Discerning the catalytic mechanism of Staphylococcus aureus sortase A with QM/MM free energy calculations

Sortases are key virulence factors in Gram-positive bacteria. These enzymes embed surface proteins in the cell wall through a transpeptidation reaction that involves recognizing a penta-peptide “sorting signal” in a target protein, cleaving it, and covalently attaching it to a second substrate that is later inserted into the cell wall. Although well studied, several aspects of the mechanism by which sortases perform these functions remains unclear. In particular, experiments have revealed two potential sorting signal binding motifs: a “Threonine-Out” (Thr-Out) structure in which the catalytically critical threonine residues protrudes into solution, and a “Threonine-In” (Thr-In) configuration in which this residue inserts into the binding site. To determine which of these is the biologically relevant state, we have performed a series of conventional and hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics simulations of the Staphylococcus aureus sortase A (SrtA) enzyme bound to a sorting signal substrate. Through the use of multi-dimensional metadynamics, our simulations were able to both map the acylation mechanism of SrtA in the Thr-In and Thr-Out states, as well as determine the free energy minima and barriers along these reactions. Results indicate that in both states the catalytic mechanisms are similar, however the free energy barriers are lower in the Thr-In configuration, suggesting that Thr-In is the catalytically relevant state. This has important implications for advancing our understanding of the mechanisms of sortase enzymes, as well we for future structure based drug design efforts aimed at inhibiting sortase function in vivo.     

QM/MM investigation of the reaction rates of substrates of 2,3-dimethylmalate lyase: A catabolic protein isolated from Aspergillus niger

Aspergillus niger is an industrially important microorganism used in the production of citric acid. It is a common cause of food spoilage and represents a health issue for patients with compromised immune systems. Recent studies on Aspergillus niger have revealed details on the isocitrate lyase (ICL) superfamily and its role in catabolism, including (2R, 3S)-dimethylmalate lyase (DMML). Members of this and related lyase super families are of considerable interest as potential treatments for bacterial and fungal infections, including Tuberculosis. In our efforts to better understand this class of protein, we investigate the catalytic mechanism of DMML, studying five different substrates and two different active site metals configurations using molecular dynamics (MD) and hybrid quantum mechanics/molecular mechanics (QM/MM) calculations. We show that the predicted barriers to reaction for the substrates show good agreement with the experimental k_cat values. This results help to confirm the validity of the proposed mechanism and open up the possibility of developing novel mechanism based inhibitors specifically for this target.     

Mechanistic insights into the catalytic reaction of plant allene oxide synthase (pAOS) via QM and QM/MM calculations

QM cluster and QM/MM protein models have been employed to understand aspects of the reaction mechanism of plant allene oxide synthase (pAOS). In this study we have investigated two reaction mechanisms for pAOS. The standard pAOS mechanism was contrasted with an alternative involving an additional active site molecule which has been shown to facilitate proton coupled electron transfer (PCET) in related systems. Firstly, we found that the results from QM/MM protein model are comparable with those from the QM cluster model, presumably due to the large active site used. Furthermore, the results from the QM cluster model show that the Fe^III and Fe^IV pathways for the standard mechanism have similar energetic and structural properties, indicating that the reaction mechanism may well proceed via both pathways. However, while the PCET process is facilitated by an additional active site bound water in other related families, in pAOS it is not, suggesting this type of process is not general to all closely related family members.     

QM/MM modeling of the hydrolysis and transfructosylation reactions of fructosyltransferase from Aspergillus japonicas,an enzyme that produces prebiotic fructooligosaccharide

