Free energy calculations elucidate substrate binding,gating mechanism,and tolerance‐promoting mutations in herbicide target 4‐hydroxyphenylpyruvate dioxygenase

4‐Hydroxyphenylpyruvate dioxygenase (HPPD) catalyzes the second reaction in the tyrosine catabolism and is linked to the production of cofactors plastoquinone and tocopherol in plants. This important biological role has put HPPD in the focus of current herbicide design efforts including the development of herbicide‐tolerant mutants. However, the molecular mechanisms of substrate binding and herbicide tolerance have yet to be elucidated. In this work, we performed molecular dynamics simulations and free energy calculations to characterize active site gating by the C‐terminal helix H11 in HPPD. We compared gating equilibria in Arabidopsis thaliana (At) and Zea mays (Zm) wild‐type proteins retrieving the experimentally observed preferred orientations from the simulations. We investigated the influence of substrate and product binding on the open–closed transition and discovered a ligand‐mediated conformational switch in H11 that mediates rapid substrate access followed by active site closing and efficient product release through H11 opening. We further studied H11 gating in At mutant HPPD, and found large differences with correlation to experimentally measured herbicide tolerance. The computational findings were then used to design a new At mutant HPPD protein that showed increased tolerance to six commercially available HPPD inhibitors in biochemical in vitro experiments. Our results underline the importance of protein flexibility and conformational transitions in substrate recognition and enzyme inhibition by herbicides.     

Influence of water activity on the enantioselective esterification of (R,S)-ibuprofen by crosslinked crystals of Candida antarctica lipase B in organic solvent media.

The lipase B from Candida antarctica was purified from a commercial source and crystallized. The microcrystals were crosslinked using glutaraldehyde. The crosslinked crystals were then used to catalyze the esterification of (R,S)-ibuprofen with dodecanol in octane at various water activities. As for the commercial preparation immobilized on acrylic resin, Novozym 435, low water activities foster better enantioselectivity, and the maximum reaction rates are obtained for a(W) = 0.1.     

Optimization of a two‐step process comprising lipase catalysis and thermal cyclization improves the efficiency of synthesis of six‐membered cyclic carbonate from trimethylolpropane and dimethylcarbonate

Six‐membered cyclic carbonates are potential monomers for phosgene and/or isocyanate free polycarbonates and polyurethanes via ring‐opening polymerization. A two‐step process for their synthesis comprising lipase‐catalyzed transesterification of a polyol, trimethylolpropane (TMP) with dimethylcarbonate (DMC) in a solvent‐free system followed by thermal cyclization was optimized to improve process efficiency and selectivity. Using full factorial designed experiments and partial least squares (PLS) modeling for the reaction catalyzed by Novozym®435 (N435; immobilized Candida antarctica lipase B), the optimum conditions for obtaining either high proportion of monocarbonated TMP and TMP‐cyclic‐carbonate (3 and 4), or dicarbonated TMP and monocarbonated TMP‐cyclic‐carbonate (5 and 6) were found. The PLS model predicted that the reactions using 15%–20% (w/w) N435 at DMC:TMP molar ratio of 10–30 can reach about 65% total yield of 3 and 4 within 10 h, and 65%–70% total yield of 5 and 6 within 32–37 h, respectively. High consistency between the predicted results and empirical data was shown with 66.1% yield of 3 and 4 at 7 h and 67.4% yield of 5 and 6 at 35 h, using 18% (w/w) biocatalyst and DMC:TMP molar ratio of 20. Thermal cyclization of the product from 7 h reaction, at 110°C in the presence of acetonitrile increased the overall yield of cyclic carbonate 4 from about 2% to more than 75% within 24 h. N435 was reused for five consecutive batches, 10 h each, to give 3+4 with a yield of about 65% in each run. © 2012 American Institute of Chemical Engineers Biotechnol. Prog., 2013.     

