Enniatin B and Valinomycin as Ion Carriers: An Empirical Force Field Analysis

Abstract

The alkali-ion binding properties of two natural depsipeptide ion carriers, enniatin B (EnB) and valinomycin (VM), are examined and compared by the empirical force field method. While VM has been shown to bind preferentially K⁺, Rb⁺, and Cs⁺ over Na⁺ in most solvents, EnB is considerably less specific.

We find that EnB forms two kinds of complexes, internal and external. In internal complexes, the ion binds to all six carbonyl oxygens, while in external ones, only three oxygens, preferentially those of the D-hydroxy-isovaleryl residues, are bound. The size of the internal cavity is best suited for Na⁺, while K⁺ and Rb⁺ squeeze in asymmetrically by distorting the molecule, and Cs⁺ not at all. External binding is much less specific. Since internal complexes possess much higher strain energies than external ones, the latter may be at least as stable as the former, even in fairly non-polar solvents.

VM is calculated to bind only internally, and with much less strain energy than EnB. The size of its internal cavity is well suited for binding the ions K⁺, Rb⁺, and Cs⁺, but is too big for Na⁺. The difference between the binding energies of Na⁺ and K⁺ is much smaller than that between the corresponding hydration enthalpies, thus explaining the binding preference for the latter ion.     

Coenzyme F430 as a possible catalyst for the reductive dehalogenation of chlorinated C1 hydrocarbons in methanogenic bacteria

Corrinoids, such as aquocobalamin, methylcobalamin, and (cyanoaquo)cobinamide, catalyze the reductive dehalogenation of CCl4 with titanium(III) citrate as the electron donor [Krone et al. (1989) Biochemistry 28, 4908-4914]. We report here that this reaction is also effectively mediated by the nickel-containing porphinoid, coenzyme F430, found in methanogenic bacteria. Chloroform, methylene chloride, methyl chloride, and methane were detected as intermediates and products. Ethane was formed in trace amounts, and several as yet unidentified nonvolatile compounds were also generated. The rate of dehalogenation decreased in the series of CCl4, CHCl3, and CH2Cl2. With coenzyme F430 as the catalyst, the reduction of CH3Cl to CH4 proceeded more than 50 times faster than with aquocobalamin. Cell suspensions of Methanosarcina barkeri were found to catalyze the reductive dehalogenation of CCl4 with CO as the electron donor (E'0 = -0.524 V). Methylene chloride was the main end product. The kinetics of CHCl3 and CH2Cl2 formation from CCl4 were similar to those with coenzyme F430 or aquocobalamin as catalysts and titanium(III) citrate as the reductant.     

Nickel requirement and factor F430 content of methanogenic bacteria.

Methanobacterium thermoautotrophicum has been reported to require nickel for growth and to contain high concentrations of a nickel tetrapyrrole designated factor F430. In this communication it is shown that all methanogenic bacteria investigated incorporated nickel during growth and also synthesized factor F430. This was also true for Methanobrevibacter smithii, which is dependent on acetate as a carbon source, and for Methanosarcina barkeri growing on acetate or methanol as energy sources. Other bacteria, including Acetobacterium woodii and Clostridium thermoaceticum, contained no factor F430. It is further shown that two yellow nickel-containing degradation products were formed from factor F430 when heated at pH 7. This finding explains why several forms of factor F430 were found in methanogenic bacteria when a heat step was employed in the purification procedure.     

Discovery of Multiple Modified F430 Coenzymes in Methanogens and Anaerobic Methanotrophic Archaea Suggests Possible New Roles for F430 in Nature

