Towards understanding the origins of the different specificities of binding the reduced (NADPH) and oxidised (NADP+) forms of nicotinamide adenine dinucleotide phosphate coenzyme to dihydrofolate reductase

Lactobacillus casei dihydrofolate reductase (DHFR) binds more than a thousand times tighter to NADPH than to NADP⁺. The origins of the difference in binding affinity to DHFR between NADPH and NADP⁺ are investigated in the present study using experimental NMR data and hybrid density functional, B3LYP, calculations. Certain protein residues (Ala 6, Gln 7, Ile 13 and Gly 14) that are directly involved in hydrogen bonding with the nicotinamide carboxamide group show consistent differences in ¹H and ¹⁵N chemical shift between NADPH and NADP⁺ in a variety of ternary complexes. B3LYP calculations in model systems of protein-coenzyme interactions show differences in the H-bond geometry and differences in charge distribution between the oxidised and reduced forms of the nicotinamide ring. GIAO isotropic nuclear shieldings calculated for nuclei in these systems reproduce the experimentally observed trends in magnitudes and signs of the chemical shifts. The experimentally observed reduction in binding of NADP⁺ compared with NADPH results partly from NADP⁺ having to change its nicotinamide amide group from a cis- to a trans-conformation on binding and partly from the oxidised nicotinamide ring of NADP⁺ being unable to take up its optimal hydrogen bonding geometry in its interactions with protein residues.     

Sensitive assay for nicotinamide adenine dinucleotide phosphate and its reduced form based on the bioluminescence method

An assay of reduced nicotinamide adenine dinucleotide phosphate (NADPH) by bioluminescence was investigated and applied for NADP⁺. The NADP⁺ is first reduced by glucose-6-phosphate dehydrogenase and then assayed in a mixture containing a NADPH/flavin mononucleotide oxidoreductase which in turn activates luciferase. Many interferences were observed and the method was modified accordingly. NADP⁺ and NADPH can be assayed separately or simultaneously within the range 1–100 pmol, which is sufficiently sensitive to be applied to biological materials. Many details and precautions must be taken into consideration.     

Reverse Phase Liquid Chromatographic Analysis of the Impurities in Nicotinamide Adenine Dinucleotides

Abstract

Commercial preparations of several different nicotine adenine dinucleotides were examined by liquid chromatography on an octadecylsilane column (Margolis, S., et al., Clin. Chem., 22, 1322, 1976). Seven impurity peaks were detected in NADP⁺, eight in NADPH, and five in NAD⁺. The estimated purity of NADP⁺ from different commercial suppliers varied from 89 to 95 percent. For NADPH the purity ranged from 77.5 to 96 percent and for NAD⁺ from 90 to 93.5 percent. Preparations of NAD⁺ contained AMP, ADPR, nicotinamide, and two unidentified impurities. The impurities found in NADP⁺ and NADPH preparations did not correspond to compounds that we could identify. Four of the impurity peaks found in NADPH form under acidic storage conditions. Five of the impurity peaks observed in NADP⁺ and three of the impurity peaks in NAD⁺ form as products of alkali-catalyzed rearrangements.     

Density Functional Studies of Coenzyme NADPH and Its Oxidized Form NADP+: Structures,UV–Vis Spectra,and the Oxidation Mechanism of NADPH

