NADH

NADH, short for nicotinamide adenine dinucleotide, is a non-protein organic substance which contains a vitamin or a mineral which combined with a specific protein forms an active enzyme system; this is why it is known as a coenzyme. In fact, its most common name is Coenzyme 1 because it is arguably the most important supplement which can be obtained by diet. NADH is present when the reaction between oxygen and glucose takes place in the mitochondria of each cell, and it is NADH that provides a spark for energy to come out of the reaction, known as respiration. In this way, without NADH the human body dies because it cannot accumulate energy. The levels of NADH in the body gradually decrease with age which leads to an energy poor state – the primary cause of aging. NADH is very important for elderly people because it promotes tissue regeneration and retards cell death. Its most common effect lies in the treatment and prevention of Alzheimer’s disease and dementia, because it improves cognitive functions and memory. NADH supplementation has also proved effective in the treatment of chronic fatigue syndrome as well as insomnia.

Mech Ageing Dev. 2005 Jun-Jul;126(6-7):705-9. Epub 2005 Feb 12.
Longevity-associated NADH dehydrogenase subunit-2 polymorphism and serum electrolyte levels in middle-aged obese Japanese men.
Kokaze A, Ishikawa M, Matsunaga N, Yoshida M, Makita R, Satoh M, Teruya K, Sekiguchi K, Masuda Y, Harada M, Uchida Y, Takashima Y.
Department of Public Health, Kyorin University School of Medicine, 6-20-2 Shinkawa, Mitaka-shi, Tokyo 181-8611, Japan.

Mitochondrial DNA 5178 cytosine/adenine polymorphism, which is also called NADH dehydrogenase subunit-2 237 leucine/methionine (ND2-237 Leu/Met) polymorphism is associated with Japanese longevity. This polymorphism is widely associated with blood pressure, serum lipid levels, hematological parameters, intraocular pressure, and serum protein fraction levels. However, there have been no reports on the association between ND2-237 Leu/Met polymorphism and serum electrolyte levels. To investigate this relationship, we performed an association study in 321 healthy middle-aged Japanese men. Crude data showed that serum sodium levels and serum chloride levels were significantly lower in men with ND2-237 Met than in those with ND2-237 Leu (P=0.021 and 0.003, respectively). Cigarette consumption and body mass index were significantly and positively associated with serum chloride levels (P=0.002 and 0.008, respectively) and hemoglobin levels were significantly and negatively associated with them (P=0.007) in ND2-237 Leu genotypic men. In men with ND2-237 Met, only hemoglobin levels were significantly and negatively associated with serum chloride levels (P=0.025). After adjusting for covariates, only in male obese (body mass index>/=25) subjects, serum sodium and chloride levels remained significantly lower, and serum calcium levels appeared to be significantly higher in ND2-237 Met than in ND2-237 Leu (P=0.013, <0.001, and 0.046, respectively). Longevity-associated NADH dehydrogenase subunit-2 polymorphism may influence serum electrolyte levels in middle-aged obese Japanese men.

Free Radic Biol Med. 2005 Jun 1;38(11):1484-90.
Pyruvate but not lactate prevents NADH-induced myoglobin oxidation.
Olek RA, Antosiewicz J, Popinigis J, Gabbianelli R, Fedeli D, Falcioni G.
Department of Bioenergetics, Jedrzej Sniadecki University School of Physical Education, Wiejska 1, 80-336 Gdansk, Poland.

In this work, we investigated the influence of NADH on the redox state of myoglobin and the roles of pyruvate and lactate in this process. NADH increased the autoxidation rate of myoglobin. Both a drop in pH and partial deoxygenation markedly stimulated the autoxidation process and the influence of NADH. A correlation between met-Mb formation rate and NADH oxidation rate was always observed. The increased rate of Mb autoxidation caused by NADH was inhibited by catalase and pyruvate but not by l-lactate. The antioxidant activity versus H(2)O(2) of both pyruvate and lactate was evidenced by chemiluminescence experiments. The antioxidant activity of lactate disappeared completely in the presence of myoglobin or apo-myoglobin, whereas it was only reduced for pyruvate. These results could be of interest in preventing autoxidation of myoglobin that can contribute to ischemia-reperfusion injury during infarction or high-intensity exercise.

