Structural insights of the MenD from Escherichia coli reveal ThDP affinity

MenD (2-succinyl-5-enolpyruvyl-6-hydroxy-3-cyclohexadiene-1-carboxylate) synthase belongs to the superfamily of thiamin diphosphate-dependent decarboxylases, which converts isochorismate and 2-oxoglutarate to SHCHC, pyruvate, and carbon dioxide. Here, we report the first crystal structure of apo-MenD from Escherichia coli determined in tetragonal crystal form. The subunit displays the typical three-domain structure observed for ThDP-dependent enzymes. Analytical gel filtration shows that EcMenD behaves as a dimer as well as a tetramer. Circular dichroism and isothermal calorimetry results confirm EcMenD dependency on ThDP, which concomitantly helps to stabilize with better configuration.     

Purification and crystallization of benzoylformate decarboxylase.

A new large-scale purification method for benzoylformate decarboxylase from Pseudomonas putida has allowed us to undertake an X-ray crystallographic study of the enzyme. The previously observed instability of the enzyme was overcome by addition of 100 microM thiamine pyrophosphate to buffers used in the purification. The final enzyme preparation was more than 97% pure, as determined by denaturing gel electrophoresis and densitometry. The mobility of the enzyme on a gel filtration column indicates that it is a tetramer of 57-kDa subunits. Large, single crystals of benzoylformate decarboxylase were grown from solutions of buffered polyethylene glycol 400, pH 8.5. The crystals diffract to beyond 1.6 A resolution and are stable for days to X-ray radiation. Analysis of X-ray data from the crystals, along with the newly determined quaternary structure, identifies the space group as I222. The unit cell dimensions are a = 82 A, b = 97 A, c = 138 A. An average Vm value for the crystals is consistent with one subunit per asymmetric unit. The subunits of the tetramer must be arranged with tetrahedral 222 symmetry.     

Mandelate pathway of Pseudomonas putida: sequence relationships involving mandelate racemase, (S)-mandelate dehydrogenase, and benzoylformate decarboxylase and expression of benzoylformate decarboxylase in Escherichia coli

The genes that encode the five known enzymes of the mandelate pathway of Pseudomonas putida (ATCC 12633), mandelate racemase (mdlA), (S)-mandelate dehydrogenase (mdlB), benzoylformate decarboxylase (mdlC), NAD(+)-dependent benzaldehyde dehydrogenase (mdlD), and NADP(+)-dependent benzaldehyde dehydrogenase (mdlE), have been cloned. The genes for (S)-mandelate dehydrogenase and benzoylformate decarboxylase have been sequenced; these genes and that for mandelate racemase [Ransom, S. C., Gerlt, J. A., Powers, V. M., & Kenyon, G. L. (1988) Biochemistry 27, 540] are organized in an operon (mdlCBA). Mandelate racemase has regions of sequence similarity to muconate lactonizing enzymes I and II from P. putida. (S)-Mandelate dehydrogenase is predicted to be 393 amino acids in length and to have a molecular weight of 43,352; it has regions of sequence similarity to glycolate oxidase from spinach and ferricytochrome b2 lactate dehydrogenase from yeast. Benzoylformate decarboxylase is predicted to be 499 amino acids in length and to have a molecular weight of 53,621; it has regions of sequence similarity to enzymes that decarboxylate pyruvate with thiamin pyrophosphate as cofactor. These observations support the hypothesis that the mandelate pathway evolved by recruitment of enzymes from preexisting metabolic pathways. The gene for benzoylformate decarboxylase has been expressed in Escherichia coli with the trc promoter, and homogeneous enzyme has been isolated from induced cells.     

A profound computational study to prioritize the natural compound inhibitors against the P. falciparum orotidine-5-monophosphate decarboxylase enzyme

Abstract

In the current contribution, a multicomplex-based pharmacophore modeling approach was employed on the structural proteome of Plasmodium falciparum orotidine-5-monophosphate decarboxylase enzyme (PfOMPDC). Among the constructed pharmacophore models, the representative hypotheses were selected as the primary filter to screen the molecules with the complementary features responsible for showing inhibition. Thereafter, auxiliary evaluations were performed on the screened candidates via drug-likeness and molecular docking studies. Subsequently, the stability of the docked protein-ligand complexes was scrutinized by employing molecular dynamics simulations and molecular mechanics-Poisson Boltzmann surface area based free binding energy calculations. The stability the docked candidates was compared with the highly active crystallized inhibitor (3S9Y-FNU) to seek more potential candidates. All the docked molecules displayed stable dynamic behavior and high binding free energy in comparison to 3S9Y-FNU. The employed workflow resulted in the retrieval of five drug-like candidates with diverse scaffolds that may show inhibitory activity against PfOMPDC and could be further used as the novel scaffold to develop novel antimalarials.

