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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Our laboratory has concentrated on the possible regulation the benzamides and nicotinamides may have on the processes of DNA repair and apoptosis. Recent reports have suggested that both apoptosis and inflammation are regulated by the transcription factor NF-kappaB. We have initiated studies regarding the hypothesis that the benzamides and nicotinamides could inhibit the production of tumor necrosis factor alpha (TNFalpha) and the inflammatory response as well as induce apoptosis via inhibition of NF-kappaB. Our data have shown that nicotinamide and two N-substituted benzamides, metoclopramide (MCA) and 3-chloroprocainamide (3-CPA), gave dose dependent inhibition of lipopolysacharide induced TNFalpha in the mouse within the dose range of 10-500 mg/kg. Moreover, lung edema was prevented in the rat by 3 x 50 mg/kg doses of 3-CPA or MCA, and 100-200 microM doses of MCA could also inhibit NF-kappaB in Hela cells. Taken together these data strongly support the notion that benzamides and nicotinamides have potent anti-inflammatory and antitumor properties, because their primary mechanism of action is regulated by inhibition at the gene transcription level of NF-kappaB, which in turn inhibits TNFalpha and induces apoptosis.
Mol Cell Biochem 1999 Mar
PMID:Newly discovered anti-inflammatory properties of the benzamides and nicotinamides. 1033 48

The aim of this work is to study the effect of thioctamide--the commercial form of alpha lipoic acid amide--on the porphyrinogenic action of hexachlorobenzene (HCB). For this purpose, porphyria was induced in rats by chronic HCB treatment, with or without simultaneous thioctamide administration. Two different groups of rats were used as reference: one treated with vehicle (control) and the other treated with thioctamide (TO). Urine delta aminolevulic acid, porphobilinogen, and porphyrin excretions were lower in the HCB + TO treated group than in the HCB group, and the same happened with liver uroporphyrin accumulation. On the other hand, the second stage of uroporphyrinogen-decarboxylase activity was significantly higher in the HCB + TO group than in the HCB group. delta aminolevulic acid synthase activity was higher in the HCB group. Hepatic thiobarbituric acid reactive substances were lower in HCB + TO group than in HCB group. Thus, we might suggest that TO would decrease HCB effects by means of its free radical scavenging ability, and by having a direct effect on uroporphyrinogen-decarboxylase activity.
Biochem Mol Biol Int 1999 May
PMID:Effect of alpha lipoic acid amide on hexachlorobenzene porphyria. 1036 52

Drosophila alcohol dehydrogenase (DADH) is an NAD+-dependent enzyme that catalyzes the oxidation of alcohols to aldehydes/ketones. DADH is the member of the short-chain dehydrogenases/reductases family (SDR) for which the largest amount of biochemical data has been gathered during the last three decades. The crystal structures of one binary form (NAD+) and three ternary complexes with NAD+.acetone, NAD+.3-pentanone and NAD+.cyclohexanone were solved at 2.4, 2.2, 1. 4 and 1.6 A resolution, respectively. From the molecular interactions observed, the reaction mechanism could be inferred. The structure of DADH undergoes a conformational change in order to bind the coenzyme. Furthermore, upon binding of the ketone, a region that was disordered in the apo form (186-191) gets stabilized and closes the active site cavity by creating either a small helix (NAD+. acetone, NAD+.3-pentanone) or an ordered loop (NAD+.cyclohexanone). The active site pocket comprises a hydrophobic bifurcated cavity which explains why the enzyme is more efficient in oxidizing secondary aliphatic alcohols (preferably R form) than primary ones. Difference Fourier maps showed that the ketone inhibitor molecule has undergone a covalent reaction with the coenzyme in all three ternary complexes. Due to the presence of the positively charged ring of the coenzyme (NAD+) and the residue Lys155, the amino acid Tyr151 is in its deprotonated (tyrosinate) state at physiological pH. Tyr151 can subtract a proton from the enolic form of the ketone and catalyze a nucleophilic attack of the Calphaatom to the C4 position of the coenzyme creating an NAD-ketone adduct. The binding of these NAD-ketone adducts to DADH accounts for the inactivation of the enzyme. The catalytic reaction proceeds in a similar way, involving the same amino acids as in the formation of the NAD-ketone adduct. The p Kavalue of 9-9.5 obtained by kinetic measurements on apo DADH can be assigned to a protonated Tyr151 which is converted to an unprotonated tyrosinate (p Ka7.6) by the influence of the positively charged nicotinamide ring in the binary enzyme-NAD+form. pH independence during the release of NADH from the binary complex enzyme-NADH can be explained by either a lack of electrostatic interaction between the coenzyme and Tyr151 or an apparent p Kavalue for this residue higher than 10.0.
J Mol Biol 1999 Jun 04
PMID:The catalytic reaction and inhibition mechanism of Drosophila alcohol dehydrogenase: observation of an enzyme-bound NAD-ketone adduct at 1.4 A resolution by X-ray crystallography. 1036 9

