Insights into enzymatic halogenation from computational studies

The halogenases are a group of enzymes that have only come to the fore over the last 10 years thanks to the discovery and characterization of several novel representatives. They have revealed the fascinating variety of distinct chemical mechanisms that nature utilizes to activate halogens and introduce them into organic substrates. Computational studies using a range of approaches have already elucidated many details of the mechanisms of these enzymes, often in synergistic combination with experiment. This Review summarizes the main insights gained from these studies. It also seeks to identify open questions that are amenable to computational investigations. The studies discussed herein serve to illustrate some of the limitations of the current computational approaches and the challenges encountered in computational mechanistic enzymology

A Computational Methodology to Screen Activities of Enzyme Variants

We present a fast computational method to efficiently screen enzyme activity. In the presented method, the effect of mutations on the barrier height of an enzyme-catalysed reaction can be computed within 24 hours on roughly 10 processors. The methodology is based on the PM6 and MOZYME methods as implemented in MOPAC2009, and is tested on the first step of the amide hydrolysis reaction catalyzed by Candida Antarctica lipase B (CalB) enzyme. The barrier heights are estimated using adiabatic mapping and are shown to give barrier heights to within 3kcal/mol of B3LYP/6-31G(d)//RHF/3-21G results for a small model system. Relatively strict convergence criteria (0.5kcal/(mol{\AA})), long NDDO cutoff distances within the MOZYME method (15{\AA}) and single point evaluations using conventional PM6 are needed for reliable results. The generation of mutant structure and subsequent setup of the semiempirical calculations are automated so that the effect on barrier heights can be estimated for hundreds of mutants in a matter of weeks using high performance computing

Molecular Dynamics Simulations of Enzymes with Quantum Mechanical/Molecular Mechanical Potentials

S-adenosyl methionine (SAM) dependent methylation process is universally found in all branches of life. It has important implications in mammalian pathogenesis and plant metabolism. The methyl transfer is normally catalyzed by SAM-dependent methyltransferases(MTases). Two MTases are studied in this dissertation: the 1,7-dimethylxanthine methyltransferase (DXMT) which involve in plant caffeine biosynthesis, and the protein arginine methyltransferase 5(PRMT5) that participates in eukaryotic posttranslational modification. The late phase of caffeine biosynthesis starts from the substrate xanthosine and ends with the product caffeine, with theobromine as an intermediate product. DXMT is a key enzyme in this process and catalyzes two methylation steps: 1)methylation of 7-methylxanthine to form theobromine; 2)methylation of theobromine to form caffeine. The catalytic mechanism and product promiscuity of DXMT is intriguing. In Chapter 1, the quantum mechanical/molecular mechanical (QM/MM) molecular dynamics (MD) and free energy simulations were performed to explain the dual catalytic roles of DXMT. Simulation results show that a histidine residue may act as a general base catalyst during methylations. PRMTs can work as modifiers for histones and methylate the substrate arginine, thus interfering with histone code orchestration. The product specificity of PRMTs refers to their ability to produce either symmetric di-methylarginine(SDMA), asymmetric di-methylarginine(ADMA) or mono-methylarginine(MMA). Understanding the product specificity of PRMTs is important since different methylations may cause distinctive, even inverse biological consequences. PRMT5 produces SDMA, as compared to PRMT1 and PRMT3 that produce ADMA. In Chapter 2, simulations of PRMT5 have drawn a theoretical insight into the catalytic difference between SDMA and ADMA. Neddylation is a type of eukaryotic Ubiquitin-like (UBL) protein modification that is essential in cell division and development. Unlike ubiquitin and other small ubiquitin-like modifiers which target variety of protein substrates, the UBL NEDD8 is highly selective on modifying cullin proteins and contributes to 10% ~20% of all cellular ubiquitination and ubiquitination-like modification. In Chapter 3, the crystal structure of a trapped E3-E2 ̴ NEDD8-CUL1 intermediate was used for modeling, and simulations were applied to investigate the catalytic mechanism of NEDD8 transfer from E2 to the substrate. Some important insights were observed that may be used to understand the functional properties of the enzyme

