Publications

• Mutations in NNT encoding nicotinamide nucleotide transhydrogenase cause familial glucocorticoid deficiency.

  Meimaridou E., Kowalczyk J., Guasti L., Hughes C.R., Wagner F., Frommolt P., Nurnberg P., Mann N.P., Banerjee R., Saka H.N., Chapple J.P., King P.J., Clark A.J., Metherell L.A.

  Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human ... >> More

  Using targeted exome sequencing, we identified mutations in NNT, an antioxidant defense gene, in individuals with familial glucocorticoid deficiency. In mice with Nnt loss, higher levels of adrenocortical cell apoptosis and impaired glucocorticoid production were observed. NNT knockdown in a human adrenocortical cell line resulted in impaired redox potential and increased reactive oxygen species (ROS) levels. Our results suggest that NNT may have a role in ROS detoxification in human adrenal glands. << Less

  Nat. Genet. 44:740-742(2012) [PubMed] [EuropePMC]

• Proton translocating nicotinamide nucleotide transhydrogenase from E. coli. Mechanism of action deduced from its structural and catalytic properties.

  Bizouarn T., Fjellstrom O., Meuller J., Axelsson M., Bergkvist A., Johansson C., Goran Karlsson B., Rydstrom J.

  Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of ... >> More

  Transhydrogenase couples the stereospecific and reversible transfer of hydride equivalents from NADH to NADP(+) to the translocation of proton across the inner membrane in mitochondria and the cytoplasmic membrane in bacteria. Like all transhydrogenases, the Escherichia coli enzyme is composed of three domains. Domains I and III protrude from the membrane and contain the binding site for NAD(H) and NADP(H), respectively. Domain II spans the membrane and constitutes at least partly the proton translocating pathway. Three-dimensional models of the hydrophilic domains I and III deduced from crystallographic and NMR data and a new topology of domain II are presented. The new information obtained from the structures and the numerous mutation studies strengthen the proposition of a binding change mechanism, as a way to couple the reduction of NADP(+) by NADH to proton translocation and occurring mainly at the level of the NADP(H) binding site. << Less

  Biochim Biophys Acta 1457:211-228(2000) [PubMed] [EuropePMC]

• X-ray structure of domain I of the proton-pumping membrane protein transhydrogenase from Escherichia coli.

  Johansson T., Oswald C., Pedersen A., Toernroeth S., Okvist M., Karlsson B.G., Rydstroem J., Krengel U.

  The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by ... >> More

  The dimeric integral membrane protein nicotinamide nucleotide transhydrogenase is required for cellular regeneration of NADPH in mitochondria and prokaryotes, for detoxification and biosynthesis purposes. Under physiological conditions, transhydrogenase couples the reversible reduction of NADP+ by NADH to an inward proton translocation across the membrane. Here, we present crystal structures of the NAD(H)-binding domain I of transhydrogenase from Escherichia coli, in the absence as well as in the presence of oxidized and reduced substrate. The structures were determined at 1.9-2.0 A resolution. Overall, the structures are highly similar to the crystal structure of a previously published NAD(H)-binding domain, from Rhodospirillum rubrum transhydrogenase. However, this particular domain is unique, since it is covalently connected to the integral-membrane part of transhydrogenase. Comparative studies between the structures of the two species reveal extensively differing surface properties and point to the possible importance of a rigid peptide (PAPP) in the connecting linker for conformational coupling. Further, the kinetic analysis of a deletion mutant, from which the protruding beta-hairpin was removed, indicates that this structural element is important for catalytic activity, but not for domain I:domain III interaction or dimer formation. Taken together, these results have important implications for the enzyme mechanism of the large group of transhydrogenases, including mammalian enzymes, which contain a connecting linker between domains I and II. << Less

  J. Mol. Biol. 352:299-312(2005) [PubMed] [EuropePMC]

  This publication is cited by 1 other entry.

• Involvement of histidine-91 of the beta subunit in proton translocation by the pyridine nucleotide transhydrogenase of Escherichia coli.

  Glavas N.A., Hou C., Bragg P.D.

  The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli. This ami ... >> More

  The pyridine nucleotide transhydrogenase (EC 1.6.1.1) carries out transmembrane proton translocation coupled to transfer of a hydride equivalent between NAD+ and NADP+. Mutations were made in histidine-91 of the beta subunit of the pyridine nucleotide transhydrogenase of Escherichia coli. This amino acid is the only conserved charged residue in the transmembrane domains of this enzyme and thus potentially is involved in proton translocation by the transhydrogenase. The mutant beta H91N retained 80% of the hydride transfer activity while proton translocation was reduced to 7%. This behavior is consistent with a role for beta His91 in the proton translocation pathway. Other mutations at this residue affected the conformation of the enzyme. Thus, the enzyme in mutants beta H91C, beta H91T, and beta H91S was unable to undergo the conformational change that occurred on binding of the substrates NADP+ or NADPH. By contrast, the enzyme in the beta H91K mutant was present in the NADP(H)-induced conformation even in the absence of these substrates. Further evidence for the linkage between beta His91 and the conformation of the beta subunit was obtained by labeling the transmembrane domain of the beta subunit with [14C]N,N'-dicyclohexylcarbodiimide (DCCD). Labeling occurred most readily with the enzyme of beta H91K. It is concluded that beta His91 is a component of the proton translocation pathway of the transhydrogenase and that its state of protonation is probably linked to conformational changes induced by binding/debinding of substrates during the catalytic cycle of the enzyme. << Less

  Biochemistry 34:7694-7702(1995) [PubMed] [EuropePMC]

• The high-resolution structure of the NADP(H)-binding component (dIII) of proton-translocating transhydrogenase from human heart mitochondria.