Methane is a potent greenhouse gas that is generated and consumed in anaerobic environments through the energy metabolism of methanogens and anaerobic methanotrophic archaea (ANME), respectively. Coenzyme F₄₃₀ is essential for methanogenesis, and a structural variant of F₄₃₀, 17²-methylthio-F₄₃₀ (F₄₃₀-2), is found in ANME and is presumably essential for the anaerobic oxidation of methane. Here we use liquid chromatography–high-resolution mass spectrometry to identify several new structural variants of F₄₃₀ in the cell extracts of selected methanogens and ANME. Methanocaldococcus jannaschii and Methanococcus maripaludis contain an F₄₃₀ variant (denoted F₄₃₀-3) that has an M⁺ of 1,009.2781. This mass increase of 103.9913 over that of F₄₃₀ corresponds to C₃H₄O₂S and is consistent with the addition of a 3-mercaptopropionate moiety bound as a thioether followed by a cyclization. The UV absorbance spectrum of F₄₃₀-3 was different from that of F₄₃₀ and instead matched that of an F₄₃₀ derivative where the 17³ keto moiety had been reduced. This is the first report of a modified F₄₃₀ in methanogens. In a search for F₄₃₀-2 and F₄₃₀-3 in other methanogens and ANME, we have identified a total of nine modified F₄₃₀ structures. One of these compounds may be an abiotic oxidative product of F₄₃₀, but the others represent naturally modified versions of F₄₃₀. This work indicates that F₄₃₀-related molecules have additional functions in nature and will inspire further research to determine the biochemical role(s) of these variants and the pathways involved in their biosynthesis.     

Coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg belongs to the superfamily of adenylate-forming enzymes.

Depending on the reduction-oxidation state of the cell, some methanogenic bacteria synthesize or hydrolyze 8-hydroxyadenylylated coenzyme F420 (coenzyme F390). These two reactions are catalyzed by coenzyme F390 synthetase and hydrolase, respectively. To gain more insight into the mechanism of the former reaction, coenzyme F390 synthetase from Methanobacterium thermoautotrophicum Marburg was purified 89-fold from cell extract to a specific activity of 0.75 mumol.min-1.mg of protein-1. The monomeric enzyme consisted of a polypeptide with an apparent molecular mass of 41 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. ftsA, the gene encoding coenzyme F390 synthetase, was cloned and sequenced. It encoded a protein of 377 amino acids with a predicted M(r) of 43,280. FtsA was found to be similar to domains found in the superfamily of peptide synthetases and adenylate-forming enzymes. FtsA was most similar to gramicidin S synthetase II (67% similarity in a 227-amino-acid region) and sigma-(L-alpha-aminoadipyl)-L-cysteine-D-valine synthetase (57% similarity in a 193-amino-acid region). Coenzyme F390 synthetase, however, holds an exceptional position in the superfamily of adenylate-forming enzymes in that it does not activate a carboxyl group of an amino or hydroxy acid but an aromatic hydroxyl group of coenzyme F420.     

Structure of Three-Way DNA Junctions 1. Non-Planar DNA Geometry

Abstract

Three-way junctions were obtained by annealing two synthetic DNA-oligomers. One of the strands contains a short palindrome sequence, leading to the formation of a hairpin with four base pairs in the stem and four bases in the loop. Another strand is complementary to the linear arms of the first hairpin-containing strand. Both strands were annealed to form a three-way branched structure with sticky ends on the linear arms. The branched molecules were ligated, and the ligation mixture was analysed on a two-dimensional gel in conditions which separated linear and circular molecules. Analysis of 2D-electrophoresis data shows that circular molecules with high mobility are formed. Formation of circular molecules is indicative of bends between linear arms. We estimate the magnitude of the angle between linear arms from the predominant size of the circular molecules formed. When the junction-to-junction distance is 20–21 bp, trimers and tetramers are formed predominately, giving an angle between linear arms as small as 60–90°. Rotation of the hairpin position in the three- way junction allowed us to measure angles between other arms, yielding similar values. These results led us to conclude that the three-way DNA junction possesses a non-planar pyramidal geometry with 60–90° between the arms. Computer modeling of the three-way junction with 60° pyramidal geometry showed a predominantly B-form structure with local distortions at the junction points that diminish towards the ends of the helices. The size distributions of circular molecules are rather broad indicating a dynamic flexibility of three-way DNA junctions.     