Biophys Chem. 2005 Apr 1;115(2-3):219-24. Epub 2004 Dec 24.
Towards a new interaction enzyme:coenzyme.
Martinez-Julvez M, Tejero J, R Peregrina J, Nogues I, Frago S, Gomez-Moreno C, Medina M.
Departamento de Bioquimica y Biologia Molecular y Celular and Institute of Biocomputation and Physics of Complex Systems (BiFi), Facultad de Ciencias, Universidad de Zaragoza, 50009 Zaragoza, Spain.

Ferredoxin-NADP(+) reductase catalyses NADP(+) reduction, being specific for NADP(+)/H. To understand coenzyme specificity determinants and coenzyme specificity reversion, mutations at the NADP(+)/H pyrophosphate binding and of the C-terminal regions have been simultaneously introduced in Anabaena FNR. The T155G/A160T/L263P/Y303S mutant was produced. The mutated enzyme presents similar k(cat) values for NADPH and NADH, around 2.5 times slower than that reported for WT FNR with NADPH. Its K(m) value for NADH decreased 20-fold with regard to WT FNR, whereas the K(m) for NADPH remains similar. The combined effect is a much higher catalytic efficiency for NAD(+)/H, with a minor decrease of that for NADP(+)/H. In the mutated enzyme, the specificity for NADPH versus NADH has been decreased from 67,500 times to only 12 times, being unable to discriminate between both coenzymes. Additionally, giving the role stated for the C-terminal Tyr in FNR, its role in the energetics of the FAD binding has been analysed.

Arch Biochem Biophys. 2004 Nov 15;431(2):233-44.
Cytochrome b(5) reductase: the roles of the recessive congenital methemoglobinemia mutants P144L, L148P, and R159*.
Ainsley Davis C, Crowley LJ, Barber MJ.
Department of Biochemistry and Molecular Biology, College of Medicine, University of South Florida, Tampa, FL 33612, USA.
Recessive congenital methemoglobinemia (RCM, OMIM 250800) arises from defects in either the erythrocytic or microsomal forms of the flavoprotein, cytochrome b(5) reductase (cb(5)r) and was the first disease to be directly associated with a specific enzyme deficiency. Of the 33 verified mutations in cb(5)r that give rise to either the type I (erythrocytic) or type II (generalized) forms of RCM, three of the mutations, corresponding to P144L, L148P, and R159*, are located in a segment of the primary sequence composed of residues G143 to V171 which serves as a "hinge" or "linker" region between the FAD- and NADH-binding lobes of the protein. With the exception of R159*, which produces a truncated non-functional cb(5)r resulting in type II RCM, the type I methemoglobinemias resulting from the P144L or L148P mutations have been proposed to be due to decreased enzyme stability. Utilizing a recombinant form of the rat cb(5)r enzyme, we have generated the P144L, L148P, and P144L/L148P mutants, purified the resulting proteins to homogeneity and characterized their spectroscopic, kinetic, and thermodynamic properties. The three mutant proteins retained full complements of FAD with the P144L and L148P variants being spectroscopically indistinguishable from wild-type cb(5)r. In contrast, kinetic analyses revealed that the P144L, L148P, and P144L/L148P variants retained only 28, 31, and 8% of wild-type NADH:cytochrome b(5) reductase activity, respectively, together with significant alterations in affinity for both NADH and NAD(+). In addition, FAD oxidation-reduction potentials were 32, 19, and 65mV more positive for the mutants than the corresponding FAD/FADH(2) couple in native cb(5)r (E(0')=-272mV). Thermal and proteolytic stability measurements indicated that all three mutants were less stable than the wild-type protein while differential spectroscopy indicated altered pyridine nucleotide binding in all three variants. These results demonstrate that the "hinge" region is important in maintaining the correct orientation of the flavin- and pyridine nucleotide-binding lobes within the protein for efficient electron transfer and that the P144L and L148P mutations disrupt the normal registration of the FAD- and NADH-binding lobes resulting in altered affinities for both the physiological reducing substrate, NADH and its product, NAD(+).

Arch Biochem Biophys. 2004 Nov 1;431(1):138-44.
Detection of hydrogen peroxide with Amplex Red: interference by NADH and reduced glutathione auto-oxidation.
Votyakova TV, Reynolds IJ.
Department of Pharmacology, University of Pittsburgh, Pittsburgh, PA 15261, USA; Bakh Institute of Biochemistry, Moscow, Russia.
We report here that reduced pyridine nucleotides and reduced glutathione result in an oxidation of Amplex Red by dioxygen that is dependent on the presence of horseradish peroxidase (HRP). Concentrations of NADH and glutathione typically found in biological systems result in the oxidation of Amplex Red at a rate comparable to that produced, for example, by respiring mitochondria. The effects of NADH and glutathione in this assay system are likely to be the result of H(2)O(2) generation via a superoxide intermediate because both catalase and superoxide dismutase prevent the oxidation of Amplex Red. These results suggest caution in the assay of H(2)O(2) production in biological systems using the Amplex Red/HRP because the assay will also report the mobilization of NADH or glutathione. However, the interruption of this process by the addition of superoxide dismutase offers a simple and reliable method for establishing the source of the oxidant signal.

