Molecular modeling of formate dehydrogenase: the formation of the Michaelis complex

The formation of the reactive enzyme-substrate complex of formate dehydrogenase has been investigated by molecular dynamics techniques accounting for different conformational states of the enzyme. Simulations revealed that the transport of substrate to the active site through the substrate channel proceeds in the open conformation of enzyme due to the crucial role of the Arg284 residue acting as a vehicle. However, formate binding in the active site of the open conformation leads to the formation of a nonproductive enzyme-substrate complex. The productive Michaelis complex is formed only in the closed enzyme conformation after the substrate and coenzyme have bound, when required rigidity of the binding site and reactive formate orientation due to interactions with Arg284, Asn146, Ile122, and His332 residues is attained. Then, the high occupancy (up to 75%) of the reactive substrate-coenzyme conformation is reached, which was demonstrated by hybrid quantum mechanics/molecular mechanics simulations using various semiempirical Hamiltonians.     

Mechanistic model for prediction of formate dehydrogenase kinetics under industrially relevant conditions

Formate dehydrogenase (FDH) from Candida boidinii is an important biocatalyst for the regeneration of the cofactor NADH in industrial enzyme‐catalyzed reductions. The mathematical model that is currently applied to predict progress curves during (semi‐)batch reactions has been derived from initial rate studies. Here, it is demonstrated that such extrapolation from initial reaction rates to performance during a complete batch leads to considerable prediction errors. This observation can be attributed to an invalid simplification during the development of the literature model. A novel mechanistic model that describes the course and performance of FDH‐catalyzed NADH regeneration under industrially relevant process conditions is introduced and evaluated. Based on progress curve instead of initial reaction rate measurements, it was discriminated from a comprehensive set of mechanistic model candidates. For the prediction of reaction courses on long time horizons (>1 h), decomposition of NADH has to be considered. The model accurately describes the regeneration reaction under all conditions, even at high concentrations of the substrate formate and thus is clearly superior to the existing model. As a result, for the first time, course and performance of NADH regeneration in industrial enzyme‐catalyzed reductions can be accurately predicted and used to optimize the cost efficiency of the respective processes. © 2009 American Institute of Chemical Engineers Biotechnol. Prog., 2010     

Molecular modeling of green fluorescent protein: structural effects of chromophore deprotonation

Molecular dynamics (MD) simulations were carried out to study the conformational rearrangement induced by deprotonation of the fluorescent chromophore in GFP, as well as the associated changes in the hydrogen-bonding network. For both the structures with either a neutral or an anionic chromophore, it was found that the beta-barrel was stable and rigid, and the conformation of the chromophore was consistent with the available x-ray structure. The conformational change in Thr203 due to deprotonation was also found to be consistent with the three-state isomerization model. Although GFP is highly fluorescent, denatured-GFP is nonfluorescent, indicating that the environment of the protein plays an important role in its fluorescence behavior. Our MD simulations, which explore the effect of the protein shell on the conformation of the chromophore, find the flexibility of the central chromophore to be significantly restricted due to the rigid nature of the protein shell. The hydrogen-bonding between the chromophore and neighboring residues was also shown to contribute to the chromophore rigidity. In addition to the MD studies, quantum mechanics/molecular mechanics (QM/MM) ONIOM calculations were carried out to investigate the effect of the beta-barrel on the internal rotation in the chromophore. Along with providing quantitative values for torsional rotation barriers about the bridging bond in the chromophore, the ONIOM calculations also validate our MD force field parameters.     

Knowledge-based modeling of the D-lactate dehydrogenase three-dimensional structure

A three-dimensional structure of the NAD-dependent D -lactate dehydrogenase of Lactobacillus bulgaricus is modeled using the structure of the formate dehydrogenase of Pseudomonas sp. as template. Both sequences share only 22% of identical residues. Regions for knowledge-based modeling are defined from the structurally conserved regions predicted by multiple alignment of a set of related protein sequences with low homology. The model of the D -LDH subunit shows, as for the formate dehydrogenase, an α/β structure, with a catalytic domain and a coenzyme binding domain. It points out the catalytic histidine (His-296) and supports the hypothetical catalytic mechanism. It also suggests that the other residues involved in the active site are Arg-235, possibly involved in the binding of the carboxyl group of the pyruvate, and Phe-299, a candidate for stabilizing the methyl group of the substrate. © 1995 Wiley-Liss, Inc.     

