Improvement of stability of nitrile hydratase via protein fragment swapping

Nitrile hydratase (NHase), which catalyzes the hydration of nitriles to amides, is the key enzyme for the production of amides in industries. However, the poor stability of this enzyme under the reaction conditions is a drawback of its industrial application. In this study, we aimed to improve the stability of NHase (PpNHase) from Pseudomonas putida NRRL-18668 using a homologous protein fragment swapping strategy. One thermophilic NHase fragment from Comamonas testosteroni 5-MGAM-4D and two fragments from Pseudonocardia thermophila JCM3095 were selected to swap the corresponding fragments of PpNHase. Seven chimeric NHases were designed using STAR (site targeted amino recombination) software and molecular dynamics to determine the crossover sites for fragment recombination. All constructed chimeric NHases showed 1.4- to 3.5-fold enhancement in thermostability and six of them become more tolerant to high-concentration product. Notably, one of these NHases, 3AB, exhibited a 1.4 ± 0.05-fold increase in activity compared to the wild-type PpNHase. Circular dichroism spectrum analysis and homology modeling revealed that the 3AB slightly differed in secondary structure from wild-type PpNHase. The 3AB constructed in this study is useful for further industrial application, and the method for designing the chimeric protein using homologous protein fragment swapping without a decrease in activity may be a strategy to improve the stability of other enzymes.     

Crystal structure of cobalt-containing nitrile hydratase.

The crystal structure of cobalt-containing nitrile hydratase from Pseudonocardia thermophila JCM 3095 at 1.8 A resolution revealed the structure of the noncorrin cobalt at the catalytic center. Two cysteine residues (alphaCys(111) and alphaCys(113)) coordinated to the cobalt were posttranslationally modified to cysteine-sulfinic acid and to cysteine-sulfenic acid, respectively, like in iron-containing nitrile hydratase. A tryptophan residue (betaTrp(72)), which may be involved in substrate binding, replaced the tyrosine residue of iron-containing nitrile hydratase. The difference seems to be responsible for the preference for aromatic nitriles rather than aliphatic ones of cobalt-containing nitrile hydratase.     

Mechanistic study of palladium-catalyzed telomerization of 1,3-butadiene with methanol

The Pd-catalyzed telomerization in the presence of phosphine and carbene ligands has been computed. It is shown that the C–C coupling of the less stable complex A with one trans- and one cis-butadiene in syn orientation forms the most stable intermediate B and is favorable both kinetically and thermodynamically. Protonation of B leads to equilibrium of the two most stable isomers of intermediate C. The overall regioselectivity is favored thermodynamically.      

The effect of a tightly bound water molecule on scaffold diversity in the computer-aided de novo ligand design of CDK2 inhibitors

We have determined the effects that tightly bound water molecules have on the de novo design of cyclin-dependent kinase-2 (CDK2) ligands. In particular, we have analyzed the impact of a specific structural water molecule on the chemical diversity and binding mode of ligands generated through a de novo structure-based ligand generation method in the binding site of CDK2. The tightly bound water molecule modifies the size and shape of the binding site and we have found that it also imposed constraints on the observed binding modes of the generated ligands. This in turn had the indirect effect of reducing the chemical diversity of the underlying molecular scaffolds that were able to bind to the enzyme satisfactorily.      

Molecular models of protein kinase 6 from Plasmodium falciparum

Cyclin-dependent kinases (CDKs) have been identified as potential targets for development of drugs, mainly against cancer. These studies generated a vast library of chemical inhibitors of CDKs, and some of these molecules can also inhibit kinases identified in the Plasmodium falciparum genome. Here we describe structural models for Protein Kinase 6 from P. falciparum (PfPK6) complexed with Roscovitine and Olomoucine. These models show clear structural evidence for differences observed in the inhibition, and may help designing inhibitors for PfPK6 generating new potential drugs against malaria. Figure Ribbon diagram of PfPK6 complexed with a roscovitine and b olomoucine     