A Mechanistic Study Of The Enzymatic Excision Mechanism In AP Endonuclease (APE1)

DNA with an abasic site is a cyto-toxic intermediate in the base excision repair (BER) pathway that is handled by the enzyme Apuridinic/Apyrimidinic endonuclease (APE1) [99, 56, 168, 90]. Several kinetics and thermodynamics aspects of the mechanism by which the APE1 enzyme processes its abasic DNA substrate have been discussed in this thesis. APE1 is an endonuclease that is it cleaves the DNA backbone at a non-terminal site, here at the abasic site. To obtain eminent insight about the catalytic role of amino acid residues and magnesium ions which are representatively recognized in active sites of endonuclease enzymes, quantum mechanical calculations of reaction pathways based on various cluster models mimicking such active sites of endonuclease enzymes have been performed and subsequently discussed in section 4. In this light our results underline the importance of an enzymatic active site architecture in the catalytic reaction, given the substrate is properly positioned. As a side-effect, we were able to evaluate the semi empirical method DFTB3/3OB [132] by comparison of reaction pathways calculating in different cluster models with the same reaction pathways calculations on the DFT level of theory (B3LYP/6- 31G(d,p)). Comparison of the obtained mechanisms and barriers obtained by DFT and DFTB for the minimalistic (cluster) models may nominate DFTB with reasonable accuracy and computational cost as a potential candidate for quantum method in hybrid QM/MM (quantum mechanics/molecular mechanics) approaches for phosphodiester hydrolysis in an enzymatic environment. However in the “reductionist approach” employed to evaluate multiple plausible types of reaction mechanism, the protein’s flexibility and heterogeneous electrostatic environment of protein residues is not taken into account. To enable reaction pathway caculations of the DNA cleavage mechanism in the full APE1 enzyme a model of a reaction-competent APE1-DNA reactant complex has been built, based on available crystal structure data and mutagenesis experiments from the literature [168, 133, 99, 92]. To remedy the lack of lucid information about structural details of APE1/DNA substrate bounded to Mg2+ ion molecular dynamic simulation together with pKa calculations for important amino acid residues in the active-site of the enzyme have been carried out (see section 5). To investigate the potential effect of metal ion binding on the stabilization of the active site in the Ape1-DNA substrate complex, the number and position of the metal ion(s) have been varied and single point mutations of vital active-site residues in the substrate complex of Ape1-DNA have been simulated. Taken together, the most likely model for an Ape1-DNA substrate complex has one Mg2+-ion located at binding site D and His309 in protonated form. At the D site, the metal ion may play a catalytic role in leaving group departure. This scenario allows Tyr171 and His309 to form hydrogen-bonds to the phosphate group that may help DNA binding as well as stabilizing a pentacovalent transition state/intermediate. Asn212 acts in properly positioning the nucleophile which can then transfer a proton to Asp210. A combined QM/MM approach wherein the active site was treated quantum mechanically and the remaining enzyme classically empowered us to investigate the enzymatic reaction pathways of phosphodiester backbone cleavage by APE1 enzyme and enabled us to depict a more realistic picture of the enzyme’s functions in abasic DNA cleavage (see section 6). Opposed to the “reductionist approach”, the QM/MM approach is