Nicotinamide Adenine Dinucleotide Binding Site Research Articles

Dual Oxidase System Genes Defects in Children With Congenital Hypothyroidism.

The objectives of this study were to analyze the distribution of dual oxidase (DUOX) system genes (containing DUOX2, DUOX1, DUOXA2, and DUOXA1) variants in children with congenital hypothyroidism (CH) and their phenotypes. Target region sequencing technology was performed on DUOX system genes among 606 CH subjects covering all the exon and intron regions. Detailed clinical data were collected for statistical analysis. A total of 95 suspected pathogenic variants were detected in the DUOX system genes, showing a 39.11% rate in variant carrying (237/606). DUOX2 had the highest rate in this study. There were statistical differences in maximum adjusted dose and current dose of levothyroxine between the DUOX system genes nonmutated group with the mutated group (both Ps < 0.001). The cases in the DUOX system genes mutated group were more likely to develop into transient CH (χ 2 = 23.155, P < 0.001) and more likely to manifested as goiter or gland-in-situ (χ 2 = 66.139, P < 0.001). In addition, there was no significant difference in clinical characteristics between DUOX system genes monoallelic and non-monoallelic. Although 20% of the variants affected the functional domain regions (EF hand, flavin adenine dinucleotide and nicotinamide adenine dinucleotide binding sites), there was no significant effect on the phenotype severity whether the variation is located in the functional domain regions. Our results showed the high variation rate of DUOX2 in the DUOX system genes among Chinese CH patients. The complex genotype-phenotype relationship of DUOX system genes broadened the understanding of CH phenotype spectrum.

Predicting flavin and nicotinamide adenine dinucleotide-binding sites in proteins using the fragment transformation method.

We developed a computational method to identify NAD- and FAD-binding sites in proteins. First, we extracted from the Protein Data Bank structures of proteins that bind to at least one of these ligands. NAD-/FAD-binding residue templates were then constructed by identifying binding residues through the ligand-binding database BioLiP. The fragment transformation method was used to identify structures within query proteins that resembled the ligand-binding templates. By comparing residue types and their relative spatial positions, potential binding sites were identified and a ligand-binding potential for each residue was calculated. Setting the false positive rate at 5%, our method predicted NAD- and FAD-binding sites at true positive rates of 67.1% and 68.4%, respectively. Our method provides excellent results for identifying FAD- and NAD-binding sites in proteins, and the most important is that the requirement of conservation of residue types and local structures in the FAD- and NAD-binding sites can be verified.

Computer Simulations of NAD Channeling between GAPDH and LDH

Functional protein-protein interactions are essential for many physiological processes and may play important roles in substrate channeling, coenzyme transfer, and compartmentation in glycolysis. Herein, Brownian dynamics (BD) elucidates the interactions between the glycolytic enzymes glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and lactate dehydrogenase (LDH) and the transfer of the cofactor nicotinamide adenine dinucleotide (NAD) between LDH and GAPDH. BD channeling simulation results strongly depend on choice of reactive atom set. When the reactive atoms set comprise of atoms in the vicinity of the NAD binding site of either enzyme, short NAD trajectories between enzyme subunits occur. If the reactive atoms set were chosen from atoms belonging to NAD binding sites, the efficiency of reaching LDH decreased significantly, and even the shortest trajectories spent time equilibrating with solvent before binding the next active site. Transfer of NAD from GAPDH to LDH is sensitive to overall structure of enzyme-enzyme complex. Small variations in orientation of one enzyme relative to the other cause changes in channeling efficiency. The process of NAD release from GAPDH and LDH binding sites was studied with steered molecular dynamics. The GAPDH/LDH complex with two LDH subunits facing two GAPDH subunits was chosen as initial structure. Six NAD molecules were included - two molecules in GAPDH active subunits and 4 NAD molecules bound to each LDH subunit. External forces were applied to O3 NAD atom of each NAD molecule. MD trajectories (2 ns) with external force (≤ 1000 pN) were able to pull the NAD out of GAPDH, but not out of LDH. Such strong binding of NAD by LDH is because the nicotinic moiety is buried deep inside LDH subunit globule. Additional studies are needed to confirm the hypothesis that intermolecular contacts soften GAPDH subunit and its affinity to NAD reduces.