Fructosyltransferases (FTs) act on sucrose by cleaving the β-(2 → 1) linkage, releasing glucose, and then transferring the fructosyl group to an acceptor molecule. These enzymes are capable of producing prebiotic fructooligosaccharides (FOSs) that are of industrial interest. While several FOS-synthesizing enzymes FTs have been investigated, their catalytic mechanism is not yet fully understood, especially the molecular details of how FOS are enzymatically synthesized from sucrose. Here, we present a comparative quantum mechanics/molecular mechanics (QM/MM) study on the hydrolysis and transfructosylation reactions catalyzed by A. japonicus FT using sucrose as donor and acceptor substrates. It is shown that the hydrolysis and transfructosylation reactions of the enzyme seem to be competitive with similar potential energy profiles. For all studied reaction steps, the fructosyl ring bound in the −1 position was observed to have a ⁴E conformation in the oxocarbonium ion-like transition state. Based on the SCC-DFTB/MM simulations of sucrose complexes of wildtype and D191A mutant FT, Asp191 is shown to be responsible for the productive sugar conformation (at subsite −1) required for catalysis. A key interaction, Asp119⋯nucleophile⋯1–OH (substrate), is proposed to facilitate the formation of fructosyl-enzyme intermediate. This is the first computational study for understanding the FOS synthesis process, and it can be applicable to related FOS-synthesizing enzymes.     

MD and QM/MM study on catalytic mechanism of a FAD-dependent enzyme ORF36: For nitro sugar biosynthesis

The catalytic mechanism of a FAD-dependent nitrososynthase (ORF36) was studied with molecular dynamics (MD) and quantum mechanical/molecular mechanical (QM/MM) methods. Residues Leu160 and Phe374 play an important role during the FAD binding with ORF36. Similar phenylalanine/leucine pair was found in the other two enzymes of this family. For the second oxidation step of ORF36 toward thymidine diphosphate-l-epi-vancosamine, three elementary catalytic steps were found: a hydroxylation step, a hydrogen back-transfer step and a hydroxyl group elimination step. The hydroxylation step is found to be the rate-determining step with an energy barrier of 26.3 kcal/mol under the B3LYP/cc-pVTZ//CHARMM22 level. Two possible pathways for the second oxidation step are carefully investigated. Our simulations indicate that an oxygen atom from the coenzyme FADHOOH is inserted into the product. In addition, the electrostatic influence of 17 individual residues and five neighboring water molecules on the rate-determining step was estimated. The results indicate that groups Gly132/Ala133/Leu134, Met375/Gln376 and a water fence play a key role in facilitating the rate-determining step. On the other hand, residues Leu160, Val161 and Ser162 are found to be critical to suppress the rate-determining step. Our results lead to further understanding of the detailed catalytic pathways for nitro sugar biosynthesis.     

Theoretical study of the hydrolysis mechanism of 2-pyrone-4,6-dicarboxylate (PDC) catalyzed by LigI

2-Pyrone-4,6-dicarboxylate lactonase (LigI) is the first identified enzyme from amidohydrolase superfamily that does not require a divalent metal ion for catalytic activity. It catalyzes the reversible hydrolysis of 2-pyrone-4,6-dicarboxylate (PDC) to 4-oxalomesaconate (OMA) and 4-carboxy-2-hydroxymuconate (CHM) in the degradation of lignin. In this paper, a combined quantum mechanics and molecule mechanics (QM/MM) approach was employed to study the reaction mechanism of LigI from Sphingomonas paucimobilis. According to the results of our calculations, the whole catalytic reaction contains three elementary steps, including the nucleophilic attack, the cleavage of CO of lactone (substrate) and the intramolecular proton transfer. The intermediate has two intramolecular proton transfer pathways, due to which, two final hydrolysis products can be obtained. The energy profile indicates that 4-carboxy-2-hydroxymuconate (CHM) is the main hydrolysis product, therefore, the isomerization between 4-carboxy-2-hydroxymuconate (CHM) and 4-oxalomesaconate (OMA) is suggested to occur in solvent. During the catalytic reaction, residue Asp248 acts as a general base to activate the hydrolytic water molecule. Although His31, His33 and His180 do not directly participate in the chemical process, they play assistant roles by forming electrostatic interactions with the substrate and its involved species in activating the carbonyl group of the substrate and stabilizing the intermediates and transition states.     