Structural heterogeneity and purification of protein-free F430 from the cytoplasm of Methanobacterium thermoautotrophicum

F430 is the nickel containing tetrapyrrole cofactor of S-methyl coenzyme M methylreductase, the enzyme that catalyzes the final step of methane production by methanogenic bacteria: the reduction of S-methyl coenzyme M (H3CSCH2CH2SO3-) to methane and coenzyme M (HSCH2CH2SO3-). The protein-free F430 obtained from the cytosol of Methanobacterium thermoautotrophicum, strain delta H, exists predominantly in two isomeric forms that differ in relative stereochemical disposition of acid side chains at the 12 and 13 positions of the macrocycle periphery (Pfaltz, A., Livingston, D. A., Jaun, B., Diekert, G., Thauer, R. K., and Eschenmoser, A. (1985) Helv. Chim. Acta 68, 1338-1358). A simple one-step chromatographic procedure for the large-scale separation of these isomers is described. X-ray absorption spectroscopic studies show that F430 (i.e. the native isomer) is 6-coordinate with long nickel-ligand bonds (approximately 2.1 A), suggesting an approximately planar macrocycle. In contrast, the 12,13-diepimer exhibits a 4-coordinate, square-planar structure with short nickel-nitrogen bonds (approximately 1.9 A), suggesting a ruffled macrocycle. Previous reports, based on other x-ray absorption spectroscopic data, of static disorder in F430 Ni-N distances are shown to be incorrect due to sample heterogeneity. The optical spectrum of F430 (whether purified from the protein-free cytosol or extracted at high ionic strength from the holoenzyme) differs significantly from that of the 12,13-diepimer. The optical spectral differences are correlated with the alterations in coordination number and geometry of the central nickel ion in the two F430 isomers.     

Force Field Dependence of NMR-Based,Restrained Molecular Dynamics DNA Structure Calculations Including an Analysis of the Influence of Residual Dipolar Coupling Restraints

Abstract

Restrained molecular dynamics is widely used to calculate DNA structures from NMR data. Here, results of an in silico experiment show that the force field can be significant compared to the NMR restraints in driving the final structures to converge. Specifically, we observed that i) the influence of the force field leads to artificially tight convergence within final families of structures and ii) the precision and character of resulting structures depend on the choice of force field used in the calculations. A canonical B-DNA model was used as a target structure. Distances, dihedral angles, and simulated residual dipolar couplings were measured in the target structure and used as restraints. X-PLOR and Discover, which use force fields developed for CHARMM and AMBER programs, respectively, were tested and found to produce different final structures despite the use of identical distance and dihedral restraints. Incorporation of residual dipolar coupling restraints in X-PLOR improves convergence with the target structure and between families of structures indicating that the force field dependence can potentially be overcome if residual dipolar coupling restraints are employed.     

An Evaluation of the MM+ Force Field

Hyperchem′s MM+ force field, based on Allinger′s MM2 is described and evaluated with respect to other MM2 variants, in terms of rotation barriers, conformational energy differences, and conjugation. Its ability to take missing parameters into account is also evaluated with respect to the Dreiding force field. This evaluation also intends to clearly separate the two different force fields MM+(91) and MM+(^**) hiding under the MM+ denomination. It is shown that, whereas MM+ proves to be generally robust, caution must be the rule when dealing with conjugated molecules, particularly with heteroaromatics.     

Homology Modelling of a Newly Discovered Thioredoxin Protein and Analysis of the Force Field and Electrostatic Properties

Thioredoxin is a small protein (Mr approximately 12,000) found in all living cells from archaebacteria to humans. The active site is highly conserved and has two redox-active cysteine residues in the sequence: -Trp-Cys-Gly-Pro-Cys-. Besides the function of the reduced form as a powerful protein disulfide oxidoreductase, thioredoxin is known to regulate and activate different target enzymes, i.e. ribonucleotide reductase and the mitochondrial 2-oxoacid dehydrogenase multienzyme complexes. Despite the high degree of homology between thioredoxin proteins from different species, there exists a strong variation in the capability of activating target enzymes. This is yet unexplainable, since there still exists no model of a thioredoxin/receptor complex.On the basis of the recently determined amino acid sequence of the thioredoxin Trx2 from rat mitochondria, which is known to be highly efficient in activating mitochondrial 2-oxoacid dehydrogenase multienzyme complexes, we construct the 3-D structure of this protein by homology modelling methods, using the X-ray structures of thioredoxin from E. coli and human as background information. We analyze the differences in the electrostatic properties of the different protein structures and show, that despite the observed homology between the primary sequences, the dipole moment of the protein structures shows significant variations, which might lead to deviations with respect to the binding to the target protein. Using the AMBER 4.0 program package we further investigate and compare the force field energies of the different thioredoxin structures.Electronic Supplementary Material available.     