Docking studies suggest ligand-specific δ-opioid receptor conformations

An automated docking procedure was used to study binding of a series of δ-selective ligands to three models of the δ-opioid receptor. These models are thought to represent the three ligand-specific receptor conformations. Docking results are in agreement with point mutation studies and suggest that different ligands—agonists and antagonists—may bind to the same binding site under different receptor conformations. Docking to different receptor models (conformations) also suggests that by changing to a receptor-specific conformation, the receptor may open or close different binding sites to other ligands. Figure  Ligands 5 (green) and 6 (orange) in bindingpocket BP1 of the R1 δ-opioid receptor model     

Effects of Nitriles and Amides on the Growth and Nitrile Hydratase Activity of the Rhodococcus sp. Strain gt1

Effects of some nitriles and amides, as well as glucose and ammonium, on the growth and the nitrile hydratase (EC 4.2.1.84) activity of the Rhodococcus sp. strain gt1 isolated from soil were studied. The activity of nitrile hydratase mainly depended on the carbon and nitrogen supply to cells. The activity of nitrile hydratase was high in the presence of glucose and ammonium at medium concentrations and decreased at concentrations of glucose of more than 0.3%. Saturated unsubstituted aliphatic nitriles and amides were found to be a good source of nitrogen and carbon. However, the presence of nitriles and amides in the medium was not absolutely necessary for the expression of the activity of nitrile hydratase of the Rhodococcus sp. strain gt1.     

Isolation,identification and characterization of <Emphasis Type="Italic">Bacillus subtilis</Emphasis> ZJB-063, a versatile nitrile-converting bacterium

Strain ZJB-063, a versatile nitrile-amide-degrading strain, was newly isolated from soil in this study. Based on morphology, physiological tests, Biolog and the 16S rDNA sequence, strain ZJB-063 was identified as Bacillus subtilis. ZJB-063 exhibited nitrilase activity without addition of inducers, indicating that the nitrilase in B. subtilis ZJB-063 is constitutive. Interestingly, the strain exhibited nitrile hydratase and amidase activity with the addition of ɛ-caprolactam. Moreover, the substrate spectrum altered with the alteration of enzyme systems due to the addition of ɛ-caprolactam. The constitutive nitrilase was highly specific for arylacetonitriles, while the nitrile hydratase/amidase in B. subtilis ZJB-063 could not only hydrolyze arylacetonitriles but also other nitriles including some aliphatic nitriles and heterocyclic nitriles. Despite comparatively low activity, the amidase of hydratase/amidase system was effective in converting amides to acids. The versatility of this strain in the hydrolysis of various nitriles and amides makes it a potential biocatalyst in organic synthesis.     

CHIH-DFT determination of the molecular structure and IR and UV spectra of solanidine

Solanidine is the steroidal aglycon of some potato glycoalkaloids and a very important precursor for the synthesis of hormones and some pharmacologically active compounds. In this work, we make use of a new chemistry model within Density Functional Theory, called CHIH-DFT, to calculate the molecular structure of solanidine, as well to predict its infrared and ultraviolet spectra. The calculated values are compared with the experimental data available for this molecule as a means of validation of our proposed chemistry model. Figure Molecular structure of solanidine calculated with the CHIH(small) model chemistry     

A computational approach to the synthesis of 1,3,5-thiadiazinane-2-thiones in aqueous medium: theoretical evidence for water-promoted heterocyclization

Based on experimental evidence and DFT studies, a probable cyclization route to 1,3,5-thiadiazinanes-2-thiones in aqueous medium is proposed. Experimental facts suggest the formation of a {[hydroxymethyl (substituted) carbamothioyl] sulfanyl}methanol intermediate via reaction of dithiocarbamate (DTC) and formaldehyde. Nucleophilic addition of glycine to this intermediate generates an adduct that undergoes intramolecular heterocyclization via an S_N2 reaction. Computational calculations predict an active role of water in the reaction mechanism that promotes intramolecular cyclization. Figure Energy profile of the proposed reaction mechanism for the synthesis of thiadiazinane-2-thione ring 11 in aqueous medium from a (hydroxymethylcarbamothioyl)sulfanylmethanol intermediate, 9     