Molecular dynamics simulations give insight into the conformational change,complex formation,and electron transfer pathway for cytochrome P450 reductase

Cytochrome P450 reductase (CYPOR) undergoes a large conformational change to allow for an electron transfer to a redox partner to take place. After an internal electron transfer over its cofactors, it opens up to facilitate the interaction and electron transfer with a cytochrome P450. The open conformation appears difficult to crystallize. Therefore, a model of a human CYPOR in the open conformation was constructed to be able to investigate the stability and conformational change of this protein by means of molecular dynamics simulations. Since the role of the protein is to provide electrons to a redox partner, the interactions with cytochrome P450 2D6 (2D6) were investigated and a possible complex structure is suggested. Additionally, electron pathway calculations with a newly written program were performed to investigate which amino acids relay the electrons from the FMN cofactor of CYPOR to the HEME of 2D6. Several possible interacting amino acids in the complex, as well as a possible electron transfer pathway were identified and open the way for further investigation by site directed mutagenesis studies.     

Molecular modeling of an antigenic complex between a viral peptide and a class I major histocompatibility glycoprotein.

Computer simulation of the conformations of short antigenic peptides (5-10 residues) either free or bound to their receptor, the major histocompatibility complex (MHC)-encoded glycoprotein H-2 Ld, was employed to explain experimentally determined differences in the antigenic activities within a set of related peptides. Starting for each sequence from the most probable conformations disclosed by a pattern-recognition technique, several energy-minimized structures were subjected to molecular dynamics simulations (MD) either in vacuo or solvated by water molecules. Notably, antigenic potencies were found to correlate to the peptides propensity to form and maintain an overall alpha-helical conformation through regular i,i + 4 hydrogen bonds. Accordingly, less active or inactive peptides showed a strong tendency to form i,i + 3 hydrogen bonds at their N-terminal end. Experimental data documented that the C-terminal residue is critical for interaction of the peptide with H-2 Ld. This finding could be satisfactorily explained by a 3-D Q.S.A.R. analysis postulating interactions between ligand and receptor by hydrophobic forces. A 3-D model is proposed for the complex between a high-affinity nonapeptide and the H-2 Ld receptor. First, the H-2 Ld molecule was built from X-ray coordinates of two homologous proteins: HLA-A2 and HLA-Aw68, energy-minimized and studied by MD simulations. With HLA-A2 as template, the only realistic simulation was achieved for a solvated model with minor deviations of the MD mean structure from the X-ray conformation. Water simulation of the H-2 Ld protein in complex with the antigenic nonapeptide was then achieved with the template-derived optimal parameters. The bound peptide retains mainly its alpha-helical conformation and binds to hydrophobic residues of H-2 Ld that correspond to highly polymorphic positions of MHC proteins. The orientation of the nonapeptide in the binding cleft is in accordance with the experimentally determined distribution of its MHC receptor-binding residues (agretope residues). Thus, computer simulation was successfully employed to explain functional data and predicts alpha-helical conformation for the bound peptide.     

Molecular simulation of pyrroloquinoline quinine-dependent glycerol dehydrogenase in Gluconobacter oxydans

During the fermentation process from glycerol to 1,3-dihydroxyacetone (DHA) by Gluconobacter oxydans, the increase in the concentration of glycerol shows obvious inhibition on the cell growth and DHA production. Researches on the interaction mechanism between glycerol and glycerol dehydrogenase (sldha) are important to improve the conversion rate from glycerol to DHA and to enhance the strains tolerance to glycerol. At present, the 3D structure of sldha is still unknown. So we analysed the 3D structure and then found the binding sites of glycerol with sldha. In the present study, we constructed the 3D structure of sldha by the homology modelling method based on Modeller 9v6 software. Four proteins, 1yiqA, 1kb0A, 1kv9A and 1lrwA, from Protein Data Bank were chosen as templates, since they have the highest similarities with sldha in Protein Data Bank which is 38%, 37%, 39% and 38%, respectively. The molecular dynamics simulation of constructed 3D structure of sldha by Gromacs 4.0.5 was carried out. Finally, the binding sites of Ala715 and H719 were found through the molecular docking simulation between glycerol and sldha by using Autodock 4.2.     

Estimating the energetic contribution of hydrogen bonding to the stability of Candida methylica formate dehydrogenase by using double mutant cycle

An homology model of Candida methylica formate dehydrogenase (cm FDH) was constructed based on the Pseudomonas sp. 101 formate dehydrogenase (ps FDH) structure. In wild type cm FDH, Thr169 and Thr226 can form hydrogen bonds with each other. We measured the interaction energy between the two threonines independent of other interactions in the proteins by using a so-called double mutant cycle and assessing the protein stability from the concentration of guanidine hydrochloride needed to denature 50% of the molecules. We conclude that the hydrogen bonds stabilize the wild type protein by -4 kcal mol(-1).     