Arch Biochem Biophys. 2004 Nov 1;431(1):55-62.
Human myeloperoxidase catalyzes an oscillating peroxidase-oxidase reaction.
Brasen JC, Lunding A, Olsen LF.
CelCom, Institute of Biochemistry and Molecular Biology, Syddansk Universitet, Campusvej 55, DK-5230 Odense M, Denmark.
We have studied the peroxidase-oxidase reaction catalyzed by human myeloperoxidase in an open system where both substrates-molecular oxygen and NADH-are supplied continuously to the reaction mixture. The reaction shows oscillatory kinetics at pH values around 5, provided that the reaction medium in addition to the enzyme and the substrates also contains an aromatic electron mediator such as melatonin or 4-hydroxybenzoic acid and chloride ions at concentrations >1mM. The experimental findings can be simulated by a detailed model of the reaction. The results are important for our understanding of oxidant production in neutrophils.

Bioorg Med Chem. 2004 Nov 1;12(21):5525-5532.
Effects of new ubiquinone-imidazo[2,1-b]thiazoles on mitochondrial complex I (NADH-ubiquinone reductase) and on mitochondrial permeability transition pore.
Andreani A, Granaiola M, Leoni A, Locatelli A, Morigi R, Rambaldi M, Recanatini M, Lenaz G, Fato R, Bergamini C.
Dipartimento di Scienze Farmaceutiche, Universita' di Bologna, Via Belmeloro 6, 40126 Bologna, Italy. In this work we describe the synthesis of a series of imidazo[2,1-b]thiazoles and 2,3-dihydroimidazo[2,1-b]thiazoles connected by means of a methylene bridge to CoQ(0). These compounds were tested as specific inhibitors of the NADH:ubiquinone reductase activity in mitochondrial membranes. The imidazothiazole system when bound to the quinone ring in place of the isoprenoid lateral side chain, may increase the inhibitory effect (with an IC(50) for NADH-Q(1) activity ranging between 0.25 and 0.96μM) whereas the benzoquinone moiety seems to lose the capability to accept electrons from complex I as indicated by very low maximal velocity elicited by the compounds tested. Moreover the low rotenone sensitivity for almost all of these compounds suggests that they are only partially able to interact with the physiological ubiquinone-reduction site. The compounds were investigated for the capability of increasing the permeability transition of the inner mitochondrial membrane in isolated mitochondria. Unlike CoQ(0), which is considered a mitochondrial membrane permeability transition inhibitor, the new compounds were inducers.

J Bacteriol. 2004 Nov;186(21):7364-8.
Diversion of the Metabolic Flux from Pyruvate Dehydrogenase to Pyruvate Oxidase Decreases Oxidative Stress during Glucose Metabolism in Nongrowing Escherichia coli Cells Incubated under Aerobic, Phosphate Starvation Conditions.
Moreau PL.
Mailing address: CNRS-LCB, 31 Chemin J. Aiguier, 13009 Marseille, France. moreau@ibsm.cnrs-mrs.fr.
Ongoing aerobic metabolism in nongrowing cells may generate oxidative stress. It is shown here that the levels of thiobarbituric acid-reactive substances (TBARSs), which measure fragmentation products of oxidized molecules, increased strongly at the onset of starvation for phosphate (P(i)). This increase in TBARS levels required the activity of the histone-like nucleoid-structuring (H-NS) protein. TBARS levels weakly increased further in DeltaahpCF mutants deficient in alkyl hydroperoxide reductase (AHP) activity during prolonged metabolism of glucose to acetate. Inactivation of pyruvate oxidase (PoxB) activity decreased the production of acetate by half and significantly increased the production of TBARS. Overall, these data suggest that during incubation under aerobic, P(i) starvation conditions, metabolic flux is diverted from the pyruvate dehydrogenase (PDH) complex (NAD dependent) to PoxB (NAD independent). This shift may decrease the production of NADH and in turn the adventitious production of H(2)O(2) by NADH dehydrogenase in the respiratory chain. The residual low levels of H(2)O(2) produced during prolonged incubation can be scavenged efficiently by AHP. However, high levels of H(2)O(2) may be reached transiently at the onset of stationary phase, primarily because H-NS may delay the metabolic shift from PDH to PoxB.