DFT investigation of the 1-octene metathesis reaction mechanism with the Phobcat precatalyst

The productive self-metathesis reaction of 1-octene in the presence of the Phobcat precatalyst [RuCl₂(Phoban-Cy)₂(=CHPh)] using density functional theory was investigated and compared to the Grubbs 1 precatalyst [RuCl₂(PCy₃)₂(=CHPh)]. At the GGA-PW91/DNP level, the geometry optimization of all the participating species and the PES scans of the various activation and catalytic cycles in the dissociative mechanism were performed. The formation of the catalytically active heptylidene species is kinetically and thermodynamically favored, while the formation of trans-tetradecene is thermodynamically favored.      

ONIOM DFT/PM3 calculation on the interaction between STI-571 and abelson tyrosine kinase

The ONIOM2 (B3LYP/6–31G (d, p): PM3) and B3LYP/6–31G (d, p) methods were applied to investigate the interaction between STI-571 and abelson tyrosine kinase binding site. The complex of N-[4-methyl-3-(4-pyridin-3-yl-pyrimidin-2-ylamino)- phenyl]-benzamide (part of STI-571) and related 16 amino acid residues were found at B3LYP/6–31G (d, p) level to have hydrogen bonds and π....π stacking interaction, their binding energy via HAF optimization was −20.4 kcal mol⁻¹. The results derived from this study agreed well with the reported observation. Figure Optimized structure of STI-571 and Thr315 in abelson tyrosine kinase based on ONIOM2 method     

Ligation of Aza bases to the AgF<Subscript>2</Subscript> molecule: a theoretical study

We have analyzed the electronic structure and chemical bonding for molecular adducts of the Ag(II)F₂ molecule with various aza Lewis bases including ammonia, nitriles, secondary amines, and their derivatives exhibiting various degrees of fluorination. Density functional theory calculations indicate that a progressive shift occurs of the spin density from the Ag center towards the coordinating nitrogen atoms of aza ligands, as the ligation energy increases. Chemistry of Ag(II) might be extended with little effort beyond the known aza connections, to include nitriles, perfluorinated nitriles and perfluorinated amines. Figure Properties of a variety of novel adducts of the AgF₂ molecule with two aza bases (L), possible precursors of the AgF₂L₂ extended solids, were assessed by the DFT calculations Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. This work is dedicated to memory of Wojciech Ochmański, unforgettable person, good-hearted man, whose craftsmanship in work was second-to-none.     

Experimental and theoretical investigations of spectroscopic properties of azobenzene derivatives in solution

The UV-Vis spectra of series of polymethylmethacrylate (PMMA) copolymers with attached trans-azobenzene derivatives were measured in 1,1,2-trichloroethane. In order to gain some insight into the recorded spectra, the quantum chemical calculations were performed for the substituted azobenzenes using both configuration interaction with single excitations method (CIS) as well as density functional theory (DFT) with B3LYP and PBE0 functionals. The calculations were performed in solvent. In particular, we found that the PBE0 excitation energies are in very good agreement with the experimental data. Figure The plots of orbital contour surfaces for molecule II. The molecular orbitals were calculated at the PBE0/6-311++G(d,p) level of theory. The upper plot presents contour surface of HOMO and the lower presents contour surface of LUMO. Shown are the contour surfaces of orbital amplitude 0.04 (red) and -0.04 (blue)     

A density functional study towards substituent effects on anion sensing with urea receptors

Effects of substituents on anion binding in different urea based receptors have been examined using density functional (B3LYP/6-311+G**) level of theory. The complexes formed by a variety of substituted urea with a halide anion (fluoride) and an oxy-anion (acetate) have been calculated. The stronger complexes were predicted for receptors with fluoride ion than that of acetate ion, however, in water the preference was found to be reversed. The pK _a calculations showed the preferred sites of deprotonation for positional isomers, while interacting with anions. The position of the substituent in the receptor, however, could change the preferred sites of deprotonation compared to the site predicted with pK _a values.      