Density functional theory has been used to study the biologically important coenzyme NADPH and its oxidized form NADP⁺. It was found that free NADPH prefers a compact structure in gas phase and exists in more extended geometries in aqueous solution. Ultraviolet–visible absorption spectra in aqueous solution were calculated for NADPH with an explicit treatment of 100 surrounding water molecules in combination with the COSMO solvation model for bulk hydration effects. The obtained spectra using the B3LYP hybrid density functional agree quite well with experimental data. The changes of Gibbs free energies ΔG in reactions of NADPH with O₂ observed experimentally in cardiovascular and in chemical systems, that is, NADPH + 2 ³O₂ → NADP⁺ + 2 O₂⁻ + H⁺ and NADPH + ¹O₂ + H⁺ → NADP⁺ + H₂O₂, respectively, were calculated. The NADPH oxidation reaction in the cardiovascular system cannot proceed without activation since the obtained ΔG is positive. The reaction of NADPH in the chemical system with singlet oxygen was found to proceed in two ways, each consisting of two steps, that is, NADPH firstly reacts with ¹O₂ barrierlessly to form NADP⁺ and HO₂⁻, from which H₂O₂ is formed in a spontaneous reaction with H⁺, or ¹O₂ and H⁺ initially form ¹HO₂⁺, which further reacts with NADPH to yield NADP⁺ and H₂O₂. © 2019 The Authors. Journal of Computational Chemistry published by Wiley Periodicals, Inc.     

Recycling of NADP+ using immobilizedE. coli and glucose-6-phosphate dehydrogenase

Recycling of NADP⁺ using immobilized wholeEscherichia coli cells as source of respiratory chain, glucose-6-phosphate, and soluble yeast glucose-6-phosphate dehydrogenase (1.1.1.49) is described. NADP⁺ was recycled more than 10-fold. We demonstrated NADPH respiration at pH 5.8 inE. coli membrane vesicles. The respiratory chain was involved most probably in NADPH oxidation.

1. The respiratory activity is localized at the level of the inner bacterial membrane. The active site for NADPH facing the cytoplasm.
2. NADPH respiration is inhibited by 10 mM cyanide, similar to the conditions of inhibition of NADH respiration.
3. NADPH dehydrogenase activity seems to be the limiting step of the respiratory chain:K _M for NADPH respiration and NADPH dehydrogenase activity are similar. The pH optima for these two activities are also comparable (around pH 5.8). Furthermore, the following properties are rather in favor of a common NADH dehydrogenase and NADPH dehydrogenase activity (1.6.99.2).

o| li](1)|At saturating concentrations of NADH and NADPH, neither respiration nor dehydrogenase activities were additive. li](2)|Similar heat inactivation kinetics were observed for NADH and NADPH dehydrogenase-activity. Protection against heat inactivation was obtained for the two activities with NAD⁺, NADP⁺, NADH, and NADPH. All these results suggested the possibility of recycling of NADP⁺ under similar conditions to those previously described for NAD⁺ (Burstein et al., 1981). It becomes thus possible to use various NAD⁺ and NADP⁺-dependent dehydrogenases in enzyme technology.     

Analysis of NAD(P) and NAD(P)H cofactors by means of imprinted polymers associated with Au surfaces:: A surface plasmon resonance study

Crosslinked films consisting of the acrylamide-acrylamidophenylboronic acid copolymer that are imprinted with recognition sites for β-nicotinamide adenine dinucleotide (NAD⁺), β-nicotinamide adenine dinucleotide phosphate NADP⁺, and their reduced forms (NAD(P)H), are assembled on Au-coated glass supports. The binding of the oxidized cofactors NAD⁺ or NADP⁺ or the reduced cofactors NADH or NADPH to the respective imprinted sites results in the swelling of the polymer films through the uptake of water. Surface plasmon resonance (SPR) spectroscopy is employed to follow the binding of the different cofactors to the respective imprinted sites. The imprinted recognition sites reveal selectivity towards the association of the imprinted cofactors. The method enables the analysis of the NAD(P)⁺ and NAD(P)H cofactors in the concentration range of 1×10⁻⁶ to 1×10⁻³ M. The cofactor-imprinted films associated with the Au-coated glass supports act as active interfaces for the characterization of biocatalyzed transformations that involve the cofactor-dependent enzymes. This is exemplified with the characterization of the biocatalyzed oxidation of lactate to pyruvate in the presence of NAD⁺ and lactate dehydrogenase using the NADH-imprinted polymer film.     