Decoding the structural events in substrate‐gating mechanism of eukaryotic prolyl oligopeptidase using normal mode analysis and molecular dynamics simulations

Prolyl oligopeptidase (POP) is a serine protease, unique for its ability to cleave various small oligopeptides shorter than 30 amino acids. POP is an important drug target since it is implicated in various neurological disorders. Although there is structural evidence that bacterial POPs undergo huge interdomain movements and acquire an “open” state in the substrate‐unbound form, hitherto, no crystal structure is available in the substrate‐unbound domain‐open form of eukaryotic POPs. Indeed, there is no difference between the substrate‐unbound/bound states of eukaryotic POPs. This raises unanswered questions about whether difference in the substrate access pathway exists between bacterial and eukaryotic POPs. Here, we have used normal mode analysis and molecular dynamics to unravel the mechanism of substrate entry in mammalian POPs, which has been debated until now. Motions observed using normal modes of porcine and bacterial POPs were analyzed and compared, augmented by molecular dynamics of these proteins. Identical to bacterial POPs, interdomain opening was found to be the possible pathway for the substrate‐gating in mammals as well. On the basis of our analyses and evidences, a mechanistic model of substrate entry in POPs has been proposed. Up‐down movement of N‐terminal hydrolase domain resulted in twisting motion of two domains, followed by the conformational changes of interdomain loop regions, which facilitate interdomain opening. Similar to bacterial POPs, an open form of porcine POP is also proposed with domain‐closing motion. This work has direct implications for the development of novel inhibitors of mammalian POPs to understand the etiology of various neurological diseases. Proteins 2014; 82:1428–1443. © 2014 Wiley Periodicals, Inc.     

Recognition of LD motifs by the focal adhesion targeting domains of focal adhesion kinase and proline‐rich tyrosine kinase 2‐beta: Insights from molecular dynamics simulations

The focal adhesion kinase (FAK) and the proline‐rich tyrosine kinase 2‐beta (PYK2) are implicated in cancer progression and metastasis and represent promising biomarkers and targets for cancer therapy. FAK and PYK2 are recruited to focal adhesions (FAs) via interactions between their FA targeting (FAT) domains and conserved segments (LD motifs) on the proteins Paxillin, Leupaxin, and Hic‐5. A promising new approach for the inhibition of FAK and PYK2 targets interactions of the FAK domains with proteins that promote localization at FAs. Advances toward this goal include the development of surface plasmon resonance, heteronuclear single quantum coherence nuclear magnetic resonance (HSQC‐NMR) and fluorescence polarization assays for the identification of fragments or compounds interfering with the FAK‐Paxillin interaction. We have recently validated this strategy, showing that Paxillin mimicking polypeptides with 2 to 3 LD motifs displace FAK from FAs and block kinase‐dependent and independent functions of FAK, including downstream integrin signaling and FA localization of the protein p130Cas. In the present work we study by all‐atom molecular dynamics simulations the recognition of peptides with the Paxillin and Leupaxin LD motifs by the FAK‐FAT and PYK2‐FAT domains. Our simulations and free‐energy analysis interpret experimental data on binding of Paxillin and Leupaxin LD motifs at FAK‐FAT and PYK2‐FAT binding sites, and assess the roles of consensus LD regions and flanking residues. Our results can assist in the design of effective inhibitory peptides of the FAK‐FAT: Paxillin and PYK2‐FAT:Leupaxin complexes and the construction of pharmacophore models for the discovery of potential small‐molecule inhibitors of the FAK‐FAT and PYK2‐FAT focal adhesion based functions.     

Simulating the fidelity and the three Mg mechanism of pol η and clarifying the validity of transition state theory in enzyme catalysis

Pol η belongs to the important Y family of DNA polymerases that can catalyze translesion synthesis across sites of damaged DNA. This activity involves the reduced fidelity of Pol η for 8‐oxo‐7,8‐dhyedro‐2′‐deoxoguanosin(8‐oxoG). The fundamental interest in Pol η has grown recently with the demonstration of the importance of a 3rd Mg2+ ion. The current work explores both the fidelity of Pol η and the role of the 3rd metal ion, by using empirical valence bond (EVB) simulations. The simulations reproduce the observed trend in fidelity and shed a new light on the role of the 3rd metal ion. It is found that this ion does not lead to a major catalytic effect, but most probably plays an important role in reducing the product release barrier. Furthermore, it is concluded, in contrast to some implications, that the effect of this metal does not violate transition state theory, and the evaluation of the catalytic effect must conserve the molecular composition upon moving from the reactant to the transition state. Proteins 2017; 85:1446–1453. © 2017 Wiley Periodicals, Inc.     

Is the large plasmid of 120 MDa in Shigella sonnei composed of two DNA segments—90 and 30 MDa?