Coordination chemistry of F430. Axial ligation equilibrium between square-planar and bis-aquo species in aqueous solution

X-ray absorption spectroscopic characterization of axial ligand coordination to factor F430, the nickel-tetrapyrrole cofactor of the S-methyl-coenzyme M (CH3SCoM) methyl reductase enzyme from methanogenic bacteria, is presented. The nickel of isolated F430 is hexacoordinate at 10 K in aqueous solution (as is the enzyme-bound cofactor), whereas the epimerized and ring-oxidized derivatives of F430 have four-coordinate nickel. Reduction of the ring-oxidized derivative, F560, with dithionite yields F430 in its native configuration, with axial ligands indistinguishable from those present when the cofactor is obtained directly from the holoenzyme. Thus, we conclude that the axial ligands to F430 in aqueous solution are water molecules. Analysis of the nickel extended x-ray absorption fine structure is consistent with this conclusion. Resonance Raman spectra obtained at room temperature contain features characteristic of both 4- and 6-coordinate forms of the cofactor. We have found that the resonance Raman, optical, and x-ray absorption spectra of aqueous solutions of F430 are temperature-dependent due to a ligand-binding equilibrium involving the square-planar and 6-coordinate bis-aquo forms of the cofactor. At low temperatures (less than 250 K) the 6-coordinate form predominates, whereas higher temperature solutions contain both 4- and 6-coordinate species in a dynamic equilibrium. Similar behavior is observed in other weakly coordinating solvents such as methanol and ethanol. The 4-coordinate form is predominant in solvents with strong electron-withdrawing substituents such as 2,2,2-trifluoroethanol and 2-mercaptoethanol. The relevance of this facile ligand exchange to the active site structure and enzymatic mechanism of the parent enzyme is discussed.     

The magnetic properties of the nickel cofactor F430 in the enzyme methyl-coenzyme M reductase of Methanobacterium thermoautotrophicum.

Preparations of gamma-aminobutyrate (GABA)/benzodiazepine receptor from pig cerebral cortex are composed of three major bands of polypeptides (51, 55 and 57 kDa) which are purified in a ratio of approx. 2:1:1 respectively. Treatment of purified receptor preparations with cyclic AMP-dependent protein kinase resulted in major incorporation of 32P into the 55 kDa band only. The maximum incorporation achieved was 0.6 mol of 32P/mol of 55 kDa polypeptide. The phosphorylated receptor subunit (beta-subunit) displays the same apparent Mr as a band labelled irreversibly with the GABA receptor agonist [3H]muscimol. The two nonphosphorylated subunit polypeptides (51 and 57 kDa) are each labelled irreversibly with [3H]flunitrazepam and are recognized by anti-peptide antibodies specific for alpha-subunits.     

Evidence for the involvement of corrinoids and factor F430 in the reductive dechlorination of 1,2-dichloroethane by Methanosarcina barkeri.

Cobalamin and the native and diepimeric forms of factor F430 catalyzed the reductive dechlorination of 1,2-dichloroethane (1,2-DCA) to ethylene or chloroethane (CA) in a buffer with Ti(III) citrate as the electron donor. Ethylene was the major product in the cobalamin-catalyzed transformation, and the ratio of ethylene to CA formed was 25:1. Native F430 and 12,13-di-epi-F430 produced ethylene and CA in ratios of about 2:1 and 1:1, respectively. Cobalamin dechlorinated 1,2-DCA much faster than did factor F430. Dechlorination rates by all three catalysts showed a distinct pH dependence, correlated in a linear manner with the catalyst concentration and doubled with a temperature increase of 10 degrees C. Crude and boiled cell extracts of Methanosarcina barkeri also dechlorinated 1,2-DCA to ethylene and CA with Ti(III) citrate as the reductant. The catalytic components in boiled extracts were heat and oxygen stable and had low molecular masses. Fractionation of boiled extracts by a hydrophobic interaction column revealed that part of the dechlorinating components had a hydrophilic and part had a hydrophobic character. These chemical properties of the dechlorinating components and spectral analysis of boiled extracts indicated that corrinoids or factor F430 was responsible for the dechlorinations. The ratios of 3:1 to 7:1 of ethylene and CA formed by cell extracts suggested that both cofactors were concomitantly active.     

