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.     

Structural Snapshots of α‐1,3‐Galactosyltransferase with Native Substrates: Insight into the Catalytic Mechanism of Retaining Glycosyltransferases

Glycosyltransferases (GTs) are a key family of enzymes that catalyze the synthesis of glycosidic bonds in all living organisms. The reaction involves the transfer of a glycosyl moiety and can proceed with retention or inversion of the anomeric configuration. To date, the catalytic mechanism of retaining GTs is a topic of great controversy, particularly for those enzymes containing a putative nucleophilic residue in the active site, for which the occurrence of a double‐displacement mechanism has been suggested. We report native ternary complexes of the retaining glycosyltransferase α‐1,3‐galactosyltransferase (α3GalT) from Bos taurus , which contains such a nucleophile in the active site, in a productive mode for catalysis in the presence of its sugar donor UDP‐Gal, the acceptor substrate lactose, and the divalent cation cofactor. This new experimental evidence supports the occurrence of a front‐side substrate‐assisted S_Ni‐type reaction for α3GalT, and suggests a conserved common catalytic mechanism among retaining GTs.     

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.     

Quantum Mechanics/Molecular Mechanics Study on the Oxygen Binding and Substrate Hydroxylation Step in AlkB Repair Enzymes

AlkB repair enzymes are important nonheme iron enzymes that catalyse the demethylation of alkylated DNA bases in humans, which is a vital reaction in the body that heals externally damaged DNA bases. Its mechanism is currently controversial and in order to resolve the catalytic mechanism of these enzymes, a quantum mechanics/molecular mechanics (QM/MM) study was performed on the demethylation of the N¹‐methyladenine fragment by AlkB repair enzymes. Firstly, the initial modelling identified the oxygen binding site of the enzyme. Secondly, the oxygen activation mechanism was investigated and a novel pathway was found, whereby the catalytically active iron(IV)–oxo intermediate in the catalytic cycle undergoes an initial isomerisation assisted by an Arg residue in the substrate binding pocket, which then brings the oxo group in close contact with the methyl group of the alkylated DNA base. This enables a subsequent rate‐determining hydrogen‐atom abstraction on competitive σ‐ and π‐pathways on a quintet spin‐state surface. These findings give evidence of different locations of the oxygen and substrate binding channels in the enzyme and the origin of the separation of the oxygen‐bound intermediates in the catalytic cycle from substrate. Our studies are compared with small model complexes and the effect of protein and environment on the kinetics and mechanism is explained.     

Revised mechanism of carboxylation of ribulose‐1,5‐biphosphate by rubisco from large scale quantum chemical calculations

Here, we describe a computational approach for studying enzymes that catalyze complex multi‐step reactions and apply it to Ribulose 1,5‐bisphosphate carboxylase–oxygenase (Rubisco), the enzyme that fixes atmospheric carbon dioxide within photosynthesis. In the 5‐step carboxylase reaction, the substrate Ribulose‐1,5‐bisphosphate (RuBP) first binds Rubisco and undergoes enolization before binding the second substrate, CO₂. Hydration of the RuBP.CO₂ complex is followed by C C bond scission and stereospecific protonation. However, details of the roles and protonation states of active‐site residues, and sources of protons and water, remain highly speculative. Large‐scale computations on active‐site models provide a means to better understand this complex chemical mechanism. The computational protocol comprises a combination of hybrid semi‐empirical quantum mechanics and molecular mechanics within constrained molecular dynamics simulations, together with constrained gradient minimization calculations using density functional theory. Alternative pathways for hydration of the RuBP.CO₂ complex and associated active‐site protonation networks and proton and water sources were investigated. The main findings from analysis of the resulting energetics advocate major revision to existing mechanisms such that: hydration takes place anti to the CO₂; both hydration and C C bond scission require early protonation of CO₂ in the RuBP.CO₂ complex; C C bond scission and stereospecific protonation reactions are concerted and, effectively, there is only one stable intermediate, the C3‐gemdiolate complex. Our main conclusions for interpreting enzyme kinetic results are that the gemdiolate may represent the elusive Michaelis–Menten‐like complex corresponding to the empirical K_m (=K_c) with turnover to product via bond scission concerted with stereospecific protonation consistent with the observed catalytic rate. © 2018 Wiley Periodicals, Inc.     