Communicated by Ramaswamy H. Sarma     

Understanding interaction and dynamics of water molecules in the epoxy via molecular dynamics simulation

The robust structural integrity of the epoxy plays an important role in ensuring the long-term service life of its applications, which is affected by the absorbed moisture. In order to understand the mechanism of the moisture effect, the knowledge of the interaction and dynamics of the water molecules inside the epoxy is of great interest. Molecular dynamics simulation is used in this work to investigate the structure and bonding behaviour of the water molecules in the highly cross-linked epoxy network. When the moisture concentration is low, the water molecules are well dispersed in the cross-linked structure and located in the vicinity of the epoxy functional groups, which predominantly form the hydrogen bond (H-bond) with the epoxy network, resulting in the low water mobility in the epoxy. At the high concentration, the water favourably forms the large cluster due to the predominant water–water H-bond interaction, and the water molecules diffuse primarily inside the cluster, which leads to the high water mobility and the accelerated H-bond dynamics. The variation of the bonding behaviour and dynamics of the water molecules reported here could be exploited to understand the material change and predict the long-term performance of the epoxy-based products during the intended service life.     

Understanding the basis of a class of paradoxical mutations in AraC through simulations

Most mutations at position 15 in the N‐terminal arm of the regulatory protein AraC leave the protein incapable of responding to arabinose and inducing the proteins required for arabinose catabolism. Mutations at other positions of the arm do not have this behavior. Simple energetic analysis of the interactions between the arm and bound arabinose do not explain the uninducibility of AraC with mutations at position 15. Extensive molecular dynamics (MD) simulations, carried out largely on the Open Science Grid, were done of the wild‐type protein with and without bound arabinose and of all possible mutations at position 15, many of which were constructed and measured for this work. Good correlation was found for deviation of arm position during the simulations and inducibility as measured in vivo of the same mutant proteins. Analysis of the MD trajectories revealed that preservation of the shape of the arm is critical to inducibility. To maintain the correct shape of the arm, the strengths of three interactions observed to be strong in simulations of the wild‐type AraC protein need to be preserved. These interactions are between arabinose and residue 15, arabinose and residues 8–9, and residue 13 and residue 15. The latter interaction is notable because residues L9, Y13, F15, W95, and Y97 form a hydrophobic cluster which needs to be preserved for retention of the correct shape. Proteins 2013. © 2012 Wiley Periodicals, Inc.     

Understanding the effect of tert-butanol on Candida antarctica lipase B using molecular dynamics simulations

The use of organic solvents as reaction media for enzymatic reactions has many advantages. Several organic solvents have been proposed as reaction media, especially for transesterifications using Candida antarctica lipase B (CalB). Among organic solvents, tert-butanol is associated with an enhanced conversion rate in bio-diesel production. Thus, it is necessary to understand the effect of tert-butanol on CalB to explain the high-catalytic efficiency compared with the reaction in other hydrophilic organic solvents. In this study, the effects of tert-butanol on the structure of CalB were investigated by MD simulations. The overall flexibility was increased in the presence of tert-butanol. The substrate entrance and the binding pocket size of CalB in tert-butanol were maintained as in TIP3P water. The distance between the catalytic residues of CalB in tert-butanol indicated a higher likelihood of forming hydrogen bonds. These structural analyses could be useful for understanding the effect of tert-butanol on lipase transesterification.     

Evaporation and Condensation at a Liquid Surface of Methanol

Abstract

Evaporation and condensation processes at a liquid surface of methanol were investigated at room temperature with a microcanonical molecular dynamics computer simulation technique. The condensation coefficient (the number ratio of condensed molecules to incident ones) was estimated by comparing two types of autocorrelation functions, and found to be less than unity, which is in qualitative agreement with experiments. A variety of complex dynamic phenomena were observed at the surface.     

Understanding discrimination by the ribosome: stability testing and groove measurement of codon-anticodon pairs

The ribosome must discriminate between correct and incorrect tRNAs with sufficient speed and accuracy to sustain an adequate rate of cell growth. Here, we report the results of explicit solvent molecular dynamics simulations, which address the mechanism of discrimination by the ribosome. The universally conserved 16S rRNA base A1493 and the kink in mRNA between A and P sites amplify differences in stability between cognate and near-cognate codon-anticodon pairs. Destabilization by the mRNA kink also provides a geometric explanation for the higher error rates observed for mismatches in the first codon position relative to mismatches in the second codon position. For more stable near-cognates, the repositioning of the universally conserved bases A1492 and G530 results in increased solvent exposure and an uncompensated loss of hydrogen bonds, preventing correct codon-anticodon-ribosome interactions from forming.     