Design and synthesis of thiourea based receptor containing naphthalene as oxalate selective sensor

The thiourea based receptor containing naphthalene groups (1), has been successfully designed and synthesized for application as an oxalate receptor. A density functional theory at B3LYP/6-31G(d,p) level of theory has been applied to predict the binding ability between 1 and selected anions, i.e., oxalate, malonate, succinate, glutarate, dihydrogen phosphate, and hydrogen sulphate. Calculation results point out that receptor 1 shows the strongest interaction to oxalate ion with the binding free energy of 172.48 kcal mol⁻¹. The recognition ability of 1 to the selected anions has been also investigated by means of the absorption and emission techniques. Experimental results are in excellent agreement with the calculation data in which receptor 1 shows highly selective for oxalate ion over the other anions with logβ of 3.82 (0.02) M⁻¹ by means of the size of binding cavity.      

3D-QSAR studies of Dipeptidyl peptidase IV inhibitors using a docking based alignment

Dipeptidyl peptidase IV (DPP-IV) deactivates the incretin hormones GLP-1 and GIP by cleaving the penultimate proline or alanine from the N-terminal (P1-position) of the peptide. Inhibition of this enzyme will prevent the degradation of the incretin hormones and maintain glucose homeostasis; this makes it an attractive target for the development of drugs for diabetes. This paper reports 3D-QSAR analysis of several DPP-IV inhibitors, which were aligned by the receptor-based technique. The conformation of the molecules in the active site was obtained through docking methods. The QSAR models were generated on two training sets composed of 74 and 25 molecules which included phenylalanine, thiazolidine, and fluorinated pyrrolidine analogs. The 3D-QSAR models are robust with statistically significant r², q², and values. The CoMFA and CoMSIA models were used to design some new inhibitors with several fold higher binding affinity. Figure The CoMFA contours around molecule D1T155 (a) steric contours - favored (green); disfavored (yellow) (b) electrostatic contours - electropositive (blue); electronegative (red)     

Molecular dynamics study of the initial stages of catalyzed single-wall carbon nanotubes growth: force field development

Effective force fields for Ni-C interactions developed by Yamaguchi and Maruyama for the formation of metallofullerenes are modified to simulate the catalyzed growth of single-wall carbon nanotubes on Ni_n clusters with n >20, and the reactive empirical bond order Brenner potential for C-C interactions is also revised to include the effect of the metal atoms on such interactions. Figure Force field parameters for carbon-metal interactions obtained from DFT calculations in small clusters.     

Cold-active enzymes studied by comparative molecular dynamics simulation

Enzymes from cold-adapted species are significantly more active at low temperatures, even those close to zero Celsius, but the rationale of this adaptation is complex and relatively poorly understood. It is commonly stated that there is a relationship between the flexibility of an enzyme and its catalytic activity at low temperature. This paper gives the results of a study using molecular dynamics simulations performed for five pairs of enzymes, each pair comprising a cold-active enzyme plus its mesophilic or thermophilic counterpart. The enzyme pairs included α-amylase, citrate synthase, malate dehydrogenase, alkaline protease and xylanase. Numerous sites with elevated flexibility were observed in all enzymes; however, differences in flexibilities were not striking. Nevertheless, amino acid residues common in both enzymes of a pair (not present in insertions of a structure alignment) are generally more flexible in the cold-active enzymes. The further application of principle component analysis to the protein dynamics revealed that there are differences in the rate and/or extent of opening and closing of the active sites. The results indicate that protein dynamics play an important role in catalytic processes where structural rearrangements, such as those required for active site access by substrate, are involved. They also support the notion that cold adaptation may have evolved by selective changes in regions of enzyme structure rather than in global change to the whole protein. Figure Collective motions in C^α atoms of the active site of cold-active xylanase Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible for authorized users.