Kinetic study of 2‐propanol and benzyl alcohol oxidation by alkaline hexacyanoferrate(III) catalyzed by a terpyridyl ruthenium complex

The complex (Trpy)RuCl₃ (Trpy = 2,2′:6′,2″‐terpyridine) reacts with alkaline hexacyanoferrate(III) to form a terpyridyl ruthenium(IV)‐oxo complex that catalyzes the oxidation of 2‐propanol and benzyl alcohol by alkaline hexacyanoferrate(III). The reaction kinetics of this catalytic oxidation have been studied photometrically. The reaction rate shows a first‐order dependence on [RU(IV)], a zero‐order dependence on [hexacyanoferrate(III)], a fractional order in [substrate], and a fractional inverse order in [HO⁻]. The kinetic data suggest a reaction mechanism in which the catalytic species and its protonated form oxidize the uncoordinated alcohol in parallel slow steps. Isotope effects, substituent effects, and product studies suggest that both species oxidize alcohol through similar pericyclic processes. The reduced catalytic intermediates react rapidly with hexacyanoferrate(III) and hydroxide to reform the unprotonated catalytic species. © 2000 John Wiley & Sons, Inc. Int J Chem Kinet 32: 760–770, 2000     

Nitrogen‐doped C60 as a robust catalyst for CO oxidation

The O₂ activation and CO oxidation on nitrogen‐doped C₅₉N fullerene are investigated using first‐principles calculations. The calculations indicate that the C₅₉N fullerene is able to activate O₂ molecules resulting in the formation of superoxide species ( ) both kinetically and thermodynamically. The active superoxide can further react with CO to form CO₂ via the Eley–Rideal mechanism by passing a stepwise reaction barrier of only 0.20 eV. Ab initio molecular dynamics (AIMD) simulation is carried out to evidence the feasibility of the Eley–Rideal mechanism. In addition, the second CO oxidation takes place with the remaining atomic O without any activation energy barrier. The full catalytic reaction cycles can occur energetically favorable and suggest a two‐step Eley–Rideal mechanism for CO oxidation with O₂ catalyzed by the C₅₉N fullerene. The catalytic properties of high percentage nitrogen‐doped fullerene (C₄₈N₁₂) is also examined. This work contributes to designing higher effective carbon‐based materials catalysts by a dependable theoretical insight into the catalytic properties of the nitrogen‐doped fullerene. © 2017 Wiley Periodicals, Inc.     

Insights into the synthesis of lipopolysaccharide and antibiotics through the structures of two retaining glycosyltransferases from family GT4

Glycosyltransferases (GTs) catalyze the synthesis of the myriad glycoconjugates that are central to life. One of the largest families is GT4, which contains several enzymes of therapeutic significance, exemplified by WaaG and AviGT4. WaaG catalyses a key step in lipopolysaccharide synthesis, while AviGT4, produced by Streptomyces viridochromogenes, contributes to the synthesis of the antibiotic avilamycin A. Here we present the crystal structure of both WaaG and AviGT4. The two enzymes contain two "Rossmann-like" (beta/alpha/beta) domains characteristic of the GT-B fold. Both recognition of the donor substrate and the catalytic machinery is similar to other retaining GTs that display the GT-B fold. Structural information is discussed with respect to the evolution of GTs and the therapeutic significance of the two enzymes.     

A QM/MM Investigation of the Catalytic Mechanism of Metal‐Ion‐Independent Core 2 β1,6‐N‐Acetylglucosaminyltransferase

β1,6‐GlcNAc‐transferase (C2GnT) is an important controlling factor of biological functions for many glycoproteins and its activity has been found to be altered in breast, colon, and lung cancer cells, in leukemia cells, in the lymhomonocytes of multiple sclerosis patients, leukocytes from diabetes patients, and in conditions causing an immune deficiency. The result of the action of C2GnT is the core 2 structure that is essential for the further elongation of the carbohydrate chains of O‐glycans. The catalytic mechanism of this metal‐ion‐independent glycosyltransferase is of paramount importance and is investigated here by using quantum mechanical (QM) (density functional theory (DFT))/molecular modeling (MM) methods with different levels of theory. The structural model of the reaction site used in this report is based on the crystal structures of C2GnT. The entire enzyme–substrate system was subdivided into two different subsystems: the QM subsystem containing 206 atoms and the MM region containing 5914 atoms. Three predefined reaction coordinates were employed to investigate the catalytic mechanism. The calculated potential energy surfaces discovered the existence of a concerted S_N2‐like mechanism. In this mechanism, a nucleophilic attack by O6 facilitated by proton transfer to the catalytic base and the separation of the leaving group all occur almost simultaneously. The transition state for the proposed reaction mechanism at the M06‐2X/6‐31G** (with diffuse functions on the O1′, O5′, O_Glu, and O6 atoms) level was located at C1? O6=1.74 Å and C1? O1=2.86 Å. The activation energy for this mechanism was estimated to be between 20 and 29 kcal mol^?1, depending on the method used. These calculations also identified a low‐barrier hydrogen bond between the nucleophile O6H and the catalytic base Glu320, and a hydrogen bond between the N‐acetamino group and the glycosidic oxygen of the donor in the TS. It is proposed that these interactions contribute to a stabilization of TS and participate in the catalytic mechanism.     