J Microbiol Methods. 2004 Nov;59(2):271-81.
Identification by fluorescence spectroscopy of lactic acid bacteria isolated from a small-scale facility producing traditional dry sausages.
Ammor S, Yaakoubi K, Chevallier I, Dufour E.
Unite de Recherche Typicite des Produits Alimentaires, ENITA-CF, Site de Marmilhat, 63370, Lempdes, France.
Three different fluorescence spectra were recorded following excitation at 250 nm (aromatic amino acids+nucleic acids, AAA+NA), 316 nm (NADH) and 380 nm (FAD) for 20 type strain collections of lactic acid bacteria (LAB). Evaluation of the data using principal component analysis and factorial discriminant analysis showed a good discrimination of considered LAB at the genus, species and genus-species level. AAA+NA fluorophores showed the highest percentage of good classification. From AAA+NA spectra recorded on LAB isolated from a small-scale facility producing traditional dry sausages, we succeeded to identify 28 of 29 wild strains. This method allowed us to discriminate between Lactobacillus sakei subsp. carnosus and Lactobacillus sakei subsp. sakei. Thus, intrinsic fluorescence is an economical and powerful tool for the identification of wild LAB isolated from meat and meat products.

Microbes Infect. 2004 Nov;6(13):1163-70.
Oral streptococcal glyceraldehyde-3-phosphate dehydrogenase mediates interaction with Porphyromonas gingivalis fimbriae.
Maeda K, Nagata H, Nonaka A, Kataoka K, Tanaka M, Shizukuishi S.
Department of Preventive Dentistry, Osaka University Graduate School of Dentistry, 1-8 Yamadaoka, Suita, Osaka 565-0871, Japan.
Interaction of Porphyromonas gingivalis with plaque-forming bacteria is necessary for its colonization in periodontal pockets. Participation of Streptococcus oralis glyceraldehyde-3-phosphate dehydrogenase (GAPDH) and P. gingivalis fimbriae in this interaction has been reported. In this investigation, the contribution of various oral streptococcal GAPDHs to interaction with P. gingivalis fimbriae was examined. Streptococcal cell surface GAPDH activity was measured by incubation of a constant number of streptococci with glyceraldehyde-3-phosphate and analysis for the conversion of NAD(+) to NADH based on the absorbance at 340 nm. Coaggregation activity was measured by a turbidimetric assay. Cell surface GAPDH activity was correlated with coaggregation activity (r = 0.854, P < 0.01) with Spearman's rank correlation coefficient. S. oralis ATCC 9811 and ATCC 10557, Streptococcus gordonii G9B, Streptococcus sanguinis ATCC 10556, and Streptococcus parasanguinis ATCC 15909 exhibited high cell surface GAPDH activity and coaggregation activity; consequently, their cell surface GAPDHs were extracted with mutanolysin and purified on a Cibacron Blue Sepharose column. Subsequently, their DNA sequences were elucidated. Purified GAPDHs bound P. gingivalis recombinant fimbrillin by Western blot assay, furthermore, their DNA sequences displayed a high degree of homology with one another. Moreover, S. oralis recombinant GAPDH inhibited coaggregation between P. gingivalis and the aforementioned five streptococcal strains in a dose-dependent manner. These results suggest that GAPDHs of various plaque-forming streptococci may be involved in their attachment to P. gingivalis fimbriae and that they may contribute to P. gingivalis colonization.