  White S.A., Peake S.J., McSweeney S., Leonard G., Cotton N.P.J., Jackson J.B.

  <h4>Background</h4>Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The protein comprises three subunits termed dI, dII and dIII. The dII com ... >> More

  <h4>Background</h4>Transhydrogenase, located in the inner membranes of animal mitochondria and the cytoplasmic membranes of bacteria, couples the transfer of reducing equivalents between NAD(H) and NADP(H) to proton pumping. The protein comprises three subunits termed dI, dII and dIII. The dII component spans the membrane. The dI component, which contains the binding site for NAD(+)/NADH, and the dIII component, which has the binding site for NADP(+)/NADPH, protrude from the membrane. Proton pumping is probably coupled to changes in the binding affinities of dIII for NADP(+) and NADPH.<h4>Results</h4>The first X-ray structure of the NADP(H)-binding component, dIII, of human heart transhydrogenase is described here at 2.0 A resolution. It comprises a single domain resembling the classical Rossmann fold, but NADP(+) binds to dIII with a reversed orientation. The first betaalphabetaalphabeta motif of dIII contains a Gly-X-Gly-X-X-Ala/Val 'fingerprint', but it has a different function to that in the classical Rossmann structure. The nicotinamide ring of NADP(+) is located on a ridge where it is exposed to interaction with NADH on the dI subunit. Two distinctive features of the dIII structure are helix D/loop D, which projects from the beta sheet, and loop E, which forms a 'lid' over the bound NADP(+).<h4>Conclusions</h4>Helix D/loop D interacts with the bound nucleotide and loop E, and probably interacts with the membrane-spanning dII. Changes in ionisation and conformation in helix D/loop D, resulting from proton translocation through dII, are thought to be responsible for the changes in affinity of dIII for NADP(+) and NADPH that drive the reaction. << Less

  Structure 8:1-12(2000) [PubMed] [EuropePMC]

• Cloning and expression of the transhydrogenase gene of Escherichia coli.

  Clarke D.M., Bragg P.D.

  Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclea ... >> More

  Based on the rationale that Escherichia coli cells harboring plasmids containing the pnt gene would contain elevated levels of enzyme, we have isolated three clones bearing the transhydrogenase gene from the Clarke and Carbon colony bank. The three plasmids were subjected to restriction endonuclease analysis. A 10.4-kilobase restriction fragment which overlapped all three plasmids was cloned into the PstI site of plasmid pUC13. Examination of several deletion derivatives of the resulting plasmid and subsequent treatment with exonuclease BAL 31 revealed that enhanced transhydrogenase expression was localized within a 3.05-kilobase segment. This segment was located at 35.4 min in the E. coli genome. Plasmid pDC21 conferred on its host 70-fold overproduction of transhydrogenase. The protein products of plasmids carrying the pnt gene were examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis of membranes from cells containing the plasmids. Two polypeptides of molecular weights 50,000 and 47,000 were coded by the 3.05-kilobase fragment of pDC11. Both polypeptides were required for expression of transhydrogenase activity. << Less

  J Bacteriol 162:367-373(1985) [PubMed] [EuropePMC]

• The soluble and membrane-bound transhydrogenases UdhA and PntAB have divergent functions in NADPH metabolism of Escherichia coli.

  Sauer U., Canonaco F., Heri S., Perrenoud A., Fischer E.

  Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiological function that could potentially transfer electron ... >> More

  Pentose phosphate pathway and isocitrate dehydrogenase are generally considered to be the major sources of the anabolic reductant NADPH. As one of very few microbes, Escherichia coli contains two transhydrogenase isoforms with unknown physiological function that could potentially transfer electrons directly from NADH to NADP+ and vice versa. Using defined mutants and metabolic flux analysis, we identified the proton-translocating transhydrogenase PntAB as a major source of NADPH in E. coli. During standard aerobic batch growth on glucose, 35-45% of the NADPH that is required for biosynthesis was produced via PntAB, whereas pentose phosphate pathway and isocitrate dehydrogenase contributed 35-45% and 20-25%, respectively. The energy-independent transhydrogenase UdhA, in contrast, was essential for growth under metabolic conditions with excess NADPH formation, i.e. growth on acetate or in a phosphoglucose isomerase mutant that catabolized glucose through the pentose phosphate pathway. Thus, both isoforms have divergent physiological functions: energy-dependent reduction of NADP+ with NADH by PntAB and reoxidation of NADPH by UdhA. Expression appeared to be modulated by the redox state of cellular metabolism, because genetic and environmental manipulations that increased or decreased NADPH formation down-regulated pntA or udhA transcription, respectively. The two transhydrogenase isoforms provide E. coli primary metabolism with an extraordinary flexibility to cope with varying catabolic and anabolic demands, which raises two general questions: why do only a few bacteria contain both isoforms, and how do other organisms manage NADPH metabolism? << Less

  J Biol Chem 279:6613-6619(2004) [PubMed] [EuropePMC]

• Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli.

  Clarke D.M., Bragg P.D.

  The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation ... >> More

  The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated. << Less

  Eur J Biochem 149:517-523(1985) [PubMed] [EuropePMC]