Structure Of NADH Research Articles

Experimental and theoretical study on the interactions between dopamine hydrochloride and nicotinamide

The interactions between dopamine hydrochloride (DH) and nicotinamide (NA) in aqueous solution were studied using CV, NMR and quantum chemistry calculations. According to the cyclic voltammetry and the 1H NMR spectra, it was found that the hydrogen bond and π-π stacking between DH and NA were the main interactions, for which the predominant interaction varied with the ratio of NA to DH. The structures of NA-DH+ complexes were optimized by the quantum chemistry calculation, which confirmed above interactions, as well as the existence of CH-π interaction in NA-DH mixtures.

Using Cryo-EM to Map Small Ligands on Dynamic Metabolic Enzymes: Studies with Glutamate Dehydrogenase.

Cryo-electron microscopy (cryo-EM) methods are now being used to determine structures at near-atomic resolution and have great promise in molecular pharmacology, especially in the context of mapping the binding of small-molecule ligands to protein complexes that display conformational flexibility. We illustrate this here using glutamate dehydrogenase (GDH), a 336-kDa metabolic enzyme that catalyzes the oxidative deamination of glutamate. Dysregulation of GDH leads to a variety of metabolic and neurologic disorders. Here, we report near-atomic resolution cryo-EM structures, at resolutions ranging from 3.2 Å to 3.6 Å for GDH complexes, including complexes for which crystal structures are not available. We show that the binding of the coenzyme NADH alone or in concert with GTP results in a binary mixture in which the enzyme is in either an “open” or “closed” state. Whereas the structure of NADH in the active site is similar between the open and closed states, it is unexpectedly different at the regulatory site. Our studies thus demonstrate that even in instances when there is considerable structural information available from X-ray crystallography, cryo-EM methods can provide useful complementary insights into regulatory mechanisms for dynamic protein complexes.

Reduction of Quinones by NADH Catalyzed by Organoiridium Complexes

Reduction of Quinones by NADH Catalyzed by Organoiridium Complexes

Structures of NADH and NAD+bound 3α-hydroxysteroid dehydrogenase fromPseudomonassp. B-0831

Structures of NADH and NAD⁺bound 3α-hydroxysteroid dehydrogenase from<i>Pseudomonas</i>sp. B-0831

The structure of NADH in the enzyme dTDP-d-glucose dehydratase (RmlB).

The structure of Streptococcus suis serotype type 2 dTDP-d-glucose 4,6-dehydratase (RmlB) has been determined to 1.5 A resolution with its nicotinamide coenzyme and substrate analogue dTDP-xylose bound in an abortive complex. During enzyme turnover, NAD(+) abstracts a hydride from the C4' atom of dTDP-glucose-forming NADH. After elimination of water, hydride is then transferred back to the C6' atom of dTDP-4-keto-5,6-glucosene-regenerating NAD(+). Single-crystal spectroscopic studies unambiguously show that the coenzyme has been trapped as NADH in the crystal. Electron density clearly demonstrates that in contrast to native structures of RmlB where a flat nicotinamide ring is observed, the dihydropyridine ring of the reduced cofactor in this complex is found as a boat. The si face, from which the pro-S hydride is transferred, has a concave surface. Ab initio electronic structure calculations demonstrate that the presence of an internal hydrogen bond, between the amide NH on the nicotinamide ring and one of the oxygen atoms on a phosphate group, stabilizes this distorted conformation. Additionally, calculations show that the hydride donor ability of NADH is influenced by the degree of bending in the ring and may be influenced by an active-site tyrosine residue (Tyr 161). These results demonstrate the ability of dehydratase enzymes to fine-tune the redox potential of NADH through conformational changes in the nicotinamide ring.

Studies on the structure of NADH:ubiquinone oxidoreductase complex: Topography of the subunits of the iron-sulfur protein component

Resolution of the mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) by chaotropic agents result in the separation of three building blocks of the enzyme, designated FP (flavoprotein), IP (iron-sulfur protein), and HP (hydrophobic protein). FP contains three subunits of M r 51, 24, and 9 kDa; one FMN; and two iron-sulfur clusters. Immunochemical studies with monospecific antibodies to the FP subunits have indicated that all three subunits of FP protrude from the inner mitochondrial membrane on the matrix side, whereas no reactive epitopes from these subunits were found exposed on the cytosolic side [A.-L. Han, T. Yagi, and Y. Hatefi (1988) Arch. Biochem. Biophys. 267, 490–496] . IP contains six subunits of M r 75, 49, 30, 18, 15, and 13 kDa and four iron-sulfur clusters. In the present study, immunochemical experiments (enzyme-linked immunosorbent assays and 125I-protein A labeling) were carried out with monospecific antibodies to the above IP subunits and with bovine Complex I, submitochondrial particles, mitoplasts, and intact mitochondria as sources of antigens. Results have indicated that all six IP subunits protrude from the inner mitochondrial membrane into the matrix, and that the 75-kDa subunit, and possibly the 15-kDa subunit, protrude in mitoplasts from the cytosolic side as well. No epitopes reactive toward the monospecific antibodies to the 49-, 30-, 18-, and 13-kDa subunits were detected in mitoplasts.