Molecular modeling studies of substrate binding by penicillin acylase

Molecular modeling has revealed intimate details of the mechanism of binding of natural substrate, penicillin G (PG), in the penicillin acylase active center and solved questions raised by analysis of available X-ray structures, mimicking Michaelis complex, which substantially differ in the binding pattern of the PG leaving group. Three MD trajectories were launched, starting from PDB complexes of the inactive mutant enzyme with PG (1FXV) and native penicillin acylase with sluggishly hydrolyzed substrate analog penicillin G sulfoxide (1GM9), or from the complex obtained by PG docking. All trajectories converged to a similar PG binding mode, which represented the near-to-attack conformation, consistent with chemical criteria of how reactive Michaelis complex should look. Simulated dynamic structure of the enzyme-substrate complex differed significantly from 1FXV, resembling rather 1GM9; however, additional contacts with residues bG385, bS386, and bN388 have been found, which were missing in X-ray structures. Combination of molecular docking and molecular dynamics also clarified the nature of extremely effective phenol binding in the hydrophobic pocket of penicillin acylase, which lacked proper explanation from crystallographic experiments. Alternative binding modes of phenol were probed, and corresponding trajectories converged to a single binding pattern characterized by a hydrogen bond between the phenol hydroxyl and the main chain oxygen of bS67, which was not evident from the crystal structure. Observation of the trajectory, in which phenol moved from its steady bound to pre-dissociation state, mapped the consequence of molecular events governing the conformational transitions in a coil region a143-a146 coupled to substrate binding and release of the reaction products. The current investigation provided information on dynamics of the conformational transitions accompanying substrate binding and significance of poorly structured and flexible regions in maintaining catalytic framework.     

米曲霉木糖醇脱氢酶的分子模建与对接

[目的]研究米曲霉木糖醇脱氢酶基因的结构与功能.[方法]克隆测序来源于米曲霉的木糖醇脱氢酶(XDH)基因,利用Swiss-MODEL和Modeller对XDH进行三级结构模建,通过PROCHECK和Prosa2003对得到的4个目标模型进行评价,从中得到一个最佳模型.在同源建模的基础上,通过分子对接软件MolsoftICM-Pro,对辅因子进行对接,预测了XDH与NAD+、Zn2+作用的相关残基.寻找底物木糖醇与XDH结合的可能活性口袋,用Molsoft模拟XDH与木糖醇的对接,预测了酶与底物作用的关键氨基酸残基.[结果]结构分析显示,米曲霉XDH含有醇脱氢酶家族锌指纹结构和典型醇脱氢酶Rossmann折叠的辅酶结合域,属于Medium-chain脱氢酶(MDR)家族.通过对接研究,预测了XDH与NAD+之间形成氢键的氨基酸有Asp206、Arg211、Ser255、Ser301和Arg303,这些氨基酸位于结合域,与Zn2+形成氢键的氨基酸有His72和Glu73,位于催化域,与天然底物木糖醇形成氢键的氨基酸有Ile46、Ile349、Lys350和Thr351,位于催化域.[结论]所得信息对XDH分子定向改造、拓展米曲霉工业应用范围有重要意义.     

Monomerization alters the dynamics of the lid region in Campylobacter jejuni CstII: an MD simulation study

CstII, a bifunctional (α2,3/8) sialyltransferase from Campylobacter jejuni, is a homotetramer. It has been reported that mutation of the interface residues Phe121 (F121D) or Tyr125 (Y125Q) leads to monomerization and partial loss of enzyme activity, without any change in the secondary or tertiary structures. MD simulations of both tetramer and monomer, with and without bound donor substrate, were performed for the two mutants and WT to understand the reasons for partial loss of activity due to monomerization since the active site is located within each monomer. RMSF values were found to correlate with the crystallographic B-factor values indicating that the simulations are able to capture the flexibility of the molecule effectively. There were no gross changes in either the secondary or tertiary structure of the proteins during MD simulations. However, interface is destabilized by the mutations, and more importantly the flexibility of the lid region (Gly152-Lys190) is affected. The lid region accesses three major conformations named as open, intermediate, and closed conformations. In both Y121Q and F121D mutants, the closed conformation is accessed predominantly. In this conformation, the catalytic base His188 is also displaced. Normal mode analysis also revealed differences in the lid movement in tetramer and monomer. This provides a possible explanation for the partial loss of enzyme activity in both interface mutants. The lid region controls the traffic of substrates and products in and out of the active site, and the dynamics of this region is regulated by tetramerization. Thus, this study provides valuable insights into the role of loop dynamics in enzyme activity of CstII.     