Binding mode exploration of LuxR-thiazolidinedione analogues,e-pharmacophore-based virtual screening in the designing of LuxR inhibitors and its biological evaluation

Master quorum sensing (QS) regulator LuxR of Vibrio harveyi is a unique member of the TetR protein superfamily. Recent studies have demonstrated the contribution of thiazolidinedione analogues in blocking QS by decreasing the DNA-binding ability of LuxR. However, the precise mechanism of thiazolidinedione analogues binding to LuxR is still unclear. In the present study, molecular docking combined with molecular dynamics (MD) simulations was performed to understand the mechanism of ligand binding to the protein. The binding pattern of thiazolidinedione analogues showed strong hydrogen bonding interactions with the amine group (NH) of polar amino acid residue Asn133 and carbonyl (C=O) interaction with negatively charged amino acid residue Gln137 in the binding site of LuxR. The stability of the protein–ligand complexes was confirmed by running 50 ns of MD simulations. Further, the four-featured pharmacophore hypothesis (AHHD) consists of one acceptor (A), two hydrophobic regions (HH) and one donor (D) group was used to screen compounds from ChemBridge database. The identified hit molecules were shown to have excellent pharmacokinetic properties under the acceptable range. Based on the computational studies, ChemBridge_5343641 was selected for in vitro assays. The 1-(4-chlorophenoxy)-3-[(4,6-dimethyl-2-pyrimidinyl)thio]-2-propanol (ChemBridge_5343641) showed significant reduction in bioluminescence in a dose-dependent manner. In addition, ChemBridge_5343641 inhibits biofilm formation and motility in V. harveyi. The result from the study suggests that ChemBridge_5343641 could serve as an anti-QS molecule.     

Understanding of known drug-target interactions in the catalytic pocket of neuraminidase subtype N1

To provide detailed information and insight into the drug-target interaction, structure, solvation, and dynamic and thermodynamic properties, the three known-neuraminidase inhibitors-oseltamivir (OTV), zanamivir (ZNV), and peramivir (PRV)-embedded in the catalytic site of neuraminidase (NA) subtype N1 were studied using molecular dynamics simulations. In terms of ligand conformation, there were major differences in the structures of the guanidinium and the bulky groups. The atoms of the guanidinium group of PRV were observed to form many more hydrogen bonds with the surrounded residues and were much less solvated by water molecules, in comparison with the other two inhibitors. Consequently, D151 lying on the 150-loop (residues 147-152) of group-1 neuraminidase (N1, N4, N5, and N8) was considerably shifted to form direct hydrogen bonds with the --OH group of the PRV, which was located rather far from the 150-loop. For the bulky group, direct hydrogen bonds were detected only between the hydrophilic side chain of ZNV and residues R224, E276, and E277 of N1 with rather weak binding, 20-70% occupation. This is not the case for OTV and PRV, in which flexibility and steric effects due to the hydrophobic side chain lead to the rearrangement of the surrounded residues, that is, the negatively charged side chain of E276 was shifted and rotated to form hydrogen bonds with the positively charged moiety of R224. Taking into account all the ligand-enzyme interaction data, the gas phase MM interaction energy of -282.2 kcal/mol as well as the binding free energy (DeltaG(binding)) of -227.4 kcal/mol for the PRV-N1 are significantly lower than those of the other inhibitors. The ordering of DeltaG(binding) of PRV < ZNV < OTV agrees well with the ordering of experimental IC(50) value.     

Exploring the molecular basis of action of ring D aromatic steroidal antiestrogens

Salpichrolides are natural plant steroids that contain an unusual six‐membered aromatic ring D. We recently reported that some of these compounds, and certain analogs with a simplified side chain, exhibited antagonist effects toward the human estrogen receptor (ER), a nuclear receptor whose endogenous ligand has an aromatic A ring (estradiol). Drugs acting through the inhibition or modulation of ERs are frequently used as a hormonal therapy for ER(+) breast cancer. Previous results suggested that the aromatic D ring was a key structural motif for the observed activity; thus, this modified steroid nucleus may provide a new scaffold for the design of novel antiestrogens. Using molecular dynamics (MD) simulation we have modeled the binding mode of the natural salpichrolide A and a synthetic analog with an aromatic D ring within the ERα. These results taken together with the calculated energetic contributions associated to the different ligand‐binding modes are consistent with a preferred inverted orientation of the steroids in the ligand‐binding pocket with the aromatic ring D occupying a position similar to that observed for the A ring of estradiol. Major changes in both dynamical behavior and global positioning of H11 caused by the loss of the ligand–His524 interaction might explain, at least in part, the molecular basis of the antagonism exhibited by these compounds. Using steered MD we also found a putative unbinding pathway for the steroidal ligands through a cavity formed by residues in H3, H7, and H11, which requires only minor changes in the overall receptor conformation. Proteins 2015; 83:1297–1306. © 2015 Wiley Periodicals, Inc.     