Cyclic voltammetric and nanogravimetric studies of NADP+ redox transformations on a yeast-modified platinum electrode

The dominant voltammetric response of a yeast suspension in neutral or slightly alkaline media can be assigned to the redox transformations of nicotinamide adenine dinucleotide (NAD⁺/NADH) and nicotinamide adenine dinucleotide phosphate (NADP⁺/NADPH). By immobilization of yeast on platinum, a stable electrode can be prepared which shows an electrocatalytic activity towards the reduction of NADP⁺ and the reoxidation of the product formed. Reversible cyclic voltammetric responses were obtained. The peak currents depend practically linearly on the NADP-Na₂ concentration and on the square root of the scan rate. The surface mass changes accompanying the redox transformations were monitored by an electrochemical quartz crystal nanobalance.     

Electrocatalytic Volleyball: Rapid Nanoconfined Nicotinamide Cycling for Organic Synthesis in Electrode Pores

In living cells, redox chains rely on nanoconfinement using tiny enclosures, such as the mitochondrial matrix or chloroplast stroma, to concentrate enzymes and limit distances that nicotinamide cofactors and other metabolites must diffuse. In a chemical analogue exploiting this principle, nicotinamide adenine dinucleotide phosphate (NADPH) and NADP⁺ are cycled rapidly between ferredoxin–NADP⁺ reductase and a second enzyme—the pairs being juxtaposed within the 5–100 nm scale pores of an indium tin oxide electrode. The resulting electrode material, denoted (FNR+E2)@ITO/support, can drive and exploit a potentially large number of enzyme‐catalysed reactions.     

Modelling Photosynthesis with ZnII‐Protoporphyrin All‐DNA G‐Quadruplex/Aptamer Scaffolds

All‐DNA scaffolds act as templates for the organization of photosystem I model systems. A series of DNA templates composed of Zn^II‐protoporphyrin IX (Zn^IIPPIX)‐functionalized G‐quadruplex conjugated to the 3′‐ or 5′‐end of the tyrosinamide (TA) aptamer and Zn^IIPPIX/G‐quadruplex linked to the 3′‐ and 5′‐ends of the TA aptamer through a four‐thymidine bridge. Effective photoinduced electron transfer (ET) from Zn^IIPPIX/G‐quadruplex to bipyridinium‐functionalized tyrosinamide, TA‐MV²⁺, bound to the TA aptamer units is demonstrated. The effectiveness of the primary ET quenching of Zn^IIPPIX/G‐quadruplex by TA‐MV²⁺ controls the efficiency of the generation of TA‐MV^+.. The photosystem‐controlled formation of TA‐MV^+. by the different photosystems dictates the secondary activation of the ET cascade corresponding to the ferredoxin‐NADP⁺ reductase (FNR)‐catalysed reduction of NADP⁺ to NADPH by TA‐MV^+., and the sequestered alcohol dehydrogenase catalysed reduction of acetophenone to 1‐phenylethanol by NADPH.     

Sequential fluorometric quantification of malic acid enantiomers by a single line flow-injection system using immobilized-enzyme reactors

A method for the sequential enantiomeric quantification of d-malate and l-malate by a single line flow-injection analysis was developed using immobilized-enzyme reactors and fluorescence detection. An immobilized d-malate dehydrogenase (d-MDH) reactor and an immobilized l-malate dehydrogenase (l-MDH) reactor were introduced into the flow line in series. Sample and coenzyme (NAD⁺ or NADP⁺) were injected into the flow line by an open sandwich method. d-Malate was selectively oxidized by d-MDH when NAD⁺ was injected with a sample. When NADP⁺ was injected with a sample, l-malate was oxidized only by l-MDH. NADH or NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of d-malate and l-malate were observed in the ranges of 1 × 10⁻⁶-1 × 10⁻⁴ M and 1 × 10⁻⁶-2 × 10⁻⁴ M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.1 mM level. This analytical method was applied to the sequential quantification of d-malate and l-malate in fruit juices and soft drinks, and the results showed good agreement with those obtained using conventional method (F-kit method).     