A water-assisted nucleophilic mechanism utilized by BphD,the meta-cleavage product hydrolase in biphenyl degradation

As members of the α/β-hydrolase superfamily, Meta-cleavage product (MCP) hydrolases generally utilize a Ser-His-Asp catalytic triad to hydrolyze the cleavage of CC bond during the aerobic catabolism of aromatic compounds by bacteria. BphD is one kind of MCP hydrolase that catalyzes the hydrolysis of 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoic acid (HOPDA) to 2-hydroxypenta-2,4-dienoic acid (HPD) and benzoate. In this article, a combined quantum mechanics and molecule mechanics (QM/MM) approach has been employed to explore the reaction mechanism of BphD from Burkholderia xenovorans LB400. On the basis of the recently resolved crystal structures, three computational models have been constructed. Our calculation results reveal that BphD utilizes a water-assisted nucleophilic mechanism, which contains acylation and deacylation stages. In acylation reaction, an active site water molecule assists the proton transfer from Ser112 to the carbanion intermediate (substrate) by forming hydrogen bonds with Ser112 and His265, and this proton transfer is in concert with the nucleophilic attack of deprotonated Ser112 on the C6-carbonyl of substrate to form the acylated intermediate. In deacylation, the Asp237-His265 dyad acts as a general base to activate the hydrolytic water, whose nucleophilic attack leads to the collapses of acyl-enzyme intermediate. The acylation and deacylation process correspond to the highest energy barriers of 21.0 and 23.9 kcal/mol, respectively. During the catalytic reaction, the active site water and Asp237-His265 dyad play an important role for each elementary steps.     

Elucidating the catalytic mechanism of β-secretase (BACE1): A quantum mechanics/molecular mechanics (QM/MM) approach

In this quantum mechanics/molecular mechanics (QM/MM) study, the mechanisms of the hydrolytic cleavage of the Met2-Asp3 and Leu2-Asp3 peptide bonds of the amyloid precursor protein (WT-substrate) and its Swedish mutant (SW) respectively catalyzed by β-secretase (BACE1) have been investigated by explicitly including the electrostatic and steric effects of the protein environment in the calculations. BACE1 catalyzes the rate-determining step in the generation of Alzheimer amyloid beta peptides and is widely acknowledged as a promising therapeutic target. The general acid-base mechanism followed by the enzyme proceeds through the following two steps: (1) formation of the gem-diol intermediate and (2) cleavage of the peptide bond. The formation of the gem-diol intermediate occurs with the barriers of 19.6 and 16.1 kcal/mol for the WT- and SW-substrate respectively. The QM/MM energetics predict that with the barriers of 21.9 and 17.2 kcal/mol for the WT- and SW-substrate respectively the cleavage of the peptide bond occurs in the rate-determining step. The computed barriers are in excellent agreement with the measured barrier of ∼18.0 kcal/mol for the SW-substrate and in line with the experimental observation that the cleavage of this substrate is sixty times more efficient than the WT-substrate.     

Assessment of a mechanism for reactive inhibition of carboxypeptidase A with QM/MM methods

The mechanism for inhibition of carboxypeptidase A (CPA) by the two enantiomers of a reactive inhibitor, N-(2-chloroethyl)-N-methylphenylalanine, has been investigated using computational methods. Quantum mechanical and molecular mechanical (QM/MM) methods have been employed to find likely enzyme binding conformations by comparison with the observed rates of inactivation of the enzyme. The study has shown that the enzyme active site appears to be flexible enough to allow the nucleophilic deactivation reactions of both the (R) and (S) forms of a model of the inhibitor to be catalysed by the Zn(II) cofactor of CPA.     