Discovery of a new metal and NAD+-dependent formate dehydrogenase from Clostridium ljungdahlii

Over the next decades, with the growing concern of rising atmospheric carbon dioxide (CO₂) levels, the importance of investigating new approaches for its reduction becomes crucial. Reclamation of CO₂ for conversion into biofuels represents an alternative and attractive production method that has been studied in recent years, now with enzymatic methods gaining more attention. Formate dehydrogenases (FDHs) are NAD(P)H-dependent oxidoreductases that catalyze the conversion of formate into CO₂ and have been extensively used for cofactor recycling in chemoenzymatic processes. A new FDH from Clostridium ljungdahlii (ClFDH) has been recently shown to possess activity in the reverse reaction: the mineralization of CO₂ into formate. In this study, we show the successful homologous expression of ClFDH in Escherichia coli. Biochemical and kinetic characterization of the enzyme revealed that this homologue also demonstrates activity toward CO₂ reduction. Structural analysis of the enzyme through homology modeling is also presented.     

Molecular modeling of the interactions of trichosanthin with four substrate analogs

Trichosanthin (TCS) is a ribosome-inactivating protein (RIP) that possesses N-glycosidase activity. It inactivates ribosomes and arrests protein synthesis by removing a specific adenine from 28S rRNA. A molecular dynamics simulated annealing method was applied to study the binding modes of TCS with substrate analogs, three oligonucleotides GAG, GAGA, and CGAGAG, based on the crystal structures of the stable complexes of TCS with NADPH and with the reaction product adenine. A water molecule proposed to be responsible for hydrolyzing the N-glycosidic bond was included in the model. All the oligoribonucleotides can dock into the active cleft of TCS without unfavorable contacts. The interaction energies between TCS and the three oligonucleotides were calculated. The interactions of TCS with NADH were also studied by a molecular dynamics simulated annealing method. The interaction energy between NADH and TCS was compared with that between NADPH and TCS, showing that the lack of 2-phosphate group leads to an energy rise of 20 kcal/mol.     

Molecular dynamics simulation of lactate dehydrogenase adsorption onto pristine and carboxylic-functionalized graphene

ABSTRACT

Lactate dehydrogenase (LDH) is a tetrameric enzyme which is composed of two subunits known as LDHA and LDHB, which are encoded by the LDHA and LDHB genes respectively. LDH catalyses the last step in anaerobic glycolysis through the reversible conversion of pyruvate to lactate via coupled oxidation of NADH cofactor. The LDHA plays an important regulatory role in anaerobic glycolysis, by catalysing the final step of the process. Therefore, it is likely that increases in the expression level of LDHA in cancer cells could facilitate the efficiency of anaerobic glycolysis. Measuring the level of serum LDHA is a key step in the diagnosis of many cancer types. In this study, the adsorption, stability, and dynamics of LDHA on the surface of pristine graphene (PG) and carboxylated graphene (COOH-Graphene) were investigated using its molecular dynamics simulation. Variations in root mean square deviation, root mean square fluctuation, solvent accessible surface area and adsorption energy of the LDHA during the simulation were calculated to analyse the effect of PG and COOH-Graphene on the overall conformation of LDHA. Results showed that the adsorption of LDHA on COOH-Graphene is mostly mediated by electrostatic interactions, whereas on the PG, both Van der Waals and π-π interactions are prominent.     

Refined models of New Delhi metallo-beta-lactamase-1 with inhibitors: an QM/MM modeling study

New Delhi metallo-beta-lactamase 1 (NDM-1) has been identified as a potential target for the treatment of multi-drug resistance bacterial infections. We used molecular docking, normal MD, SIE, QM/MM MD simulations, QM/MM GBSA binding free energy, and QM/MM GBSA alanine-scanning mutagenesis techniques to investigate interactions of the NDM-1 with 11 inhibitors (Tigecycline, BAL30072, D-captopril, Penicillin G, Ampicillin, Carbenicillin, Cephalexin, Cefaclor, Nitrocefin, Meropenem, and Imipenem). From our normal MD and QM/MM simulations, the correlation coefficients between the predicted binding free energies and experimental values are .88 and .93, respectively. Then simulations, which combined QM/MM/GBSA and alanine-scanning mutagenesis techniques, were performed and our results show that two residues (Lys211 and His250) have the strongest impact on the binding affinities of the 11 NDM-1/inhibitors. Therefore, our approach theoretically suggests that the two residues (Lys211 and His250) are responsible for the selectivity of NDM-1 associated inhibitors.     