Sequential fluorometric quantification of γ-aminobutyrate and l-glutamate using a single line flow-injection system with immobilized-enzyme reactors

A single line flow-injection system with immobilized-enzyme reactors is proposed for the sequential quantification of γ-aminobutyrate (GABA) and l-glutamate. A co-immobilized l-glutamate oxidase and catalase reactor and an immobilized GABase reactor were introduced into the flow line in series. Sample and reagent were injected into the flow line using an open sandwich method. GABA was selectively detected by GABase when α-ketoglutarate at a high concentration and NADP⁺ were injected as the reagents with a sample. When GABA at a high concentration and NADP⁺ were injected as the reagents with a sample, l-glutamate only was determined by the series of enzymatic reactions. NADPH produced by the immobilized-enzyme reactors was monitored fluorometrically at 455 nm (excitation at 340 nm). Linear relationships between the responses and concentrations of GABA or l-glutamate were observed in the ranges of 5.0 × 10⁻⁶-5.0 × 10⁻⁴ M and 1.0 × 10⁻⁵-5.0 × 10⁻⁴ M, respectively. The relative standard deviations for ten successive injections were less than 2% at the 0.5 mM level. This analytical method was applied to the sequential quantification of GABA and l-glutamate that were produced and consumed, respectively, by lactic acid bacteria, and the results showed good agreement with those obtained using liquid chromatography.     

Modelling Photosynthesis with ZnII-Protoporphyrin All-DNA G-Quadruplex/Aptamer Scaffolds

All-DNA scaffolds act as templates for the organization of photosystem I model systems. A series of DNA templates composed of Zn^II-protoporphyrin IX (Zn^IIPPIX)-functionalized G-quadruplex conjugated to the 3′- or 5′-end of the tyrosinamide (TA) aptamer and Zn^IIPPIX/G-quadruplex linked to the 3′- and 5′-ends of the TA aptamer through a four-thymidine bridge. Effective photoinduced electron transfer (ET) from Zn^IIPPIX/G-quadruplex to bipyridinium-functionalized tyrosinamide, TA-MV²⁺, bound to the TA aptamer units is demonstrated. The effectiveness of the primary ET quenching of Zn^IIPPIX/G-quadruplex by TA-MV²⁺ controls the efficiency of the generation of TA-MV^+.. The photosystem-controlled formation of TA-MV^+. by the different photosystems dictates the secondary activation of the ET cascade corresponding to the ferredoxin-NADP⁺ reductase (FNR)-catalysed reduction of NADP⁺ to NADPH by TA-MV^+., and the sequestered alcohol dehydrogenase catalysed reduction of acetophenone to 1-phenylethanol by NADPH.     

Computational algorithmic and molecular dynamics study of functional and structural impacts of non-synonymous single nucleotide polymorphisms in human DHFR gene

Human dihydrofolate reductase (DHFR) is a conserved enzyme that is central to folate metabolism and is widely targeted in pathogenic diseases as well as cancers. Although studies have reported the fact that genetic mutations in DHFR leads to a rare autosomal recessive inborn error of folate metabolism and drug resistance, there is a lack of an extensive study on how the deleterious non-synonymous SNPs (nsSNPs) disrupt its phenotypic effects. In this study, we aim at discovering the structural and functional consequences of nsSNPs in DHFR by employing a combined computational approach consisting of ten recently developed in silico tools for identification of damaging nsSNPs and molecular dynamics (MD) simulation for getting deeper insights into the magnitudes of damaging effects. Our study revealed the presence of 12 most deleterious nsSNPs affecting the native phenotypic effects, with three (R71T, G118D, Y122D) identified in the co-factor and ligand binding active sites. MD simulations also suggested that these three SNPs particularly Y122D, alter the overall structural flexibility and dynamics of the native DHFR protein which can provide more understandings into the crucial roles of these mutants in influencing the loss of DHFR function.     