The Catalytic Mechanism of the Marine‐Derived Macrocyclase PatGmac

Cyclic peptides are a class of compounds with high therapeutic potential, possessing bioactivities including antitumor and antiviral (including anti‐HIV). Despite their desirability, efficient design and production of these compounds has not been achieved to date. The catalytic mechanism of patellamide macrocyclization by the PatG macrocyclase domain has been computationally investigated by using quantum mechanics/molecular mechanics methodology, specifically ONIOM(M06/6‐311++G(2d,2p):ff94//B3LYP/6‐31G(d):ff94). The mechanism proposed herein begins with a proton transfer from Ser783 to His 618 and from the latter to Asp548. Nucleophilic attack of Ser783 on the substrate leads to the formation of an acyl–enzyme covalent complex. The leaving group Ala‐Tyr‐Asp‐Gly (AYDG) of the substrate is protonated by the substrate's N terminus, leading to the breakage of the P1?P1′ bond. Finally, the substrate's N terminus attacks the P1 residue, decomposing the acyl–enzyme complex forming the macrocycle. The formation and decomposition of the acyl–enzyme complex have the highest activation free energies (21.1 kcal mol^?1 and 19.8 kcal mol^?1 respectively), typical of serine proteases. Understanding the mechanism behind the macrocyclization of patellamides will be important to the application of the enzymes in the pharmaceutical and biotechnological industries.     

Solvent Impedes CO2 Cycloaddition on Metal–Organic Frameworks

The catalytic performance of metal–organic frameworks (MOFs) for the synthesis of cyclic carbonate from carbon dioxide and epoxides has been explored under solvent and solvent‐free conditions, respectively. It was found that MOF catalysts have significantly improved catalytic activities in solvent‐free CO₂ cycloaddition reactions than those in solvent. The mechanism was discussed with regard to the competition of solvent with substrate to adhere MOF catalysts during the reaction process.     

A quantum mechanics/molecular mechanics study on the hydrolysis mechanism of New Delhi metallo-β-lactamase-1

New Delhi metallo-β-lactamase-1 (NDM-1) has emerged as a major global threat to human health for its rapid rate of dissemination and ability to make pathogenic microbes resistant to almost all known β-lactam antibiotics. In addition, effective NDM-1 inhibitors have not been identified to date. In spite of the plethora of structural and kinetic data available, the accurate molecular characteristics of and details on the enzymatic reaction of NDM-1 hydrolyzing β-lactam antibiotics remain incompletely understood. In this study, a combined computational approach including molecular docking, molecular dynamics simulations and quantum mechanics/molecular mechanics calculations was performed to characterize the catalytic mechanism of meropenem catalyzed by NDM-1. The quantum mechanics/molecular mechanics results indicate that the ionized D124 is beneficial to the cleavage of the C–N bond within the β-lactam ring. Meanwhile, it is energetically favorable to form an intermediate if no water molecule coordinates to Zn2. Moreover, according to the molecular dynamics results, the conserved residue K211 plays a pivotal role in substrate binding and catalysis, which is quite consistent with previous mutagenesis data. Our study provides detailed insights into the catalytic mechanism of NDM-1 hydrolyzing meropenem β-lactam antibiotics and offers clues for the discovery of new antibiotics against NDM-1 positive strains in clinical studies.     