Biochemistry. 2004 Oct 26;43(42):13442-51.
Pyruvate Dehydrogenase Kinase Isoform 2 Activity Stimulated by Speeding Up the Rate of Dissociation of ADP.
Bao H, Kasten SA, Yan X, Hiromasa Y, Roche TE.
Department of Biochemistry, Kansas State University, Manhattan, Kansas 66506.
Pyruvate dehydrogenase kinase 2 (PDK2) activity is stimulated by NADH and NADH plus acetyl-CoA via the reduction and reductive acetylation of the lipoyl groups of the dihydrolipoyl acetyltransferase (E2) component. Elevated K(+) and Cl(-) were needed for significant stimulation. Stimulation substantially increased both k(cat) and the K(m) for ATP; the fractional stimulation increased with the level of ATP. With an E2 structure lacking the pyruvate dehydrogenase (E1) binding domain, stimulation of PDK2 was retained, the K(m) for E1 decreased, and the equilibrium dissociation constant for ATP increased but remained much lower than the K(m) for ATP. Stimulation of PDK2 activity greatly reduced the fraction of bound ADP. These results fit an ordered reaction mechanism with ATP binding before E1 and stimulation increasing the rate of dissociation of ADP. Conversion of all of the lipoyl groups in the E2 60mer to the oxidized form (E2(ox)) greatly reduced k(cat) and the K(m) of PDK2 for ATP. Retention over an extended period of time of a low portion of reduced lipoyl groups maintains E2 in a state that supported much higher PDK2 activity than short-term (5 min) reduction of a large portion of lipoyl groups of E2(ox), but reduction of E2(ox) produced a larger fold stimulation. Reduction and to a greater extent reductive acetylation increased PDK2 binding to E2; conversion to E2(ox) did not significantly hinder binding. We suggest that passing even limited reducing equivalents among lipoyl groups maintains E2 lipoyl domains in a conformation that aids kinase function.

J Biol Chem. 2004 Oct 22;279(43):44394-44399. Epub 2004 Aug 10.
The Human TAZ Gene Complements Mitochondrial Dysfunction in the Yeast taz1{Delta} Mutant: IMPLICATIONS FOR BARTH SYNDROME.
Ma L, Vaz FM, Gu Z, Wanders RJ, Greenberg ML.
Department of Biological Sciences, Wayne State University, Detroit, Michigan, 48202.
Barth syndrome is a genetic disorder that is caused by different mutations in the TAZ gene G4.5. The yeast gene TAZ1 is highly homologous to human TAZ, and the taz1Delta mutant has phospholipid defects similar to those observed in Barth syndrome cells, including aberrant cardiolipin species and decreased cardiolipin levels. Subcellular fractionation studies revealed that Taz1p is localized exclusively in mitochondria, which supports the theory that tafazzins are involved in cardiolipin remodeling. Because cardiolipin plays an important role in respiratory function, we measured the energy transformation and osmotic properties of isolated mitochondria from the taz1Delta mutant. Energy coupling in taz1Delta mitochondria was dependent on the rate of oxidative phosphorylation, as coupling was diminished when NADH was used as a respiratory substrate but was unaffected when ethanol was the substrate. Membrane stability was compromised in taz1Delta mitochondria exposed to increased temperature and hypotonic conditions. Mitochondria from taz1Delta also displayed decreased swelling in response to ATP, which induces the yeast mitochondrial unspecific channel, and to alamethicin, a membrane-disrupting agent. Coupling was measured in taz1Delta cells containing different splice variants of the human TAZ gene. Only the variant that restores wild type cardiolipin synthesis (lacking exon 5) restored coupling in hypotonic conditions and at elevated temperature. These findings may shed light on the mitochondrial deficiencies observed in Barth syndrome.

Biotechnol Bioeng. 2004 Oct 20;88(2):157-67.
Analysis of in vivo kinetics of glycolysis in aerobic Saccharomyces cerevisiae by application of glucose and ethanol pulses.
Visser D, van Zuylen GA, van Dam JC, Eman MR, Proll A, Ras C, Wu L, van Gulik WM, Heijnen JJ.
PURAC, P.O. Box 21, 4200 AA Gorinchem, The Netherlands. D.Visser@pura.com
This article presents the dynamic responses of several intra- and extracellular components of an aerobic, glucose-limited chemostat culture of Saccharomyces cerevisiae to glucose and ethanol pulses within a time window of 75 sec. Even though the ethanol pulse cannot perturb the glycolytic pathway directly, a distinct response of the metabolites at the lower part of glycolysis was found. We suggest that this response is an indirect effect, caused by perturbation of the NAD/NADH ratio, which is a direct consequence of the conversion of ethanol into acetaldehyde. This effect of the NAD/NADH ratio on glycolysis might serve as an additional explanation for the observed decrease of 3PG, 2PG, and PEP during a glucose pulse. The responses measured during the ethanol pulse were used to evaluate the allosteric regulation of glycolysis. Our results confirm that FBP stimulates pyruvate kinase and suggest that this effect is pronounced. Furthermore, it appears that PEP does not play an important role in the allosteric regulation of phosphofructo kinase.