Molecular dynamic modeling of CYP51B in complex with azole inhibitors

Cytochrome P450 14α-sterol demethylase (CYP51), the key enzyme in sterol biosynthesis pathway, is an important target protein of cholesterol-lowering agents, antifungal drugs, and herbicides. CYP51B enzyme is one of the CYP51 family members. In the present study, we have chosen four representative inhibitors of CYP51B: diniconazole (Din), fluconazole (Flu), tebuconazole (Teb), and voriconazole (Vor), and launched to investigate the binding features of CYP51B-inhibitor and gating characteristics via molecular docking and molecular dynamics (MD) simulations. The results show that the ranking of binding affinities among these inhibitors is Din > Teb > Vor > Flu. Din shows the strongest binding affinity, whereas Flu shows the weakest binding affinity. More importantly, based on the calculated binding free energy results, we hypothesize that the nonpolar interactions are the most important contributors, and three key residues (Thr77, Ala258, and Lys454) play crucial role in protein-inhibitor binding. Besides, residue Phe180 may play a molecular switch role in the process of the CYP51B-Teb and CYP51B-Vor binding. Additionally, Tunnel analysis results show that the major tunnel of Din, Flu, and Teb is located between helix K, FG loop, and β4 hairpin (Tunnel II).The top ranked possible tunnel (Tunnel II) corresponds to Vor exits through helix K, F and helix J. This study further revealed the CYP51B relevant structural characteristics at the atomic level as well as provided a basis for rational design of new and more efficacious antifungal agents.     

Sensitivity of molecular dynamics simulations to the choice of the X-ray structure used to model an enzymatic reaction

A subject of great practical importance that has not received much attention is the question of the sensitivity of molecular dynamics simulations to the initial X-ray structure used to set up the calculation. We have found two cases in which seemingly similar structures lead to quite different results, and in this article we present a detailed analysis of these cases. The first case is acyl-CoA dehydrogenase, and the chief difference of the two structures is attributed to a slight shift in a backbone carbonyl that causes a key residue (the proton-abstracting base) to be in a bad conformation for reaction. The second case is xylose isomerase, and the chief difference of the two structures appears to be the ligand sphere of a Mg2+ metal cofactor that plays an active role in catalysis.     

Computer modeling and molecular dynamics simulations of ligand bound complexes of bovine angiogenin: dinucleotide topology at the active site of RNase a family proteins

We have undertaken the modeling of substrate-bound structures of angiogenin. In our recent study, we modeled the dinucleotide ligand binding to human angiogenin. In the present study, the substrates CpG, UpG, and CpA were docked onto bovine angiogenin. This was achieved by overcoming the problem of an obstruction to the B1 site by the C-terminus and identifying residues that bind to the second base. The modeled complexes retain biochemically important interactions. The docked models were subjected to 1 ns of molecular dynamics, and structures from the simulation were refined by using simulated annealing. Our models explained the enzyme's specificity for both B1 and B2 bases as observed experimentally. The nature of binding of the dinucleotide substrate was compared with that of the mononucleotide product. The models of these complexes were also compared with those obtained earlier with human angiogenin. On the basis of the simulations and annealed structures, we came up with a consensus topology of dinucleotide ligands that binds to human and bovine angiogenins. This dinucleotide conformation can serve as a starting model for ligand-bound complex structures for RNase A family of proteins. We demonstrated this capability by generating the complex structure of CpA bound to eosinophil-derived neurotoxin (EDN) by fitting the consensus topology of CpA to the crystal structure of native EDN.     

Overview of simulation studies on the enzymatic activity and conformational dynamics of the GTPase Ras

Over the last 40 years, we have learnt a great deal about the Ras onco-proteins. These intracellular molecular switches are essential for the function of a variety of physiological processes, including signal transduction cascades responsible for cell growth and proliferation. Molecular simulations and free energy calculations have played an essential role in elucidating the conformational dynamics and energetics underlying the GTP hydrolysis reaction catalysed by Ras. Here we present an overview of the main lessons from molecular simulations on the GTPase reaction and conformational dynamics of this important anti-cancer drug target. In the first part, we summarise insights from quantum mechanical and combined quantum mechanical/molecular mechanical simulations as well as other free energy methods and highlight consensus viewpoints as well as remaining controversies. The second part provides a very brief overview of new insights emerging from large-scale molecular dynamics simulations. We conclude with a perspective regarding future studies of Ras where computational approaches will likely play an active role.