Selective aerobic oxidation of alcohols catalyzed by iron chloride hexahydrate/TEMPO in the presence of silica gel

An environmentally friendly and efficient process whereby FeCl₃?6H₂O/2,2,6,6‐tetramethylpiperidine N‐oxyl (TEMPO)‐catalyzed oxidation of alcohols to the corresponding aldehydes and ketones is accomplished in the presence of silica gel using molecular oxygen or air as the terminal oxidant. The electron‐deficient benzyl alcohol was smoothly oxidized to the corresponding aldehydes with up to 99% isolated yield. It was found that silica gel not only could enhance the catalytic reaction rate but also increase the selectivity for the product. The high performance of FeCl₃?6H₂O/TEMPO catalyst system in the presence of silica gel might be attributed to the surface silanol groups. UV–visible spectra analysis showed that the Fe (III)–TEMPO complex could serve as the active intermediate species in the present catalytic system. A plausible mechanism of the catalytic system is proposed. Copyright © 2012 John Wiley & Sons, Ltd.     

QM/MM simulations predict a covalent intermediate in the hen egg white lysozyme reaction with its natural substrate

Quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations indicate that the reaction of native HEWL with its natural substrate involves a covalent intermediate, in contrast to the 'textbook' mechanism for this seminal enzyme.     

Mechanism of proteolysis in matrix metalloproteinase‐2 revealed by QM/MM modeling

The mechanism of enzymatic peptide hydrolysis in matrix metalloproteinase‐2 (MMP‐2) was studied at atomic resolution through quantum mechanics/molecular mechanics (QM/MM) simulations. An all‐atom three‐dimensional molecular model was constructed on the basis of a crystal structure from the Protein Data Bank (ID: 1QIB), and the oligopeptide Ace‐Gln‐Gly～Ile‐Ala‐Gly‐Nme was considered as the substrate. Two QM/MM software packages and several computational protocols were employed to calculate QM/MM energy profiles for a four‐step mechanism involving an initial nucleophilic attack followed by hydrogen bond rearrangement, proton transfer, and C? N bond cleavage. These QM/MM calculations consistently yield rather low overall barriers for the chemical steps, in the range of 5–10 kcal/mol, for diverse QM treatments (PBE0, B3LYP, and BB1K density functionals as well as local coupled cluster treatments) and two MM force fields (CHARMM and AMBER). It, thus, seems likely that product release is the rate‐limiting step in MMP‐2 catalysis. This is supported by an exploration of various release channels through QM/MM reaction path calculations and steered molecular dynamics simulations. © 2015 Wiley Periodicals, Inc.     

Insight into the Mechanism of Hydrogenation of Amino Acids to Amino Alcohols Catalyzed by a Heterogeneous MoOx‐Modified Rh Catalyst

Hydrogenation of amino acids to amino alcohols is a promising utilization of natural amino acids. We found that MoO_x‐modified Rh/SiO₂ (Rh–MoO_x/SiO₂) is an efficient heterogeneous catalyst for the reaction at low temperature (323 K) and the addition of a small amount of MoO_x drastically increases the activity and selectivity. Here, we report the catalytic potential of Rh–MoO_x/SiO₂ and the results of kinetic and spectroscopic studies to elucidate the reaction mechanism of Rh–MoO_x/SiO₂ catalyzed hydrogenation of amino acids to amino alcohols. Rh–MoO_x/SiO₂ is superior to previously reported catalysts in terms of activity and substrate scope. This reaction proceeds by direct formation of an aldehyde intermediate from the carboxylic acid moiety, which is different from the reported reaction mechanism. This mechanism can be attributed to the reactive hydride species and substrate adsorption caused by MoO_x modification of Rh metal, which results in high activity, selectivity, and enantioselectivity.     

A Cofactor‐Substrate‐Based Supramolecular Fluorescent Probe for the Ultrafast Detection of Nitroreductase under Hypoxic Conditions

Identifying the location and expression levels of enzymes under hypoxic conditions in cancer cells is vital in early‐stage cancer diagnosis and monitoring. By encapsulating a fluorescent substrate, L‐NO₂ , within the NADH mimic‐containing metal–organic capsule Zn‐ MPB , we developed a cofactor‐substrate‐based supramolecular luminescent probe for ultrafast detection of hypoxia‐related enzymes in solution in vitro and in vivo. The host–guest structure fuses the coenzyme and substrate into one supramolecular probe to avoid control by NADH, switching the catalytic process of nitroreductase from a double‐substrate mechanism to a single‐substrate one. This probe promotes enzyme efficiency by altering the substrate catalytic process and enhances the electron transfer efficiency through an intra‐molecular pathway with increased activity. The enzyme content and fluorescence intensity showed a linear relationship and equilibrium was obtained in seconds, showing potential for early tumor diagnosis, biomimetic catalysis, and prodrug activation.     