accurate enough and computationally efficient to gain electronic level insight into the chemical reaction while taking the protein environment explicitly into account and as such allowed the contribution of individual residues in the enzyme environment to be quantified. To find the energetically most favorable pathway for phosphate hydrolysis catalyzed by APE1 enzyme, several possible reaction mechanisms were initially explored by potential energy calculations. As dynamical effects are important in the reaction progress, free energy calculations have subsequently been performed on selected pathways (based on their potential energy profile). According to our QM/MM calculated enzymatic reaction pathways of phosphodiester backbone cleavage by the APE1 enzyme, the dissociative type of mechanism is the most favorable pathway. Herein an important role is played by a metal ligated water molecule that donates a proton to the leaving group while the cleaved sugar backbone migrates toward the Mg2+ ion. This allows a water molecule, activated by proton transfer to Asp210, to attack the phosphorous atom of abasic DNA as nucleophile. The APE1 enzyme as an indispensable key player in BER has been discussed in atomic resolution in this thesis, Our efforts promote the understanding of catalytic and dynamical features of the APE1 enzyme in the abasic DNA cleavage mechanism, including effects of pH and single-site mutations. The detailed insight thus gained may be helpful in designing inhibitors for APE1 as a potential drug target in cancer chemotherapy.Abasische DNA ist ein cytotoxisches Intermediat des Basenexizions Reparaturmechanismus (BER), welches durch das Enzym apuridinische/apyrimidinische Endonuklease (APE1) [99, 56, 168, 90] weiter prozessiert wird. In dieser These wurden verschiedene kinetische und thermodynamische Aspekte des Mechanismus, durch welchen APE1 Enzyme ihr abasisches DNA Substrat umwandeln, diskutiert. APE1 ist eine Endonuklease, welche das DNA Rückgrat an nicht-terminaler Position spaltet, in diesem Fall an der abasischen Position. Um tiefere Einsicht in die katalytische Funktion von Aminosäure-Seitenketten und Magnesiumionen, welche representativ für das aktive Zentrum von Endonukleasen stehen, zu erhalten, wurden quantummechanische Berechnungen von Reaktionswegen basierend auf verschiedenen Cluster-Modellen, die das aktive Zentrum von Endonukleasen imitieren, durchgeführt und nachfolgend im Abschnitt 4 diskutiert. In diesem Licht unterstreichen unsere Ergebnisse die Relevanz der Architektur des aktiven Zentrums für die katalytische Reaktion, vorausgesetzt das Substrat ist korrekt positioniert. Als Nebeneffekt waren wir in der Lage die semi-empirische Methode DFTB3/3OB durch den Vergleich von Reaktionswegen berechnet in verschiedenen Cluster-Modellen mit gleichen Reaktionsweg-Parametern auf DFT -Level zu evaluieren (B3LYP/6-31G(d,p)). Der Vergleich, der durch DFT und DFTB erhaltenen Mechanismen und Barrieren für die minimalistischen (Cluster) Modelle, nominiert DFTB [132] mit angemessener Genauigkeit und Rechenkosten als potentiellen Kandidaten für quantummechanische Methoden in hybrid QM/MM (Quantummechaniken/ Molekulare Mechaniken) Ansätzen für Phosphodiester-Hydrolyse in enzymatischer Umbegung. Jedoch wird im oft verwendeten „reduktionistischen Ansatz“ zur Evaluierung multipler plausibler Formen von Reaktions-Mechanismen die Fexibilität des Proteins und die heterogene elektrostatische Umgebung der Protein-Seitenketten nicht berücksichtigt. Um Reaktionsweg-Berechnungen des DNASpaltungs-Mechanismus im gesamten APE1 Enzym zu ermöglichen, wurde ein Modell eines Reaktionskompetenten APE1-DNA Edukt-Komplexes