Association between SIRT2 gene polymorphism and height in healthy, elderly Japanese subjects

T he silent information regulator factor 2 (SIR2; a nicotinamide adenine dinucleotide–dependent histone deacetylase) protein is related to life span extension in a diverse range of species. In mammals there are 7 sirtuins, all possessing a highly conserved central nicotinamide adenine dinucleotide–binding site and common catalytic domain. Sirtuin 2 (SIRT2) is the mammalian ortholog of the yeast HST2, one of the four SIR2 paralogs. Similar to SIR2, HST2 is upregulated by caloric restriction and oxidative stress and extends life span. SITR2 acts as an important regulator of cell differentiation through modulation of forkhead transcription factor 1 acetylation/phosphorylation. Forkhead transcription factor 1 is a homolog of daf-16, a key regulator of the insulin-like growth factor 1 signaling pathway, and is necessary for increasing life span. An association between chronic obstructive pulmonary disease and SIRT2 single nucleotide polymorphisms (SNPs) has been reported, but an association of the SIRT2 gene with longevity and other phenotypes has not been reported. We focused on the biological pleiotropic roles of SIRT2 and investigated their association with various laboratory and anthropometric data in elderly Japanese subjects.

Differential binding of NAD+ and NADH allows the transcriptional corepressor carboxyl-terminal binding protein to serve as a metabolic sensor.

Carboxyl-terminal binding protein (CtBP) is a transcriptional corepressor originally identified through its ability to interact with adenovirus E1A. The finding that CtBP-E1A interactions were regulated by the nicotinamide adeninine dinucleotides NAD+ and NADH raised the possibility that CtBP could serve as a nuclear redox sensor. This model requires differential binding affinities of NAD+ and NADH, which has been controversial. The structure of CtBP determined by x-ray diffraction revealed a tryptophan residue adjacent to the proposed nicotinamide adenine dinucleotide binding site. We find that this tryptophan residue shows strong fluorescence resonance energy transfer to bound NADH. In this report, we take advantage of these findings to measure the dissociation constants for CtBP with NADH and NAD+. The affinity of NADH was determined by using fluorescence resonance energy transfer. The binding of NADH to protein is associated with an enhanced intensity of NADH fluorescence and a blue shift in its maximum. NAD+ affinity was estimated by measuring the loss of the fluorescence blue shift as NADH dissociates on addition of NAD+. Our studies show a >100-fold higher affinity of NADH than NAD+, consistent with the proposed function of CtBP as a nuclear redox sensor. Moreover, the concentrations of NADH and NAD+ required for half-maximal binding are approximately the same as their concentrations in the nuclear compartment. These findings support the possibility that changes in nuclear nicotinamide adenine dinucleotides could regulate the functions of CtBP in cell differentiation, development, or transformation.

The Nicotinamide Adenine Dinucleotide-Binding Site of Chicken Liver Xanthine Dehydrogenase: Evidence for alteration of the redox potential of the flavin by NAD binding or modification of the NAD-binding site and isolation of a modified peptide

Abstract Affinity labeling of the NAD-binding site of chicken liver xanthine dehydrogenase by 5'-p-fluorosulfonylbenzoyladenosine (5'-FSBA) caused spectral perturbation around 450 nm in the same way as NAD. Reductive titration with xanthine of native xanthine dehydrogenase in the presence of NAD showed that redox potentials of the FAD/FADH. and FADH./FADH2 couples were shifted positive by NAD binding to the enzyme. The redox potentials of these couples were also shifted to some extent by modification of the NAD-binding site with 5'-FSBA. These results provide further evidence that binding of NAD to chicken liver xanthine dehydrogenase modulates the reactivity of the enzyme by shifting the redox potential of FAD. Proteolytic cleavage of the [14C]-5'-FSBA-modified enzyme yielded several domain peptides, only one of which contained radioactivity. The isolated radioactive peptide was further digested with Staphylococcus aureus protease and the 14C-labeled peptide was purified by two steps of high performance liquid chromatography. The amino acid sequence of the peptide was determined, and a reactive tyrosine residue was identified.

Evidence for the existence of a tyrosyl residue in the nicotinamide adenine dinucleotide binding site of chicken liver xanthine dehydrogenase.