A combined QM/MM study of the nucleophilic addition reaction of methanethiolate and N-methylacetamide

A combined quantum mechanical (QM) and molecular mechanical (MM) method was used to study the nucleophilic addition reaction of methanethiolate to N-methylacetamide (NMA) in the gas phase and aqueous solution. At the B3LYP/aug-cc-pVDZ//HF/6-31 + G(d) level, the ion-dipole complex was found to be the global minimum on the potential energy surface in the gas phase with a binding energy of 21.2 kcal/mol. The complex has a C-S distance of 4.33 A, and no stabilized tetrahedral intermediate was located. The computed potential of mean force in water shows that solvent effects stabilize the reactants over the tetrahedral adduct by 36.5 kcal/mol, and that the tetrahedral intermediate does not exist for the present reaction in water. The present study provides an initial step for modeling the cysteine protease hydrolysis reactions in enzymes.     

Challenges in computational studies of enzyme structure,function and dynamics

In this review we give an overview of the field of Computational enzymology. We start by describing the birth of the field, with emphasis on the work of the 2013 chemistry Nobel Laureates. We then present key features of the state-of-the-art in the field, showing what theory, accompanied by experiments, has taught us so far about enzymes. We also briefly describe computational methods, such as quantum mechanics-molecular mechanics approaches, reaction coordinate treatment, and free energy simulation approaches. We finalize by discussing open questions and challenges.     

Investigation of the rescue mechanism catalyzed by a nucleophile mutant of rice BGlu1

In the present study, the quantum mechanical/molecular mechanical (QM/MM) method was used to investigate the rescue mechanism of an E386G mutant as well as the glycosylation mechanism of the wild rice β-d-glucosidase. E386G mutant experiences an asynchronous collaborative process to glycosylate the anionic formate with an energy barrier of 22.6 kcal/mol, while the energy barrier is 25.9 kcal/mol for the wild complex. The low energy barrier of the mutated complex suggests that anionic formate might be a good nucleophile to attack the anomeric carbon atom. Both energy barriers can be lowered when the leaving departure releases from the active site, suggesting that the product release, rather than chemistry, contributes to the rate limiting in BGlu1 mutants. Structure analyses also indicate that the external nucleophile has little steric hindrance with pocket residues and adjusts freely to proceed the rescue mechanism of the mutated complex. Our calculations provide a guide for the selectivity of exogenous nucleophiles in the future study of β-glucosidase.     

Identification of a potential proton donor to the linking oxygen atom in a three-metal ion assisted catalysis pathway catalyzed by Fructose-1, 6-bisphosphatase

In this paper, the dephosphorylation mechanism of FBP to F6P catalyzed by the Fructose-1, 6-bisphosphatase (St-Fbp) from Sulfolobus tokodaii was studied using quantum mechanical/molecular mechanical (QM/MM) approach. Based on the experimental results, total five possible catalytic mechanisms (path1-path4′) were designed. The most possible dephosphorylation reaction follows a two-step mechanism (path2): a dephosphorylation process (with D12 being an base of W6 and residue K133 being the proton donor of the linking FBP:O4) and a proton exchange process (between K133 and the water W1). Furthermore, the three-step of path4 is also possible: a dephosphorylation process (with D54 being the base of W6 and residue K133 being the proton donor of the linking FBP:O4) and two proton exchange processes (first between residues D54 and D12 then between K133 and the water W1). The relative low energy of this pathway suggests that D54 might also be a base except D12. Our calculations indicate that K133 is the preferred proton donor during the breaking of the phosphate bond O4-P1, with the W1 being an alternative proton donor to access to a more stable product. Findings here give a new insight into the understanding of catalytic mechanism of FBPase.     

Theoretical study on the proton shuttle mechanism of saccharopine dehydrogenase

Saccharopine dehydrogenase (SDH) is the last enzyme in the AAA pathway of l-lysine biosynthesis. On the basis of crystal structures of SDH, the whole catalytic cycle of SDH has been studied by using density functional theory (DFT) method. Calculation results indicate that hydride transfer is the rate-limiting step with an energy barrier of 25.02 kcal/mol, and the overall catalytic reaction is calculated to be endothermic by 9.63 kcal/mol. Residue Lys77 is proved to be functional only in the process of saccharopine deprotonation until the formation of product l-lysine, and residue His96 is confirmed to take part in multiple proton transfer processes and can be described as a proton transfer station. From the point of view of energy, the SDH catalytic reaction for the synthesis of l-lysine is unfavorable compared with its reverse reaction for the synthesis of saccharopine. These results are essentially consistent with the experimental observations from pH dependence of kinetic parameters and isotope effects.     