Here we show that nicotinamide modulates the promoter activity of rat thyrotropin (TSHR) and major histocompatibility complex (MHC) class II genes in rat FRTL-5 thyroid cells, and have identified a novel mechanism for its action. TSHR and MHC class II, are potentiated through reduced expression of a common repressor of these two genes, TSEP-1 (TSHR suppressor element binding protein-1)/YB-1. Thus we show that TSHR mRNA is increased and TSHR promoter activity was concentration-dependently activated from 0 to 40 mM nicotinamide. The promoter lengths of TSHR and MHC class II containing TSEP/YB-1 binding sites were enhanced by 40 mM nicotinamide, but not the ones deleted of these binding sites. TSEP-1/YB-1 binding to the recognition sites in both TSHR and MHC class II promoters was reduced in nicotinamide-treated FRTL-5 nuclear extracts. Nicotinamide reduced the expression of TSEP-1/YB-1 mRNA and TSEP-1/YB-1 protein in the nucleus.
Mol Cell Endocrinol 1999 Mar 25
PMID:Nicotinamide potentiates TSHR and MHC class II promoter activity in FRTL-5 cells. 1037 26

We have previously shown that a range of nicotinamide containing 'biomimetic coenzymes' function as active analogues of NAD+ in the oxidation of alcohols by horse liver alcohol dehydrogenase (HLADH), despite their apparently astonishing lack of structural similarity to the natural coenzyme. The simplest structure as yet shown to exhibit activity is the biomimetic coenzyme CL4. To investigate the effect of the structure of this truncated artificial coenzyme on its activity, a range of close structural analogues of CL4 were designed, synthesized and characterized. The electrochemical reduction potentials of the analogues were strongly influenced by the nature of the groups attached to the pyridine ring. All of the analogues could be chemically reduced using sodium borohydride, to give compounds with altered UV-visible absorption and fluorescence properties. An HPLC-based assay suggested that two of the new analogues were coenzymically active in the oxidation of butan-1-ol by HLADH, with one displaying a significantly higher activity than CL4. The results demonstrate which features of the structures of the coenzymes lead to desirable electrochemical and spectroscopic properties, but suggest that the structural requirements for a functional coenzyme are quite stringent. These observations may be used to design an artificial coenzyme which combines the best features of those studied so far.
J Mol Recognit
PMID:Synthesis and properties of new coenzyme mimics based on the artificial coenzyme CL4. 1039 96

The molecular basis of the interaction of DT-diaphorase with a cytotoxic nitrobenzamide CB1954 [5-(aziridin-1-yl)-2, 4-dinitrobenzamide] and five inhibitors was investigated with wild-type DT-diaphorase (human and rat) and five mutants [three rat mutants (rY128D, rG150V, rH194D) and two human mutants (hY155F, hH161Q)]. hY155F and hH161Q were generated to evaluate a hypothesis that Tyr155 and His161 participate in the obligatory two-electron transfer reaction of the enzyme. The catalytic properties of hY155F and hH161Q were compared with a naturally occurring mutant, hP187S. Pro187 to Ser mutation disturbs the structure of the central parallel beta-sheet, resulting in a reduction of the binding affinity of the flavin-adenine dinucleotide prosthetic group. With NADH as the electron donor and menadione as the electron acceptor, the k(cat) values for the wild-type human DT-diaphorase, hY155F, hH161Q, and hP187S were measured as 66 +/- 1, 23 +/- 0, 5 +/- 0 and 8 +/- 2 x 10(3) min(-1), respectively. Because hY155F still has significant catalytic activity, the hydroxyl group on Tyr155 may not be as important as proposed. Interestingly, hY155F was found to be 3. 3 times more active than the human wild-type DT-diaphorase in the reduction of CB1954. Computer modeling based on our results suggests that CB1954 is situated in the active site, with the aziridinyl group pointing toward Tyr155 and the amide group placed near a hydrophobic pocket next to Tyr128. Dicoumarol, Cibacron blue, chrysin, 7,8-dihydroxyflavone, and phenindone are competitive inhibitors of the enzyme with respect to nicotinamide coenzymes. The binding orientations of dicoumarol, flavones, and phenindone in the active site of DT-diaphorase were predicted by results from our inhibitor-binding studies and computer modeling based on published X-ray structures. Our studies generated results that explain why dicoumarol is a potent inhibitor and binds differently from flavones and phenindone in the active site of DT-diaphorase.
Mol Pharmacol 1999 Aug
PMID:Molecular characterization of binding of substrates and inhibitors to DT-diaphorase: combined approach involving site-directed mutagenesis, inhibitor-binding analysis, and computer modeling. 1041 45