Understanding the mode of action of L-nucleosides as antiviral agents: a molecular modeling approach

Computer modeling studies have been performed on the several pairs of D- and L-nucleoside inhibitors with the HIV-1 RT model. Additionally, clinically important M184V mutation, which confers the viral resistance against 3TC and FTC, were studied by the same modeling system.     

Principal component and normal mode analysis of proteins; a quantitative comparison using the GroEL subunit

Principal component analysis (PCA) and normal mode analysis (NMA) have emerged as two invaluable tools for studying conformational changes in proteins. To compare these approaches for studying protein dynamics, we have used a subunit of the GroEL chaperone, whose dynamics is well characterized. We first show that both PCA on trajectories from molecular dynamics (MD) simulations and NMA reveal a general dynamical behavior in agreement with what has previously been described for GroEL. We thus compare the reproducibility of PCA on independent MD runs and subsequently investigate the influence of the length of the MD simulations. We show that there is a relatively poor one-to-one correspondence between eigenvectors obtained from two independent runs and conclude that caution should be taken when analyzing principal components individually. We also observe that increasing the simulation length does not improve the agreement with the experimental structural difference. In fact, relatively short MD simulations are sufficient for this purpose. We observe a rapid convergence of the eigenvectors (after ca. 6 ns). Although there is not always a clear one-to-one correspondence, there is a qualitatively good agreement between the movements described by the first five modes obtained with the three different approaches; PCA, all-atoms NMA, and coarse-grained NMA. It is particularly interesting to relate this to the computational cost of the three methods. The results we obtain on the GroEL subunit contribute to the generalization of robust and reproducible strategies for the study of protein dynamics, using either NMA or PCA of trajectories from MD simulations.     

Collective motions in proteins: a covariance analysis of atomic fluctuations in molecular dynamics and normal mode simulations.

A method is described for identifying collective motions in proteins from molecular dynamics trajectories or normal mode simulations. The method makes use of the covariances of atomic positional fluctuations. It is illustrated by an analysis of the bovine pancreatic trypsin inhibitor. Comparison of the covariance and cross-correlation matrices shows that the relative motions have many similar features in the different simulations. Many regions of the protein, especially regions of secondary structure, move in a correlated manner. Anharmonic effects, which are included in the molecular dynamics simulations but not in the normal analysis, are of some importance in determining the larger scale collective motions, but not the more local fluctuations. Comparisons of molecular dynamics simulations in the present and absence of solvent indicate that the environment is of significance for the long-range motions.     

Evidences for the action mechanism of angiotensin II and its analogs on Plasmodium sporozoite membranes

Malaria is an infectious disease responsible for approximately one million deaths annually. Oligopeptides such as angiotensin II (AII) and its analogs are known to have antimalarial effects against Plasmodium gallinaceum and Plasmodium falciparum. However, their mechanism of action is still not fully understood at the molecular level. In the work reported here, we investigated this issue by comparing the antimalarial activity of AII with that of (i) its diastereomer formed by only d ‐amino acids; (ii) its isomer with reversed sequence; and (iii) its analogs restricted by lactam bridges, the so‐called VC5 peptides. Data from fluorescence spectroscopy indicated that the antiplasmodial activities of both all‐D‐AII and all‐D‐VC5 were as high as those of the related peptides AII and VC5, respectively. In contrast, retro‐AII had no significant effect against P. gallinaceum. Conformational analysis by circular dichroism suggested that AII and its active analogs usually adopted a β‐turn conformation in different solutions. In the presence of membrane‐mimetic micelles, AII had also a β‐turn conformation, while retro‐AII was random. Molecular dynamics simulations demonstrated that the AII chains were slightly more bent than retro‐AII at the surface of a model membrane. At the hydrophobic membrane interior, however, the retro‐AII chain was severely coiled and rigid. AII was much more flexible and able to experience both straight and coiled conformations. We took it as an indication of the stronger ability of AII to interact with membrane headgroups and promote pore formation. Copyright © 2016 European Peptide Society and John Wiley & Sons, Ltd.     