Docking,molecular dynamics and QM/MM studies to delineate the mode of binding of CucurbitacinE to F-actin

CucurbitacinE (CurE) has been known to bind covalently to F-actin and inhibit depolymerization. However, the mode of binding of CurE to F-actin and the consequent changes in the F-actin dynamics have not been studied. Through quantum mechanical/molecular mechanical (QM/MM) and density function theory (DFT) simulations after the molecular dynamics (MD) simulations of the docked complex of F-actin and CurE, a detailed transition state (TS) model for the Michael reaction is proposed. The TS model shows nucleophilic attack of the sulphur of Cys257 at the β-carbon of Michael Acceptor of CurE producing an enol intermediate that forms a covalent bond with CurE. The MD results show a clear difference between the structure of the F-actin in free form and F-actin complexed with CurE. CurE affects the conformation of the nucleotide binding pocket increasing the binding affinity between F-actin and ADP, which in turn could affect the nucleotide exchange. CurE binding also limits the correlated displacement of the relatively flexible domain 1 of F-actin causing the protein to retain a flat structure and to transform into a stable “tense” state. This structural transition could inhibit depolymerization of F-actin. In conclusion, CurE allosterically modulates ADP and stabilizes F-actin structure, thereby affecting nucleotide exchange and depolymerization of F-actin.     

Binding mechanism of CDK5 with roscovitine derivatives based on molecular dynamics simulations and MM/PBSA methods

Roscovitine derivatives are potent inhibitors of cyclin-dependent kinase 5 (CDK5), but they exhibit different activities, which has not been understood clearly up to now. On the other hand, the task of drug design is difficult because of the fuzzy binding mechanism. In this context, the methods of molecular docking, molecular dynamics (MD) simulation, and binding free energy analysis are applied to investigate and reveal the detailed binding mechanism of four roscovitine derivatives with CDK5. The electrostatic and van der Waals interactions of the four inhibitors with CDK5 are analyzed and discussed. The calculated binding free energies in terms of MM-PBSA method are consistent with experimental ranking of inhibitor effectiveness for the four inhibitors. The hydrogen bonds of the inhibitors with Cys83 and Lys33 can stabilize the inhibitors in binding sites. The van der Waals interactions, especially the pivotal contacts with Ile10 and Leu133 have larger contributions to the binding free energy and play critical roles in distinguishing the variant bioactivity of four inhibitors. In terms of binding mechanism of the four inhibitors with CDK5 and energy contribution of fragments of each inhibitor, two new CDK5 inhibitors are designed and have stronger inhibitory potency.     

Mechanistic and kinetic study on the reaction of methylperoxy radical with atomic iodine

Singlet and triplet potential energy surfaces for the CH₃O₂ with I reaction have been investigated computationally to propose the reaction mechanisms and possible products. Multichannel RRKM theory and transition-state theory have been used to compute the overall and individual rate constants at 200–3000 K and 10⁻¹⁴–10¹⁴ Torr. On the singlet PES, addition-elimination, substitution and H-abstraction mechanisms are located, and the addition-elimination mechanism is dominant. At 70 Torr with N₂ as bath gas, IM1(CH₃OOI) formed by collisional stabilization is dominated at 200–300 K, whereas CH₂O and HIO are the major products at the temperatures between 350 and 3000 K; The title reaction exhibits the typical falloff behavior. The results show that temperature and pressure affect the yield of products.Furthermore, the predicted rate constants at 298 K 70 Torr of N₂ agree well with the available experimental values. On the triplet PES, the most favorable product should be CH₃I + O₂(³Σ) at atmospheric condition. Other two pathways on the triplet PES will not compete with the pathways on the singlet PES in kinetically and thermodynamically.