Studies on the structure of NADH:ubiquinone oxidoreductase complex: Topography of the subunits of the iron-sulfur flavoprotein component

A catalytic component of the bovine mitochondrial NADH:ubiquinone oxidoreductase complex (Complex I) is a soluble NADH dehydrogenase iron-sulfur flavoprotein (FP). FP is composed of three subunits of M r 51,000, 24,000, and 9,000, and contains FMN and two iron-sulfur clusters. Previous studies by others with the use of various chemical probes had suggested that, except for an access for NADH to the 51-kDa subunit, the FP polypeptides are buried within Complex I and shielded from the medium. In the present study, monospecific antibodies were raised to each of the three FP subunits, and used in conjunction with Complex I, submitochondrial particles (SMP), mitoplasts, and intact mitochondria as sources of antigens. Results of enzyme-linked immunosorbent assays and 125I-protein A labeling experiments indicated that epitopes from the 51-, 24-, and 9-kDa subunits of FP are exposed to the medium in Complex I and SMP, but not in mitoplasts and mitochondria. Appropriate enzymatic assays showed that none of the antibodies inhibited the NADH dehydrogenase activity of isolated FP or the NADH oxidase activity of SMP. These results have been discussed in relation to the structure of Neurospora Complex I deduced from membrane crystals of the isolated enzyme complex by Leonard et al. [K. Leonard, H. Haiker, and H. Weiss (1987) J. Mol. Biol. 194, 277–286] .

Structure of NADH:Q oxidoreductase studied by electron microscopy

Structure of NADH:Q oxidoreductase studied by electron microscopy

Structure of NADH:Q oxidoreductase from bovine heart mitochondria studied by electron microscopy

Two-dimensional crystalline arrays of NADH:Q oxidoreductase preparations have been obtained by microdiffusion of protein dissolved in detergent against a 15 mM sodium acetate buffer of pH 5.5 containing 10% ( w v ) ammonium sulphate. Electron microscopy was used to study the structure of negatively stained crystals. Computer-reconstructed images were obtained by the Fourier peak filtering method. The crystals have p4 symmetry and a square unit cell with dimensions of 15.2 ± 0.5 nm. The four asymmetric units in the unit cell form a single tetrameric molecule with a dimension in the third direction of 8.2 nm. It is concluded on the basis of the estimated molecular mass that each tetramer cannot contain more than only one FMN molecule. This implies that the tetramers possibly are only a part of Complex I, since there is much evidence that one functional enzyme molecule of Complex I contains two FMN molecules.

Pyridine nucleotide involvement in rat hepatic microsomal drug metabolism—III. The influence of the 1,4,5,6-tetrahydronicotinamide analogue of NADH on the NADPH kinetics of aminopyrine- N-demethylation

1,4,5,6-Tetrahydronicotinamide adenine dinucleotide (NADH 3), a structural analogue of NADH, was unable to support the demethylation of aminopyrine or the reduction of the cytochrome P450-aminopyrine complex. However, the combination of NADH 3 with NADPH stimulated the NADPH dependent reduction of the cytochrome P450-aminopyrine complex. There was no significant alteration in the apparent K m (NADPH) value, but there was an 80 per cent increase in apparent V of NADPH for NADPH-cytochrome P450-reductase (plus aminopyrine) when the kinetic constants were determined in the presence of 100 μM NADH 3. The inclusion of NADH 3 in the medium for aminopyrine demethylation also resulted in a significant increase in apparent V compared to the value obtained in the absence of NADH 3. The results suggest that the structure of NADH, as well as its capacity to donate the electron, is responsible for the NADH mediated increase in aminopyrine metabolism.

The temperature dependence of the spectroscopic properties of reduced nicotinamide adenine dinucleotide

The reduced form of nicotinamide adenine dinucleotide (NADH) is usually assayed by means of its absorption at 340 or 366 nm (1). The structure of NADH in aqueous solution is thought to be an equilibrium between a “folded” and an “unfolded” form and it is thought that the temperature sensitivity of the spectral properties of NADH reflects, at least in part, an increase in the proportion of the unfolded form with increasing temperature (2,3). In particular, the absorption spectrum of NADH is sensitive to this conformational change (1,4) but data on this variation of extinction coefficient are few (1,4). The finding (5) that there is a correlation between the stereospecificity of the dehydrogenases and the spectral shift of NADH on binding to these enzymes emphasises the need for such data. This paper deseribes the analytical significance of these changes in extinction coefficient and also estimates the enthalpy of the conformational change.