Specific detection of nicotinamide coenzymes by liquid chromatography and amperometric detection with immobilized glucose-6-phosphate dehydrogenase

A liquid chromatographic system for the specific and simultaneous detection of nicotinamide coenzymes is constructed by combining an immobilized glucose-6-phosphate dehydrogenase reactor with an amperometric system based on a phenazine methosulphate-mediated reaction, after separation on a reversed-phase column. The calibration graphs are linear from 0.05 to 20 nmol for all four coenzymes. The detection limits are 3.2, 5.2, 7.9 and 9.4 pmol for NADP⁺, NADPH, NAD⁺ and NADH, respectively. The enzyme reactor retains most of its original activity after repeated use for 2 months.     

Purification and characterization of a microbial dehydrogenase

Pseudomonas fluorescens (strain BTP9) was found to have at least two NAD(P)-dependent vanillin dehydrogenases: one is induced by vanillin, and the other is constitutive. The constitutive enzyme was purified by ammonium sulfate fractionation, gel-filtration, and Q-Sepharose chromatography. The subunit Mr value was 55,000, determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The native M _r value estimated by gelfiltration chromatography gave a value of 210,000. The enzyme made use of NAD⁺ less effectively than NADP⁺. Benzaldehyde, 4-hydroxybenzaldehyde, hexanal, and acetaldehyde were not oxidized at detectable rates in the presence of NAD⁺ or NADP⁺. The ultraviolet absorption spectrum indicated that there is no cofactor or prosthetic group bound. The vanillin oxidation reaction was essentially irreversible. The pH optimum was 9.5 and the pI of the enzyme was 4.9. Enzyme activity was not affected when assayed in the presence of salts, except FeCl₂. The enzyme was inhibited by the thiol-blocking reagents 4-chloromercuribenzoate and N-ethylmaleimide. NAD⁺ and NADP⁺ protected the enzyme against such a type of inhibition along with vanillin to a lesser extent. The enzyme exhibited esterase activity with 4-nitrophenyl acetate as substrate and was activated by low concentrations of NAD⁺ or NADP⁺. We compared the properties of the enzyme with those of some well-characterized microbial benzaldehyde dehydrogenases.     

Incorporating dynamics in E. coli dihydrofolate reductase enhances structure-based drug discovery

Escherichia coli dihydrofolate reductase (DHFR) is a long-standing target for enzyme studies. The influence of protein motion on its catalytic cycle is significant, and the conformation of the M20 loop is of particular interest. We present receptor-based pharmacophore models-an equivalent of solvent-mapping of binding hotspots-based on ensembles of protein conformations from molecular dynamics simulations of DHFR.NADPH in both the closed and open conformation of the M20 loop. The optimal models identify DHFR inhibitors over druglike non-inhibitors; furthermore, high-affinity inhibitors of E. coli DHFR are preferentially identified over general DHFR inhibitors. As expected, models resulting from simulations with DHFR in the productive conformation with a closed M20 loop have better performance than those from the open-loop simulations. Model performance improves with increased dynamic sampling, indicating that including a greater degree of protein flexibility can enhance the quest for potent inhibitors. This was compared to the limited conformational sampling seen in crystal structures, which were suboptimal for this application.     

Redox reactions of coenzymes induced by γ-irradiated sodium chloride

A spectrophotometric study is reported of the oxido-reductant reactions with the major coenzymes as NADH, NADPH, NAD⁺, NADP⁺ and FAD effected by low dose and by the energy stored in F and hole centres in -irradiated sodium chloride. The G values for the two modes of these redox reactions are compared.     