Abstract The reversibility of adhesion of 3 representative strains of oral streptococci from a phosphate-buffered suspension onto 5 different solid substrata was studied.
Streptococcus mitis T9 (surface free energy γ_b= 39 mJ · m⁻²). Streptococcus sanguis CH3 (γ_b= 95 mJ · m⁻²) and Streptococcus mutans NS (γ_b= 117 mJ · m⁻²) were selected on basis of their surface free energy. Solid substrata were employed with a surface free energy γ_s ranging from 20 mJ · m⁻² for polytetrafluorethylene to 109 mJ · m⁻² for glass. Bacterial suspensions containing 2.5 × 10⁹ cells per ml were incubated with 2 samples of each substratum. After 1 h the number of adhering bacteria was evaluated on one sample, while the second sample was kept for another hour at a 10-fold lower bacterial concentration. Bacteria with a low surface free energy desorbed only from substrata with a high surface free energy, while bacteria with a high surface free energy desorbed from substrata with a low surface free energy. Thus low energy bacterial strains adhered reversibly to high energy substrata and vice versa. Similar observations were made with polystyrene particles. Calculation of the interfacial free energy of adhesion (Δ F _adh) for each bacterial strain as well as for the polystyrene particles showed that a reversible adhesion was associated with a positive Δ F _adh, denoting unfavourable adhesion conditions upon a thermodynamic basis.     

Insights into the binding specificity of wild type and mutated wheat germ agglutinin towards Neu5Acα(2‐3)Gal: a study by in silico mutations and molecular dynamics simulations

Wheat germ agglutinin (WGA) is a plant lectin, which specifically recognizes the sugars NeuNAc and GlcNAc. Mutated WGA with enhanced binding specificity can be used as biomarkers for cancer. In silico mutations are performed at the active site of WGA to enhance the binding specificity towards sialylglycans, and molecular dynamics simulations of 20 ns are carried out for wild type and mutated WGAs (WGA1, WGA2, and WGA3) in complex with sialylgalactose to examine the change in binding specificity. MD simulations reveal the change in binding specificity of wild type and mutated WGAs towards sialylgalactose and bound conformational flexibility of sialylgalactose. The mutated polar amino acid residues Asn114 (S114N), Lys118 (G118K), and Arg118 (G118R) make direct and water mediated hydrogen bonds and hydrophobic interactions with sialylgalactose. An analysis of possible hydrogen bonds, hydrophobic interactions, total pair wise interaction energy between active site residues and sialylgalactose and MM‐PBSA free energy calculation reveals the plausible binding modes and the role of water in stabilizing different binding modes. An interesting observation is that the binding specificity of mutated WGAs (cyborg lectin) towards sialylgalactose is found to be higher in double point mutation (WGA3). One of the substituted residues Arg118 plays a crucial role in sugar binding. Based on the interactions and energy calculations, it is concluded that the order of binding specificity of WGAs towards sialylgalactose is WGA3 > WGA1 > WGA2 > WGA. On comparing with the wild type, double point mutated WGA (WGA3) exhibits increased specificity towards sialylgalactose, and thus, it can be effectively used in targeted drug delivery and as biological cell marker in cancer therapeutics. Copyright © 2014 John Wiley & Sons, Ltd.     

Thermodynamics of Loop Formation in the Denatured State of Rhodopseudomonas palustris Cytochrome c′: Scaling Exponents and the Reconciliation Problem

The observation that denatured proteins yield scaling exponents, ν, consistent with random-coil behavior and yet can also have pockets of residual or nonrandom structure has been termed the “reconciliation problem”. To provide greater insight into the denatured state of a foldable sequence, we have measured histidine-heme loop formation equilibria in the denatured state of a class II c-type cytochrome, cytochrome c′ from Rhodopseudomonas palustris. We have prepared a series of variants that provide His-heme loop stabilities, pK_loop(His), for loop sizes ranging from 10 to 111 residues at intervals of 7 to 11 residues along the sequence of the protein. We observe a scaling exponent for loop formation, ν₃, of 2.5 ± 0.3. Theoretical values for ν₃ range from 1.8 to 2.4; thus, the observed ν₃ is consistent with random-coil behavior. However, in contrast to data for loop formation as a function of loop size obtained with peptides of homogeneous sequence, we observe considerable scatter about the linear dependence of loop stability on loop size. Thus, foldable sequences behave very differently from homogeneous peptide sequences. The observed scatter suggests that there is considerable variation in the conformational properties along the backbone of a foldable sequence, consistent with alternating compact and extended regions. With regard to the reconciliation problem, it is evident that a scaling exponent consistent with a random coil is necessary but not sufficient to demonstrate random-coil behavior.