Theoretical Explanation for How SO3H‐Functionalized Ionic Liquids Promote the Conversion of Cellulose to Glucose

While the catalytic transformation of cellulose to glucose by functionalized ionic liquids (ILs) has been achieved successfully under mild conditions, insight into the fundamental molecular mechanism is still lacking. The present work presents the first attempt to address the fundamental reaction chemistry of the catalytic transformation. An enzyme‐like catalytic mechanism of ILs, in which glycosidic bond hydrolysis proceeds through a retaining mechanism and/or an inverting mechanism, is proposed. DFT calculations show that both mechanisms involve moderate barriers (<30 kcal mol^?1), which is consistent with the catalytic performance of the ILs under mild conditions (<100 °C). The “biomimetic” mechanism model proposed herein is expected to be viable for understanding the unique catalytic activity of ILs under mild conditions.     

Development of a Continuous‐Flow System for Asymmetric Hydrogenation Using Self‐Supported Chiral Catalysts

Well‐designed, self‐assembled, metal–organic frameworks were constructed by simple mixing of multitopic MonoPhos‐based ligands ( 3 ; MonoPhos=chiral, monodentate phosphoramidites based on the 1,1′‐bi‐2‐naphthol platform) and [Rh(cod)₂]BF₄ (cod=cycloocta‐1,5‐diene). This self‐supporting strategy allowed for simple and efficient catalyst immobilization without the use of extra added support, giving well‐characterized, insoluble (in toluene) polymeric materials ( 4 ). The resulting self‐supported catalysts ( 4 ) showed outstanding catalytic performance for the asymmetric hydrogenation of a number of α‐dehydroamino acids ( 5 ) and 2‐aryl enamides ( 7 ) with enantiomeric excess (ee) ranges of 94–98 % and 90–98 %, respectively. The linker moiety in 4 influenced the reactivity significantly, albeit with slight impact on the enantioselectivity. Acquisition of reaction profiles under steady‐state conditions showed 4 h and 4 i to have the highest reactivity (turnover frequency (TOF)=95 and 97 h^?1 at 2 atm, respectively), whereas appropriate substrate/catalyst matching was needed for optimum chiral induction. The former was recycled 10 times without loss in ee (95–96 %), although a drop in TOF of approximately 20 % per cycle was observed. The estimation of effective catalytic sites in self‐supported catalyst 4 e was also carried out by isolation and hydrogenation of catalyst–substrate complex, showing about 37 % of the Rh^I centers in the self‐supported catalyst 4 e are accessible to substrate 5 c in the catalysis. A continuous flow reaction system using an activated C/ 4 h mixture as stationary‐phase catalyst for the asymmetric hydrogenation of 5 b was developed and run continuously for a total of 144 h with >99 % conversion and 96–97 % enantioselectivity. The total Rh leaching in the product solution is 1.7 % of that in original catalyst 4 h .     

Inactivation of [Fe?Fe]‐hydrogenase by O2. Thermodynamics and frontier molecular orbitals analyses

The oxidation of H‐cluster in gas phase, and in aqueous enzyme phase, has been investigated by means of quantum mechanics (QM) and combined quantum mechanics–molecular mechanics (QM/MM). Several potential reaction pathways (in the above‐mentioned chemical environments) have been studied, wherein only the aqueous enzyme phase has been found to lead to an inhibited hydroxylated cluster. Specifically, the inhibitory process occurs at the distal iron (Fe_d) of the catalytic H‐cluster (which isalso the atom involved in H₂ synthesis). The processes involved in the H‐cluster oxidative pathways are O₂ binding, e^? transfer, protonation, and H₂O removal. We found that oxygen binding is nonspontaneous in gas phase, and spontaneous for aqueous enzyme phase where both Fe atoms have oxidation state II; however, it is spontaneous for the partially oxidized and reduced clusters in both phases. Hence, in the protein environment the hydroxylated H‐cluster is obtained by means of completely exergonic reaction pathway starting with proton transfer. A unifying endeavor has been carried out for the purpose of understanding the thermodynamic results vis‐à‐vis several other performed electronic structural methods, such as frontier molecular orbitals (FMO), natural bond orbital partial charges (NBO), and H‐cluster geometrical analysis. An interesting result of the FMO examination (for gas phase) is that an e^? is transferred to LUMO_α rather than to SOMO_β, which is unexpected because SOMO_β usually resides in a lower energy rather than LUMO_α for open‐shell clusters. © 2008 Wiley Periodicals, Inc. Int J Quantum Chem, 2009