gebaut, basierend auf den in der Literatur [168, 133, 99, 92]. vorhandenen Krystallstrukturdaten und Mutagenesis-Experimenten. Um den Fehlen von Informationen über strukturelle Details von APE1/DNA-Substrat gebunden an Mg2+ Ionen entgegenzuwirken, wurden Moleküldynamik-Simulationen zusammen mit pKa Berechnungen für wichtige Aminosäure-Seitenketten im aktiven Zentrum durchgeführt (siehe Abschnitt 5). Zur Untersuchung des potentiellen Effekts der Bindung von Metallionen auf die Stabilisierung des aktiven Zentrum im APE1-DNA Substratkomplex wurde die Anzahl und Position der Metallionen variiert und Punktmutationen wesentlicher Seitenketten des Substratkomplex von APE1-DNA simuliert. Detailierte Analyse dieser Simulationen versorgte uns mit angemessenen Statistiken und Informationen über strukturelle Details des aktiven Zentrums von APE1. Zusammenfassend beiinhalted das wahrscheinlichste Modell für den APE1-DNA Substratkomplex ein Mg2+-Ion lokalisiert an der Bindungsstelle D sowie ein His309 in protonierter Form. And der D Bindungsstelle könnte das Metallion eine katalytische Rolle bei der Abspaltung der Abgangsgruppe einnehmen. Dieses Szenario erlaubt Tyr171 und His309 Wasserstoffbrücken zu den Phosphatgruppen zu formen, welche bei der DNA-Bindung sowie bei der Stabilisierung eines pentakovalenten Überganszustands/ Intermediates behilflich sein könnten. Asn212 spielt bei der korrekten Positionierung des Substrats eine Rolle, welches dann ein Proton auf Asp210 übertragen kann. Ein kombinierter QM/MM Ansatz, in welchem das aktive Zentrum quantummechanisch und das übrige Enzym klassisch betrachtet wurde, ermöglichte uns die enzymatischen Reaktionswege der Phosphodiesterrückgrat-Spaltung durch APE1 zu untersuchen und befähigte uns zu einer realistischeren Abbildung der enzymatischen Funtkionen der abasischen DNA Spaltung (siehe Abschnitt 6). Im Gegensatz zum „reduktionistischen Ansatz“ ist der QM/MM Ansatz präzise genug und rechentechnisch effizient um auf elektorischem Level Einsicht in chemische Reaktionen bei explizieter Berücksichtigung der Proteinumgebung zu erhalten und erlaubt als dieses die Quantifizierung der Beiträge individueller Seitenketten der Enzym-Umgebung. Um den engergetisch favorisierten Reaktionsweg für Phosphathydrolyse katalysiert durch APE1-Enzyme zu indentifizieren, wurden mehrere Reaktionsmechanismen initial durch Potentialenergie-Berechnungen untersucht. Aufgrund der Wichtigkeit dynamischer Effekte für den Reaktionsprozess wurden Berechnungen mit freier Energie stückweise an ausgewählten Reaktionswegen durchgeführt (basierend auf ihrem potentiellen Energieprofil). Zufolge unserer durch QM/MM berechneten enzymatischen Reaktionswege der Phosphodiester RückgratSpaltung durch APE1 Enzyme ist der dissoziative Mechanismus der favorisierte Reaktionsweg. In diesem wird eine wichtige Rolle durch ein Metalliganden gebundenes Wasser-Molekül eingenommen, welches Protonen an die Abgangsgruppe überträgt während das gespaltene Zucker-Rückgrat in Richtung des Mg2+ Ions wandert. Das erlaubt einem Wasser-Molekül, aktiviert durch durch Protonentransfer zu Asp210, als Nukleophil das Phosphoratom der abasischen DNA anzugreifen. Das APE1-Enzym wurde in dieser Thesis als ein unverzichtbarer Schlüsselspieler des BER mit atomarer Auflösung diskutiert. Unsere Bemühungen fördern das Verständnis der katalytischen und dynamischen Eigenschaften des APE1 Enzyms im abasischen DNA Spaltungs-Mechanismus unter Einbeziehung von Effekten durch pH und Punktmutationen. Der hierdurch erhaltene Einblick könnte für das Design von Inhibitoren für APE1 als potentielles Ziel für Therapeutika in der Chemotherapie hilfreich sein