FEMS Microbiol Lett. 2004 Oct 15;239(2):309-18.
Heterologous expression of alkene monooxygenase components from Xanthobacter autotrophicus Py2 and reconstitution of the active complex.
Champreda V, Zhou NY, Leak DJ.
Department of Biological Sciences, Imperial College London, London SW7 2AZ, UK.
The coupling protein and ferredoxin from Xanthobacter autotrophicus Py2 alkene monooxygenase (Xamo) have been functionally expressed in both N-terminal affinity tagged fusion and native forms in Escherichia coli. However, attempts to express the NADH-oxidoreductase and oxygenase, always resulted in the production of inactive, insoluble proteins. Nevertheless, the recombinant reductase from the toluene 4-monooxygenase of Pseudomonas mendocina KR1 was found to functionally complement the Xamo system. In vitro reconstitution, using the recombinant coupling protein and other components purified from the wild type, showed that steady-state epoxidation rate and coupling efficiency were dependent on the relative concentration of Xamo components in the reaction. The optimal molar stoichiometric ratio of Xamo components was determined to be approximately 1:0.25-0.3:2:2 (oxygenase hexamer:reductase:ferredoxin:coupling protein), suggesting the formation of an efficient catalytic complex at the minimal stoichiometric ratio to saturate the probable two-fold symmetry binding sites on the oxygenase.

Biochemistry. 2004 Oct 12;43(40):12887-93.
Aminobacter aminovorans NADH:flavin oxidoreductase His140: a highly conserved residue critical for NADH binding and utilization.
Russell TR, Tu SC.
Department of Biology and Biochemistry, University of Houston, Houston, Texas 77204-5001, USA.
Homodimeric FRD(Aa) Class I is an NADH:flavin oxidoreductase from Aminobacter aminovorans. It is unusual because it contains an FMN cofactor but utilizes a sequential-ordered kinetic mechanism. Because little is known about NADH-specific flavin reductases in general and FRD(Aa) in particular, this study aimed to further explore FRD(Aa) by identifying the functionalities of a key residue. A sequence alignment of FRD(Aa) with several known and hypothetical flavoproteins in the same subfamily reveals within the flavin reductase active-site domain a conserved GDH motif, which is believed to be responsible for the enzyme and NADH interaction. Mutation of the His140 in this GDH motif to alanine reduced FRD(Aa) activity to <3%. An ultrafiltration assay and fluorescence quenching demonstrated that H140A FRD(Aa) binds FMN in the same 1:1 stoichiometric ratio as the wild-type enzyme, but with slightly weakened affinity (K(d) = 0.9 microM). Anaerobic stopped-flow studies were carried out using both the native and mutated FRD(Aa). Similar to the native enzyme, H140A FRD(Aa) was also able to reduce the FMN cofactor by NADH although much less efficiently. Kinetic analysis of anaerobic reduction measurements indicated that the His140 residue of FRD(Aa) was essential to NADH binding, as well as important for the reduction of the FMN cofactor. For the native enzyme, the cofactor reduction was followed by at least one slower step in the catalytic pathway.

Biochemistry. 2004 Oct 12;43(40):12799-808.
Rates of the phthalate dioxygenase reaction with oxygen are dramatically increased by interactions with phthalate and phthalate oxygenase reductase.
Tarasev M, Rhames F, Ballou DP.
Department of Biological Chemistry, University of Michigan, Ann Arbor, Michigan 48109-0606, USA.
The phthalate dioxygenase system, which catalyzes the dihydroxylation of phthalate to form its cis-dihydrodiol (DHD), has two components: phthalate dioxygenase (PDO), a multimer with one Rieske-type [2Fe-2S] and one Fe(II) center per monomer, and phthalate dioxygenase reductase (PDR), which contains flavin mononucleotide (FMN) and a plant-like ferredoxin [2Fe-2S] center. PDR is responsible for transferring electrons from NADH to the Rieske center of PDO, and the Rieske center supplies electrons to the mononuclear center for the oxygenation of substrate. Reduced PDO (PDO(red)) that lacks Fe(II) at the mononuclear metal site (PDO-APO) reacts slowly with O(2) (1.4 x 10(-3) s(-1) at 125 microM O(2) and 22 degrees C), presumably in a direct reaction with the Rieske center. Binding of phthalate and/or PDR(ox) to reduced PDO-APO increases the reactivity of the Rieske center with O(2). When no PDR or phthalate is present, the oxidation of the Rieske center in native PDO(red) [which contains Fe(II) at the mononuclear site] occurs in two phases (approximately 1 and 0.1 s(-1) at 125 mM O(2), 23 degrees C), both much faster than in the absence of Fe(II), presumably because in this case O(2) reacts at the mononuclear Fe(II). Addition of PDR(ox) to native PDO(red) resulted in a large fraction of the Rieske center being oxidized at 5 s(-1), and the addition of phthalate resulted in about 70% of the reaction proceeding at 42 s(-1). With both PDR(ox) and phthalate present, most of the PDO(red) (approximately 80-85%) oxidizes at 42 s(-1), with the remaining oxidizing at approximately 5 s(-1). Thus, the binding of phthalate or PDR(ox) to PDO(red) each results in greater reactivity of PDO with O(2). The presence of both the substrate and PDR was synergistic, making PDO fully catalytically active. A model that explains the observed effects is presented and discussed in terms of PDO subunit cooperativity. It is proposed that, during oxidation of reduced PDO, each of two Rieske centers on separate subunits transfers an electron to the Fe(II) mononuclear center on a third subunit. This explanation is consistent with the observed multiphasic kinetics of the oxidation of the Rieske center and is being further tested by product analysis experiments.