Five rough colony mutants of Mycobacterium smegmatis mc2155 were produced by transposon mutagenesis. The mutants were unable to synthesize glycopeptidolipids that are normally abundant in the cell wall of wild-type M. smegmatis. The glycopeptidolipids have a lipopeptide core comprising a fatty acid amide linked to a tetrapeptide that is modified with O-methylated rhamnose and O-acylated 6-deoxy talose. Compositional analysis of lipids extracted from the mutants indicated that the defect in glycopeptidolipid synthesis occurred in the assembly of the lipopeptide core. No other defects or compensatory changes in cell wall structure were detected in the mutants. All five mutants had transposon insertions in a gene encoding an enzyme belonging to the peptide synthetase family. Targeted disruption of the gene in the wild-type strain gave a phenotype identical to that of the five transposon mutants. The M. smegmatis peptide synthetase gene is predicted to encode four modules that each contain domains for cofactor binding and for amino acid recognition and adenylation. Three modules also have amino acid racemase domains. These data suggest that the common lipopeptide core of these important cell wall glycolipids is synthesized by a peptide synthetase.
Mol Microbiol 1999 Sep
PMID:Identification of a peptide synthetase involved in the biosynthesis of glycopeptidolipids of Mycobacterium smegmatis. 1051 Feb 38

Drosophila alcohol dehydrogenase belongs to the short chain dehydrogenase/reductase (SDR) family which lack metal ions in their active site. In this family, it appears that the three amino acid residues, Ser138, Tyr151 and Lys155 have a similar function as the catalytic zinc in medium chain dehydrogenases. The present work has been performed in order to obtain information about the function of these residues. To obtain this goal, the pH and temperature dependence of various kinetic coefficients of the alcohol dehydrogenase from Drosophila lebanonensis was studied and three-dimensional models of the ternary enzyme-coenzyme-substrate complexes were created from the X-ray crystal coordinates of the D. lebanonensis ADH complexed with either NAD(+) or the NAD(+)-3-pentanone adduct. The kon velocity for ethanol and the ethanol competitive inhibitor pyrazole increased with pH and was regulated through the ionization of a single group in the binary enzyme-NAD(+) complex, with a DeltaHion value of 74(+/-4) kJ/mol (18(+/-1) kcal/mol). Based on this result and the constructed three-dimensional models of the enzyme, the most likely candidate for this catalytic residue is Ser138. The present kinetic study indicates that the role of Lys155 is to lower the pKa values of both Tyr151 and Ser138 already in the free enzyme. In the binary enzyme-NAD(+) complex, the positive charge of the nicotinamide ring in the coenzyme further lowers the pKa values and generates a strong base in the two negatively charged residues Ser138 and Tyr151. With the OH group of an alcohol close to the Ser138 residue, an alcoholate anion is formed in the ternary enzyme NAD(+) alcohol transition state complex. In the catalytic triad, along with their effect on Ser138, both Lys155 and Tyr151 also appear to bind and orient the oxidized coenzyme.
J Mol Biol 1999 Nov 26
PMID:The catalytic triad in Drosophila alcohol dehydrogenase: pH, temperature and molecular modelling studies. 1061 Jul 83

DT-diaphorase is an FAD-containing enzyme capable of a two-electron reduction of ortho- and paraquinones. Nicotinamide coenzymes (NADH + H+ and NADPH + H+) serve as hydrogen sources in these reactions. The role of DT-diaphorase has been thoroughly investigated in situations when the enzyme is able to reduce exogenous and endogenous quinones, hence protecting the cells against these reactive intermediates. The enzyme has also been studied in connection with its ability to activate some quinoid cytostatics. It is surprising that DT-diaphorase has never been investigated in pigment-producing cells that are known to generate considerable amounts of ortho-quinones. Using a spectrophotometric method we could readily measure the activity of DT-diaphorase in epidermis and various cultured pigment cells. The melanocytes isolated from dark skin showed generally higher DT-diaphorase activity than those from fair skin samples. Also, darkly pigmented congenital naevus cells exhibited higher activity of this enzyme. The most striking was the high DT-diaphorase activity in melanoma cell cultures. In these cells DT-diaphorase activity could be induced by incubation of the cells with 4-hydroxyanisole. A similar effect was seen when a catechol-O-methyltransferase (COMT) inhibitor (3-(3,4-dihydroxy-5-nitrobenzylidene)-2,4-pentanedione (OR-462) was utilised. The induction was inhibited by cyclohexidine.
Cell Mol Biol (Noisy-le-grand) 1999 Nov
PMID:Study of DT-diaphorase in pigment-producing cells. 1064 8

Enzymes bind NAD(+) in extended conformations and yet NAD(+) exists in aqueous solution as a compact, folded molecule. Thus, NAD(+) conformation is environment dependent. In an attempt to investigate the effects of environmental changes on the conformation of NAD(+), a series of molecular dynamics simulations in different solvents was performed. The solvents investigated (water, DMSO, methanol and chloroform) represented changes in relative permittivity and hydrophobic character. The simulations predicted folded conformations of NAD(+) to be more stable in water, DMSO and methanol. In contrast, extended conformations of NAD(+) were observed to be more stable in chloroform. Furthermore, the extended conformations observed in chloroform were similar to conformations of NAD(+) bound to enzymes. In particular, a large separation between the aromatic rings and a strong interaction between the pyrophosphate and nicotinamide groups were observed. The implications of these observations for the recognition of NAD(+) by enzymes is discussed. It is argued that a hydrophobic environment is important for stabilizing unfolded conformations of NAD(+).
J Mol Recognit
PMID:Conformations of nicotinamide adenine dinucleotide (NAD(+)) in various environments. 1067 94


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