Unexpected Abundance of Coenzyme F420-Dependent Enzymes in Mycobacterium tuberculosis and Other Actinobacteria

Regimens targeting Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), require long courses of treatment and a combination of three or more drugs. An increase in drug-resistant strains of M. tuberculosis demonstrates the need for additional TB-specific drugs. A notable feature of M. tuberculosis is coenzyme F₄₂₀, which is distributed sporadically and sparsely among prokaryotes. This distribution allows for comparative genomics-based investigations. Phylogenetic profiling (comparison of differential gene content) based on F₄₂₀ biosynthesis nominated many actinobacterial proteins as candidate F₄₂₀-dependent enzymes. Three such families dominated the results: the luciferase-like monooxygenase (LLM), pyridoxamine 5′-phosphate oxidase (PPOX), and deazaflavin-dependent nitroreductase (DDN) families. The DDN family was determined to be limited to F₄₂₀-producing species. The LLM and PPOX families were observed in F₄₂₀-producing species as well as species lacking F₄₂₀ but were particularly numerous in many actinobacterial species, including M. tuberculosis. Partitioning the LLM and PPOX families based on an organism''s ability to make F₄₂₀ allowed the application of the SIMBAL (sites inferred by metabolic background assertion labeling) profiling method to identify F₄₂₀-correlated subsequences. These regions were found to correspond to flavonoid cofactor binding sites. Significantly, these results showed that M. tuberculosis carries at least 28 separate F₄₂₀-dependent enzymes, most of unknown function, and a paucity of flavin mononucleotide (FMN)-dependent proteins in these families. While prevalent in mycobacteria, markers of F₄₂₀ biosynthesis appeared to be absent from the normal human gut flora. These findings suggest that M. tuberculosis relies heavily on coenzyme F₄₂₀ for its redox reactions. This dependence and the cofactor''s rarity may make F₄₂₀-related proteins promising drug targets.Mycobacterium tuberculosis, the causative agent of tuberculosis (TB), is an actinobacterium that presents a number of clinical challenges. For example, due to the high frequency of drug-resistant mutants, TB antibiotic regimens require long courses of treatment and a combination of three or more separate drugs (37). Long courses of combination therapy contribute to noncompliance, which in turn has led to an increase in the occurrence of multiple-drug-resistant (MDR) and extensively drug-resistant (XDR) tuberculosis (39). There is a clear need for additional tuberculosis-specific drugs that, in combination with the current pharmacopeia, can shorten the course of treatment and increase its effectiveness.Biological features that are present in mycobacteria but rare or absent in other organisms are useful targets for treating TB. For example, mycobacteria have “mycolic” fatty acids present in their cell walls that distinguish them from all other bacteria. Four major anti-TB drugs (isoniazid, cycloserine, ethambutol, and ethionamide) are known to target enzymes involved in the biosynthesis of the mycobacterial cell wall, and others, such as pyrazinamide, caprazamycin, and caprolactams, may do so as well (34, 43).Similarly, the enzyme cofactor F₄₂₀ (Fig. ​(Fig.1),1), a deazaflavin analog of flavin mononucleotide (FMN), is absent from humans but distributed sporadically and sparsely among prokaryotes and observed universally in the mycobacteria (including being encoded by the reduced genome of Mycobacterium leprae). It has been suggested that the reduced F₄₂₀ (F₄₂₀H₂) produced by the action of the F₄₂₀-dependent glucose-6-phosphate dehydrogenase (4) under aerobic conditions may protect mycobacterial cells from macrophage-generated NO₂ (31). Moreover, Rv3547 from M. tuberculosis uses reduced F₄₂₀ in the activation of the NO₂-containing antitubercular drug candidate PA-824 (40). Overall, F₄₂₀ may confer an advantage to mycobacteria in anaerobic environments because it has a lower redox potential than NADP (5).Open in a separate windowFIG. 1.Flavonoid cofactor structures. (A) FMN. (B) Coenzyme F₄₂₀. Note that coenzyme F₄₂₀ typically contains 5 to 7 side chain glutamate residues in mycobacterial species (3).The sporadic phylogenetic distribution of F₄₂₀ provides an opportunity for the application of comparative genomic methods. We introduced partial phylogenetic profiling (PPP) to efficiently discover protein families codistributed with such patterns of biological traits (21). Unlike earlier profiling methods, PPP does not require the prior accurate determination of protein families for success. This method is well suited to the identification of F₄₂₀-dependent enzyme families, which may have distributions only partially spanning the entire profile. PPP analysis is further augmented by SIMBAL (sites inferred by metabolic background assertion labeling) (36). This technique can pinpoint sites discriminating F₄₂₀ binding from FMN binding and subsequently identify additional correlated genes that are undetectable by PPP.Here we demonstrate how comparative genomics, namely, profiling, can strongly associate sets of genes in a particular genome of interest with a biologically important trait, generating numerous experimentally testable hypotheses. This analysis has indicated a pervasive and presumably important feature of M. tuberculosis and its lifestyle. The lack of F₄₂₀-based reactions in humans or their associated gut flora and their prevalence in M. tuberculosis may provide another drug target.     