J Biol Chem. 2004 Oct 8;279(41):43098-106. Epub 2004 Jul 26.
Catalase-peroxidases (KatG) exhibit NADH oxidase activity.
Singh R, Wiseman B, Deemagarn T, Donald LJ, Duckworth HW, Carpena X, Fita I, Loewen PC.
Department of Microbiology, University of Manitoba, Winnipeg, Manitoba R3T 2N2, Canada.
Catalase-peroxidases (KatG) produced by Burkholderia pseudomallei, Escherichia coli, and Mycobacterium tuberculosis catalyze the oxidation of NADH to form NAD+ and either H2O2 or superoxide radical depending on pH. The NADH oxidase reaction requires molecular oxygen, does not require hydrogen peroxide, is not inhibited by superoxide dismutase or catalase, and has a pH optimum of 8.75, clearly differentiating it from the peroxidase and catalase reactions with pH optima of 5.5 and 6.5, respectively, and from the NADH peroxidase-oxidase reaction of horseradish peroxidase. B. pseudomallei KatG has a relatively high affinity for NADH (Km=12 microm), but the oxidase reaction is slow (kcat=0.54 min(-1)) compared with the peroxidase and catalase reactions. The catalase-peroxidases also catalyze the hydrazinolysis of isonicotinic acid hydrazide (INH) in an oxygen- and H2O2-independent reaction, and KatG-dependent radical generation from a mixture of NADH and INH is two to three times faster than the combined rates of separate reactions with NADH and INH alone. The major products from the coupled reaction, identified by high pressure liquid chromatography fractionation and mass spectrometry, are NAD+ and isonicotinoyl-NAD, the activated form of isoniazid that inhibits mycolic acid synthesis in M. tuberculosis. Isonicotinoyl-NAD synthesis from a mixture of NAD+ and INH is KatG-dependent and is activated by manganese ion. M. tuberculosis KatG catalyzes isonicotinoyl-NAD formation from NAD+ and INH more efficiently than B. pseudomallei KatG.

J Biol Chem. 2004 Oct 8;279(41):43035-45. Epub 2004 Jul 23.
A mechanism of sulfite neurotoxicity: direct inhibition of glutamate dehydrogenase.
Zhang X, Vincent AS, Halliwell B, Wong KP.
Department of Biochemistry, Faculty of Medicine, National University of Singapore, 8 Medical Drive, Singapore 117597, Singapore.
Exposure of Neuro-2a and PC12 cells to micromolar concentrations of sulfite caused an increase in reactive oxygen species and a decrease in ATP. Likewise, the biosynthesis of ATP in intact rat brain mitochondria from the oxidation of glutamate was inhibited by micromolar sulfite. Glutamate-driven respiration increased the mitochondrial membrane potential (MMP), and this was abolished by sulfite but the MMP generated by oxidation of malate and succinate was not affected. The increased rate of production of NADH from exogenous NAD+ and glutamate added to rat brain mitochondrial extracts was inhibited by sulfite, and mitochondria preincubated with sulfite failed to reduce NAD+. Glutamate dehydrogenase (GDH) in rat brain mitochondrial extract was inhibited dose-dependently by sulfite as was the activity of a purified enzyme. An increase in the Km (glutamate) and a decrease in Vmax resulting in an attenuation in Vmax/Km (glutamate) at 100 microm sulfite suggest a mixed type of inhibition. However, uncompetitive inhibition was noted with decreases in both Km (NAD+) and Vmax, whereas Vmax/Km (NAD+) remained relatively constant. We propose that GDH is one target of action of sulfite, leading to a decrease in alpha-ketoglutarate and a diminished flux through the tricarboxylic acid cycle accompanied by a decrease in NADH through the mitochondrial electron transport chain, a decreased MMP, and a decrease in ATP synthesis. Because glutamate is a major metabolite in the brain, inhibition of GDH by sulfite could contribute to the severe phenotype of sulfite oxidase deficiency in human infants.