Xanthine-NAD and NADH-methylene blue oxidoreductase activities of chicken liver xanthine dehydrogenase were inactivated by incubation with 5'-[p-(fluorosulfonyl)benzoyl]adenosine (5'-FSBA), an active site directed reagent for nucleotide binding sites. The inactivation reaction displayed pseudo-first-order kinetics. A double-reciprocal plot of inactivation velocity vs. 5'-FSBA concentration showed that 5'-FSBA and enzyme formed a complex prior to inactivation. NAD protected the enzyme from inactivation by 5'-FSBA in a competitive fashion. The modified enzyme had the same xanthine-dichlorophenolindophenol and xanthine-O2 oxidoreductase activities as the native enzyme, and on addition of xanthine to the modified enzyme, bleaching of the spectrum occurred in the visible region. The amount of radioactivity incorporated into the enzyme by incubation with [14C]-5'-FSBA was parallel to the loss of xanthine-NAD oxidoreductase activity, and the stoichiometry was 1 mol/mol of enzyme-bound FAD for complete inactivation. These results indicated that 5'-FSBA modified specifically the binding site for NAD of chicken liver xanthine dehydrogenase. The incorporated radioactivity was released slowly from 14C-labeled enzyme by incubation with dithiothreitol with concomitant restoration of catalytic activity. The modified residue responsible for inactivation was identified as a tyrosine.

Binding of nicotinamide adenine dinucleotide by the renal brush border membrane from rat kidney cortex

The characteristics of nicotinamide adenine dinucleotide (NAD) binding on brush border membranes prepared from rat renal cortex were investigated with the use of radioactively labelled NAD, [ adenine-2,8- 3H]NAD +, as a ligand. (1) We found that NAD binds on brush border membrane and that the extent of NAD binding is linearly proportional to the brush border membrane protein, and progressively increases with concentration of NAD in the medium. (2) The rate of NAD binding was dependent on temperature. At 20°C, the equilibrium binding was obtained at 15 min, while NAD binding at 0°C was slower, but the final level of binding reached at 120 min was similar to that plateau of binding observed at 20°C. Brush border membrane inactivated by heating at 95°C for 3 min did not bind NAD. Binding of NAD on brush border membranes was reversed by simple dilution or by the addition of unlabelled NAD. Both α-NAD and β-NAD stereoisomers displaced bound [ 3H]NAD. Reduced NAD (NADH) caused less displacement of bound NAD than oxidized NAD +. Adenine, nicotinamide, pyrophosphate, of 5′-AMP did not displace bound NAD. (3) The NAD binding to brush border membranes was nearly saturable, approximating saturation at 10 −4 M NAD. Kinetic analysis by Scatchard plot indicates two sets of NAD binding sites in brush border membranes: a high-affinity binding site ( K d = 1.9·10 −5 M) and a low-affinity binding site ( K d = 2.2·10 −3 M). (4) Unlike concentrative uptake of d-[ 14C]glucose by brush border membrane vesicles, binding of NAD was not dependent on the presence of an outside-in sodium gradient [Na o + > Na i +], nor was it abolished by repeated freezing and thawing of brush border membranes. Unlike d-[ 14C]glucose uptake, NAD binding by brush border membranes did not change upon decrease of intravesicular volume in hypertonic media. These observations indicate that NAD association with brush border membranes is true binding rather than intravesicular uptake of this compound. (5) The presence of specific binding sites in renal brush border membrane capable of binding of NAD with a high degree of affinity suggests that such sites may be involved in previously observed (Kempson, S.A., Colon-Otero, G., Ou, S.L., Turner, S.T. and Dousa, T.P. (1981) J. Clin. Invest. 67, 1347) modulatory effect of NAD on sodium-gradient-dependent uptake of phosphate across luminal brush border membrane of proximal tubules.