Design and SAR Analysis of Covalent Inhibitors Driven by Hybrid QM/MM Simulations

Quantum mechanics/molecular mechanics (QM/MM) hybrid technique is emerging as a reliable computational method to investigate and characterize chemical reactions occurring in enzymes. From a drug discovery perspective, a thorough understanding of enzyme catalysis appears pivotal to assist the design of inhibitors able to covalently bind one of the residues belonging to the enzyme catalytic machinery. Thanks to the current advances in computer power, and the availability of more efficient algorithms for QM-based simulations, the use of QM/MM methodology is becoming a viable option in the field of covalent inhibitor design. In the present review, we summarized our experience in the field of QM/MM simulations applied to drug design problems which involved the optimization of agents working on two well-known drug targets, namely fatty acid amide hydrolase (FAAH) and epidermal growth factor receptor (EGFR). In this context, QM/MM simulations gave valuable information in terms of geometry (i.e., of transition states and metastable intermediates) and reaction energetics that allowed to correctly predict inhibitor binding orientation and substituent effect on enzyme inhibition. What is more, enzyme reaction modelling with QM/MM provided insights that were translated into the synthesis of new covalent inhibitor featured by a unique combination of intrinsic reactivity, on-target activity, and selectivity

Simulations of Chemical Catalysis

This dissertation contains simulations of chemical catalysis in both biological and heterogeneous contexts. A mixture of classical, quantum, and hybrid techniques are applied to explore the energy profiles and compare possible chemical mechanisms both within the context of human and bacterial enzymes, as well as exploring surface reactions on a metal catalyst. A brief summary of each project follows. Project 1 — Bacterial Enzyme SpvC The newly discovered SpvC effector protein from Salmonella typhimurium interferes with the host immune response by dephosphorylating mitogen-activated protein kinases (MAPKs) with a -elimination mechanism. The dynamics of the enzyme substrate complex of the SpvC effector is investigated with a 3.2 ns molecular dynamics simulation, which reveals that the phosphorylated peptide substrate is tightly held in the active site by a hydrogen bond network and the lysine general base is positioned for the abstraction of the alpha hydrogen. The catalysis is further modeled with density functional theory (DFT) in a truncated active-site model at the B3LYP/6-31 G(d,p) level of theory. The truncated model suggested the reaction proceeds via a single transition state. After including the enzyme environment in ab initio QM/MM studies, it was found to proceed via an E1cB-like pathway, in which the carbanion intermediate is stabilized by an enzyme oxyanion hole provided by Lys104 and Tyr158 of SpvC. Project 2 — Human Enzyme CDK2 Phosphorylation reactions catalyzed by kinases and phosphatases play an indispensable role in cellular signaling, and their malfunctioning is implicated in many diseases. Ab initio quantum mechanical/molecular mechanical studies are reported for the phosphoryl transfer reaction catalyzed by a cyclin-dependent kinase, CDK2. Our results suggest that an active-site Asp residue, rather than ATP as previously proposed, serves as the general base to activate the Ser nucleophile. The corresponding transition state features a dissociative, metaphosphate-like structure, stabilized by the Mg(II) ion and several hydrogen bonds. The calculated free-energy barrier is consistent with experimental values. Project 3 — Bacterial Enzyme Anthrax Lethal Factor In this dissertation, we report a hybrid quantum mechanical and molecular mechanical study of the catalysis of anthrax lethal factor, an important first step in designing inhibitors to help treat this powerful bacterial toxin. The calculations suggest that the zinc peptidase uses the same general base-general acid mechanism as in thermolysin and carboxypeptidase A, in which a zinc-bound water is activated by Glu687 to nucleophilically attack the scissile carbonyl carbon in the substrate. The catalysis is aided by an oxyanion hole formed by the zinc ion and the side chain of Tyr728, which provide stabilization for the fractionally charged carbonyl oxygen. Project 4 — Methanol Steam Reforming on PdZn alloy Recent experiments suggested that PdZn alloy on ZnO support is a very active and selective catalyst for methanol steam reforming (MSR). Plane-wave density functional theory calculations were carried out on the initial steps of MSR on both PdZn and ZnO surfaces. Our calculations indicate that the dissociation of both methanol and water is highly activated on \ufb02at surfaces of PdZn such as (111) and (100), while the dissociation barriers can be lowered significantly by surface defects, represented here by the (221), (110), and (321) faces of PdZn. The corresponding processes on the polar Zn-terminated ZnO(0001) surfaces are found to have low or null barriers. Implications of these results for both MSR and low temperature mechanisms are discussed