J Mol Biol. 2004 Oct 8;343(1):147-55.
Structural basis for the variation in triclosan affinity to enoyl reductases.
Pidugu LS, Kapoor M, Surolia N, Surolia A, Suguna K.
Molecular Biophysics Unit, Indian Institute of Science, Bangalore 560 012.
Bacteria synthesize fatty acids in a dissociated type pathway different from that in humans. Enoyl acyl carrier protein reductase, which catalyzes the final step of fatty acid elongation, has been validated as a potential anti-microbial drug target. Triclosan is known to inhibit this enzyme effectively. Precise characterization of the mode of triclosan binding is required to develop highly specific inhibitors. With this in view, interactions between triclosan, the cofactor NADH/NAD+ and the enzyme from five different species, one plant and four of microbial origin, have been examined in the available crystal structures. A comparison of these structures shows major structural differences at the substrate/inhibitor/cofactor-binding loop. The analysis reveals that the conformation of this flexible loop and the binding affinities of triclosan to each of these enzymes are strongly correlated.

J Mol Biol. 2004 Oct 8;343(1):29-41.
Crystal structure and kinetics identify Escherichia coli YdcW gene product as a medium-chain aldehyde dehydrogenase.
Gruez A, Roig-Zamboni V, Grisel S, Salomoni A, Valencia C, Campanacci V, Tegoni M, Cambillau C.
Architecture et Fonction des Macromolecules Biologiques, UMR 6098 CNRS, 31 chemin J. Aiguier, F-13402 Marseille Cedex 20, France.

Biochim Biophys Acta. 2004 Oct 5;1680(1):60-6.
Cloning and expression of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii: evidence of the divergence of enzymes in the class of two-protein component aromatic hydroxylases.
Thotsaporn K, Sucharitakul J, Wongratana J, Suadee C, Chaiyen P.
Department of Biochemistry and Center for Excellence in Protein Structure and Function, Faculty of Science, Mahidol University, 10400 Bangkok, Thailand.
The genes encoding for the reductase and oxygenase components of p-hydroxyphenylacetate 3-hydroxylase from Acinetobacter baumannii were cloned and expressed in an E. coli system. The recombinant enzymes were purified and shown to have the same catalytic properties as the native enzyme. Sequence analysis and biochemical studies indicate that the enzyme represents a novel prototype of enzyme in the two-protein component class of aromatic hydroxylases. The C(2) component shows little similarity to other oxygenases in the same class, correlating with its uniquely broad flavin specificity. Analysis of the C(1) reductase sequence indicates that the binding sites of flavin and NADH mainly reside in the N-terminal half while the C-terminal half may be responsible for HPA-stimulation of NADH oxidation.

J Neural Transm. 2000;107(12):1475-81.
No evidence for cognitive improvement from oral nicotinamide adenine dinucleotide (NADH) in dementia.
Rainer M, Kraxberger E, Haushofer M, Mucke HA, Jellinger KA.
Memory-Clinic and Psychiatric Department, Donauspital, Sozialmedizinisches Zentrum Ost, Wein, Austria.
Reduced nicotinamide adenine dinucleotide (NADH) is advertised as an over-the-counter product or dietary supplement to treat Alzheimer's disease. We performed a 3-month open-label study with oral 10 mg/day NADH with 25 patients with mild to moderate dementia of the Alzheimer, vascular, and fronto-temporal types in addition to their current cholinomimetic drug medication. In 19 patients who completed the study, we found no evidence for any cognitive effect as defined by established psychometric tests. We conclude that NADH is unlikely to achieve cognitive improvements in an extent reported earlier, and present theoretical arguments against an effectiveness of this compound in dementia disorders.