Nicotinamide adenine dinucleotide depresses synaptic transmission in the hippocampus and has specific binding sites on the synaptic membranes

The electrical activity of transverse slices of hippocampus was used as a bioassay in which extracts of fresh brain tissue were screened for biological activity. A factor that depressed synaptic transmission was identified as nicotinamide adenine dinucleotide (NAD). This depressant action of NAD could be observed at concentrations in the range 1-10 microM and the degree of depression was monotonically related to the concentration of NAD in the bathing medium. NAD did not affect the antidromic invasion of the granule cells nor did it alter the relationship between the electrically evoked excitatory postsynaptic field potential (e.p.s.p.) and the population discharge of the granule cells (population spike). These results suggest that NAD did not affect the electrical excitability of the neuronal membranes. NAD had little effect on the sensitivity of granule cells to iontophoretically applied L-glutamate, the putative excitatory transmitter for the perforant path-granule cell pathway. Pure synaptosomal membranes, free of mitochondria, had two binding sites for NAD: a high affinity site with a Kd of 1 microM and a low affinity site with a Kd of 17 microM. These sites were similar in affinity to those of mitochondria, although the density of the high affinity sites was 5 X greater in the synaptosomal membranes. Adenosine had a relatively weak affinity for the NAD binding sites. It was concluded that NAD probably depressed synaptic transmission in the dentate gyrus by binding to sites on the presynaptic nerve terminal and reducing the amount of transmitter released by a nerve impulse. The physiological significance of this view is discussed.

Resolution and reconstitution of Rhodospirillum rubrum pyridine dinucleotide transhydrogenase: Localization of substrate binding sites

Butanedione in the presence of borate buffer reversibly inhibits Rhodospirillum rubrum chromatophore transhydrogenase complex and the separated membrane-bound and soluble factor components of the complex. NADP + completely protected against inactivation of the membrane-bound component, whereas NAD + was without effect. Soluble factor was maximally protected only partially by either NAD + or NADP +, but a mixture of the substrates afforded complete protection. NADP +-dependent association of soluble factor with factor-depleted membranes was markedly decreased after incubation of membranes with butanedione in the absence, but not in the presence, of NADP +. Soluble factor was bound to agarose-NAD and was eluted by NAD +, but not by NADP +. These results demonstrate the presence of at least three nicotinamide adenine dinucleotide binding sites on R. rubrum transhydrogenase complex, including separate NADP and NAD binding sites on soluble factor and a NADP binding site on the membrane-bound component.

Nonpolar Interactions of Inhibitors with the Nicotinamide Adenine Dinucleotide-binding Sites of L-α-Glycerophosphate Dehydrogenase

Abstract l-α-Glycerophosphate dehydrogenase was shown to be inhibited by a homologous series of aliphatic carboxylic acids ranging in size from formic to dodecanoic acid, inclusive. The inhibition obtained was observed to be competitive with respect to the coenzyme, NAD, and noncompetitive with respect to substrate. The binding of carboxylic acids to the enzyme was shown to increase with increasing chain length of these inhibitors. Multiple inhibition studies were used to show that, in binding to the dehydrogenase, the carboxylic acids utilize the pyrophosphate and hydrophobic regions of the NAD-binding sites. It is suggested that nonpolar interactions with the same hydrophobic region facilitate the binding of carboxylic acids, N1-alkylnicotinamide chlorides, and n-alkylammonium chlorides to this enzyme.

Properties of the Nicotinamide Adenine Dinucleotide-binding Sites of L-α-Glycerophosphate Dehydrogenase

Abstract l-α-Glycerophosphate dehydrogenase was demonstrated to be inhibited by a homologous series of N1-alkylnicotinamide chlorides, ranging from the N1-methyl to the N1-dodecyl derivative, inclusive. The inhibition obtained was observed to be reversible and competitive with respect to the coenzyme NAD. The binding of N1-alkylnicotinamide chlorides larger than the N1-pentyl derivative to glycerophosphate dehydrogenase was shown to be facilitated through nonpolar interactions with the enzyme. Adenosine, adenylic acid, adenosine diphosphate, and adenosine diphosphoribose were also shown to be coenzyme-competitive inhibitors of glycerophosphate dehydrogenase. The effectiveness of binding of these compounds increased with the size of the adenine derivative employed. Multiple inhibition analysis was used to demonstrate simultaneous binding of inhibitors that interact with different regions of the NAD-binding site of the enzyme. Inhibitors interacting with the same regions of the NAD-binding site were shown to mutually exclude one another from binding to the enzyme. It was suggested that the binding of NAD to glycerophosphate dehydrogenase occurs through interactions involving the adenosine moiety, the pyrophosphate grouping, and the positively charged nicotinamide ring system. The presence of a hydrophobic region near the NAD-binding site of the enzyme was indicated.