix-S6; n = 8, Calix-S8) was observed in the alcoholysis of N-acetyl-l-amino acids in methanol. The methanolysis rates of basic amino acid substrates (His, Lys, and Arg) were markedly enhanced in the presence of Calix-Sn, as compared with rates observed with p-hydroxybenzenesulfonic acid (pHBS), which is a noncyclic analogue of Calix-Sn. This catalytic effect of Calix-Sn was not observed for the methanolysis of Phe, Tyr, and Trp substrates. On the other hand, (1)H NMR experiments following the effect of Calix-Sn on N-acetyl-l-amino acid substrates in CD(3)OD showed that the spectrum of a mixture of the His substrate with Calix-Sn was significantly different from the combined spectra of the respective compounds. These changes in spectra support the formation of an inclusion complex of Calix-Sn with basic amino acids. Furthermore, it was obvious that methanolysis of the His substrate catalyzed by Calix-S4 and Calix-S6 obeyed Michaelis-Menten kinetics. These results indicate that the catalytic activity of Calix-Sn originates from its forming a complex with specific substrates (basic amino acids), similar to enzymatic reactions.     

The Claisen condensation in biology

The mechanism for carbon-carbon bond formation used in the biosynthesis of natural products such as fatty acids and polyketides is a decarboxylating Claisen condensation. The enzymes that catalyze this reaction in various bacterial systems, collectively referred to as condensing enzymes, have been intensively studied in the past several decades, and members of the family have been crystallized. The condensing enzymes share a common 3-dimensional fold, first described for the biosynthetic thiolase I that catalyzes a non-decarboxylating Claisen condensation, although they share little similarity at the amino acid level. Their active sites, however, possess significant similarities. The initiation condensing enzymes use CoA primers and possess a catalytic triad of Cys, His, Asn; and the elongating condensing enzymes that exclusively use ACP thioesters have a triad of Cys, His, His. These active site differences affect the sensitivity of the respective enzymes to the antibiotics thiolactomycin and cerulenin. Different reaction mechanisms have been proposed for the condensing enzymes. This review covers the recent structural and mechanistic data to see if a unifying hypothesis for the reaction mechanism catalyzed by this important family of enzymes can be established.     

Zinc‐Catalyzed Alkene Cyclopropanation through Zinc Vinyl Carbenoids Generated from Cyclopropenes

The zinc‐catalyzed reaction of cyclopropenes with alkenes leading to vinylcyclopropane derivatives is reported. A broad range of alkenes (including highly substituted or functionalized alkenes) is compatible with this protocol. On the basis of trapping experiments and computational studies, this cyclopropanation reaction is proposed to proceed through initial formation of an electrophilic zinc vinyl carbenoid intermediate, which may be involved in a concerted cyclopropanation reaction. The reported protocol represents an unprecedented and simple strategy for the catalytic generation of zinc vinyl carbenoids, which are promising intermediates in organic synthesis.