Improving the carboligase activity of benzoylformate decarboxylase from Pseudomonas putida by a combination of directed evolution and site-directed mutagenesis

Benzoylformate decarboxylase (BFD) from Pseudomonas putida was subjected to directed molecular evolution to generate mutants with increased carboligase activity which is a side reaction of the enzyme. After a single round of random mutagenesis mutants were isolated which exhibited a 5-fold increased carboligase activity in aqueous buffer compared to the wild-type enzyme with a high enantiomeric excess of the product (S)-2-hydroxy-1-phenyl-propanone. From the same library, mutants with enhanced carboligase activity in water-miscible organic solvents have been isolated. The selected mutants have been characterized by sequencing, revealing that all mutants carry a mutation at Leu476, which is close to the active site but does not directly interact with the active center. BFD-L476Q has a 5-fold higher carboligase activity than the wild-type enzyme. L476 was subjected to saturation mutagenesis yielding eight different mutants with up to 5-fold increased carboligase activity. Surprisingly, all L476 mutants catalyze the formation of 2-hydroxy-1-phenyl-propanone with significantly higher enantioselectivity than the wild-type enzyme although enantioselectivity was not a selection parameter. Leu476 potentially plays the role of a gatekeeper of the active site of BFD, possibly by controlling the release of the product. The biocatalyst could be significantly improved for its side reaction, the C-C bond formation and for application under conditions that are not optimized in nature.     

Understanding the determinants of stability and folding of small globular proteins from their energetics

The results of minimal model calculations indicate that the stability and the kinetic accessibility of the native state of small globular proteins are controlled by few "hot" sites. By means of molecular dynamics simulations around the native conformation, which describe the protein and the surrounding solvent at the all-atom level, an accurate and compact energetic map of the native state of the protein is generated. This map is further simplified by means of an eigenvalue decomposition. The components of the eigenvector associated with the lowest eigenvalue indicate which hot sites are likely to be responsible for the stability and for the rapid folding of the protein. The comparison of the results of the model with the findings of mutagenesis experiments performed for four small proteins show that the eigenvalue decomposition method is able to identify between 60% and 80% of these (hot) sites.     

Identification of the active site of human mitochondrial malonyl‐coenzyme a decarboxylase: A combined computational study

Malonyl‐CoA decarboxylase (MCD) can control the level of malonyl‐CoA in cell through the decarboxylation of malonyl‐CoA to acetyl‐CoA, and plays an essential role in regulating fatty acid metabolism, thus it is a potential target for drug discovery. However, the interactions of MCD with CoA derivatives are not well understood owing to unavailable crystal structure with a complete occupancy in the active site. To identify the active site of MCD, molecular docking and molecular dynamics simulations were performed to explore the interactions of human mitochondrial MCD (HmMCD) and CoA derivatives. The findings reveal that the active site of HmMCD indeed resides in the prominent groove which resembles that of CurA. However, the binding modes are slightly different from the one observed in CurA due to the occupancy of the side chain of Lys183 from the N‐terminal helical domain instead of the adenine ring of CoA. The residues 300 ? 305 play an essential role in maintaining the stability of complex mainly through hydrogen bond interactions with the pyrophosphate moiety of acetyl‐CoA. Principle component analysis elucidates the conformational distribution and dominant concerted motions of HmMCD. MM_PBSA calculations present the crucial residues and the major driving force responsible for the binding of acetyl‐CoA. These results provide useful information for understanding the interactions of HmMCD with CoA derivatives. Proteins 2016; 84:792–802. © 2016 Wiley Periodicals, Inc.     

Understanding the folding and stability of a designed WW domain protein with replica exchange molecular dynamics simulations

WW domain proteins are usually regarded as simple models for understanding the folding mechanism of β-sheet. CC45 is an artificial protein that is capable of folding into the same structure as WW domain. In this article, the replica exchange molecular dynamics simulations are performed to investigate the folding mechanism of CC45. The analysis of thermal stability shows that β-hairpin 1 is more stable than β-hairpin 2 during the unfolding process. Free energy analysis shows that the unfolding of this protein substantially proceeds through solvating the smaller β-hairpin 2, followed by the unfolding of β-hairpin 1. We further propose the unfolding process of CC45 and the folding mechanism of two β-hairpins. These results are similar to the previous folding studies of formin binding protein 28 (FBP28). Compared with FBP28, it is found that CC45 has more aromatic residues in N-terminal loop, and these residues contact with C-terminal loop to form the outer hydrophobic core, which increases the stability of CC45. Knowledge about the stability and folding behaviour of CC45 may help in understanding the folding mechanisms of the β-sheet and in designing new WW domains.