Geometry and the visual brain.

Receptive fields structure of neurons in primary visual cortex suggests that they process visual stimuli in the frequency domain, in a way similar to the frequency analysis performed in the auditory system. As a consequence, both psychophysicists and electrophysiologists have long probed the visual system using extended sine wave gratings that are well localized in the frequency domain but poorly defined in visual space. Meanwhile, how the brain processes the geometrical properties and the spatial and temporal relationships between stimulus parts has received less attention. Recent progress in visual neuroscience that uncovered long-range horizontal connections between cortical neurons and revealed the complex architecture of primary visual cortex and feedback connectivity led to new insights concerned with the processing of geometrical properties of visual stimuli in V1. This paper presents a short historical perspective of the emergence of new issues related to the cortical architecture and its functional consequences on the processing of geometrical properties.     

Biosynthesis of coenzyme F430 in methanogenic bacteria. Identification of 15,17(3)-seco-F430-17(3)-acid as an intermediate

Coenzyme F430 is a hydroporphinoid nickel complex present in all methanogenic bacteria. It is part of the enzyme system which catalyzes methane formation from methyl-coenzyme M. We describe here that under certain conditions a second nickel porphinoid accumulates in methanogenic bacteria. The compound was identified at 15,17(3)-seco-F430-17(3)-acid. The structural assignment rests on 14C-labelling experiments, fast-atom-bombardment mass spectra, 1H-NMR spectra of the corresponding hexamethyl ester, and ultraviolet/visible spectral comparison with model compounds. In cell extracts and in intact cells of methanogenic bacteria, 15,17(3)-seco-F430-17(3)-acid was converted to F430. These findings indicate that the new nickel-containing porphinoid is an intermediate in the biosynthesis of coenzyme F430.     

Optimization of the Bowtie Gap Geometry for a Maximum Electric Field Enhancement

Optimization of the geometry of a metallic bowtie gap at radio frequency is presented. We investigate the geometry of the bowtie gap including gap size, tip width, metal thickness and tip angle at macroscale to find the maximum electric field enhancement across the gap. The results indicate that 90^° bowtie with 0.06 λ gap size has the most |E _t |² enhancement. Effects of changing the permittivity and conductivity of the material across the gap are also investigated. NEC-2 simulations show that the numerical calculations agree with the experimental results. Since the design and fabrication of a plasmonic device (nanogap) at nanoscale is challenging, the results of this study can be used to estimate the best design parameters for nanogap structure. Different amounts of enhancement at different frequency ranges are explained by mode volume. The product of the mode volume and |E _t |² enhancement is constant for different gap structures and different frequencies.     

Tetrapyrrole assembly and modification into the ligands of biologically functional cofactors

Data obtained using a combination of molecular biology and NMR spectroscopy has transformed our thinking about the evolution of the biochemical machinery required for the synthesis of the vital metallopigments: haem, chlorophyll, vitamin B₁₂ and factor F₄₃₀. One of the most recent advances is the discovery of a unique dipyrromethane cofactor that is bound covalently at the active site of porphobilinogen deaminase, the key enzyme of tetrapyrrole assembly. We will also discuss how the oxidation level and chromophoric arrangement of the uroporphinoid ring, rather than its substitution pattern, provides the necessary molecular recognition for some of the later enzymes, whose function is to decorate the template by C-methylation on the way to the biologically active cofactors.