A Catalytic Mechanism for Cysteine N-Terminal Nucleophile Hydrolases, as Revealed by Free Energy Simulations

The N-terminal nucleophile (Ntn) hydrolases are a superfamily of enzymes specialized in the hydrolytic cleavage of amide bonds. Even though several members of this family are emerging as innovative drug targets for cancer, inflammation, and pain, the processes through which they catalyze amide hydrolysis remains poorly understood. In particular, the catalytic reactions of cysteine Ntn-hydrolases have never been investigated from a mechanistic point of view. In the present study, we used free energy simulations in the quantum mechanics/molecular mechanics framework to determine the reaction mechanism of amide hydrolysis catalyzed by the prototypical cysteine Ntn-hydrolase, conjugated bile acid hydrolase (CBAH). The computational analyses, which were confirmed in water and using different CBAH mutants, revealed the existence of a chair-like transition state, which might be one of the specific features of the catalytic cycle of Ntn-hydrolases. Our results offer new insights on Ntn-mediated hydrolysis and suggest possible strategies for the creation of therapeutically useful inhibitors

The Use of Multiscale Molecular Simulations in Understanding a Relationship between the Structure and Function of Biological Systems of the Brain: The Application to Monoamine Oxidase Enzymes

Computational techniques provide accurate descriptions of the structure and dynamics of biological systems, contributing to their understanding at an atomic level.Classical MD simulations are a precious computational tool for the processes where no chemical reactions take place.QM calculations provide valuable information about the enzyme activity, being able to distinguish among several mechanistic pathways, provided a carefully selected cluster model of the enzyme is considered.Multiscale QM/MM simulation is the method of choice for the computational treatment of enzyme reactions offering quantitative agreement with experimentally determined reaction parameters.Molecular simulation provide insight into the mechanism of both the catalytic activity and inhibition of monoamine oxidases, thus aiding in the rational design of their inhibitors that are all employed and antidepressants and antiparkinsonian drugs. Aging society and therewith associated neurodegenerative and neuropsychiatric diseases, including depression, Alzheimer's disease, obsessive disorders, and Parkinson's disease, urgently require novel drug candidates. Targets include monoamine oxidases A and B (MAOs), acetylcholinesterase (AChE), butyrylcholinesterase (BChE), and various receptors and transporters. For rational drug design it is particularly important to combine experimental synthetic, kinetic, toxicological, and pharmacological information with structural and computational work. This paper describes the application of various modern computational biochemistry methods in order to improve the understanding of a relationship between the structure and function of large biological systems including ion channels, transporters, receptors, and metabolic enzymes. The methods covered stem from classical molecular dynamics simulations to understand the physical basis and the time evolution of the structures, to combined QM, and QM/MM approaches to probe the chemical mechanisms of enzymatic activities and their inhibition. As an illustrative example, the later will focus on the monoamine oxidase family of enzymes, which catalyze the degradation of amine neurotransmitters in various parts of the brain, the imbalance of which is associated with the development and progression of a range of neurodegenerative disorders. Inhibitors that act mainly on MAO A are used in the treatment of depression, due to their ability to raise serotonin concentrations, while MAO B inhibitors decrease dopamine degradation and improve motor control in patients with Parkinson disease. Our results give strong support that both MAO isoforms, A and B, operate through the hydride transfer mechanism. Relevance of MAO catalyzed reactions and MAO inhibition in the context of neurodegeneration will be discussed