Selectivity analysis of 5-(arylthio)-2,4-diaminoquinazolines as inhibitors of Candida albicans dihydrofolate reductase by molecular dynamics simulations

A series of 5-(arylthio)-2,4-diaminoquinazolines are known as selective inhibitors of dihydrofolate reductase (DHFR) from Candida albicans. We have performed docking and molecular dynamics simulations of these inhibitors with C. albicans and human DHFR to understand the basis for selectivity of these agents. Study was performed on a selected set of 10 compounds with variation in structure and activity. Molecular dynamics simulations were performed at 300 K for 45 ps with equilibration for 10 ps. Trajectory data was analyzed on the basis of hydrogen bond interactions, energy of binding and conformational energy difference. The results indicate that hydrogen bonds formed between the compound and the active site residues are responsible for inhibition and higher potency. The selectivity index, i.e the ratio of I₅₀ against human DHFR to I₅₀ against fungal DHFR, is mainly determined by the conformation adapted by the compounds within the active site of two enzymes. Since the human DHFR active site is rigid, the compound is trapped in a higher energy conformation. This energy difference between the two conformations E mainly governs the selectivity against fungal DHFR. The information generated from this analysis of potency and selectivity should be useful for further work in the area of antifungal research.     

Mechanostability of the Single‐Electron‐Transfer Complexes of Anabaena Ferredoxin–NADP+ Reductase

The complexes formed between the flavoenzyme ferredoxin–NADP⁺ reductase (FNR; NADP⁺=nicotinamide adenine dinucleotide phosphate) and its redox protein partners, ferredoxin (Fd) and flavodoxin (Fld), have been analysed by using dynamic force spectroscopy through AFM. A strategy is developed to immobilise proteins on a substrate and AFM tip to optimise the recognition ability. The differences in the recognition efficiency regarding a random attachment procedure, together with nanomechanical results, show two binding models for these systems. The interaction of the reductase with the natural electron donor, Fd, is threefold stronger and its lifetime is longer and more specific than that with the substitute under iron‐deficient conditions, Fld. The higher bond probability and two possible dissociation pathways in Fld binding to FNR are probably due to the nature of this complex, which is closer to a dynamic ensemble model. This is in contrast with the one‐step dissociation kinetics that has been observed and a specific interaction described for the FNR:Fd complex.     

Hierarchical structure of the energy landscape of proteins revisited by time series analysis. I. Mimicking protein dynamics in different time scales

Time series models, which are constructed from the projections of the molecular-dynamics (MD) runs on principal components (modes), are used to mimic the dynamics of two proteins: tendamistat and immunity protein of colicin E7 (ImmE7). Four independent MD runs of tendamistat and three independent runs of ImmE7 protein in vacuum are used to investigate the energy landscapes of these proteins. It is found that mean-square displacements of residues along the modes in different time scales can be mimicked by time series models, which are utilized in dividing protein dynamics into different regimes with respect to the dominating motion type. The first two regimes constitute the dominance of intraminimum motions during the first 5 ps and the random walk motion in a hierarchically higher-level energy minimum, which comprise the initial time period of the trajectories up to 20-40 ps for tendamistat and 80-120 ps for ImmE7. These are also the time ranges within which the linear nonstationary time series are completely satisfactory in explaining protein dynamics. Encountering energy barriers enclosing higher-level energy minima constrains the random walk motion of the proteins, and pseudorelaxation processes at different levels of minima are detected in tendamistat, depending on the sampling window size. Correlation (relaxation) times of 30-40 ps and 150-200 ps are detected for two energy envelopes of successive levels for tendamistat, which gives an overall idea about the hierarchical structure of the energy landscape. However, it should be stressed that correlation times of the modes are highly variable with respect to conformational subspaces and sampling window sizes, indicating the absence of an actual relaxation. The random-walk step sizes and the time length of the second regime are used to illuminate an important difference between the dynamics of the two proteins, which cannot be clarified by the investigation of relaxation times alone: ImmE7 has lower-energy barriers enclosing the higher-level energy minimum, preventing the protein to relax and letting it move in a random-walk fashion for a longer period of time.