The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli

Bizouarn, Tania; Althage, Magnus; Pedersen, Anders; Tigerström, Anna; Karlsson, Jenny; Johansson, Christina; Rydström, Jan

doi:10.1016/s0005-2728(02)00266-9

Cited by 22 publications

(23 citation statements)

References 31 publications

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“…The association of domain II helices has been studied by crosslinking and oxidation of introduced cysteines (28,29). These experiments indicate that E. coli TH is an ␣ 2 ␤ 2 tetramer predominantly associated via ␤ subunits; 2-fold symmetric quaternary structure is consistent with the dimeric relationship of domain I components in crystals (24,26,29).…”

Section: Nicotinamide Nucleotide Transhydrogenases (Th)supporting

confidence: 56%

“…1). Cysteine mutants were introduced in E. coli TH between all predicted transmembrane ␣-helices, and fluorescence labeling experiments defined their orientation with respect to the membrane (25)(26)(27). One transmembrane helix, H5, predicted for bovine TH does not occur in the E. coli enzyme, corresponding to the division between ␣ and ␤ subunits.…”

Section: Nicotinamide Nucleotide Transhydrogenases (Th)mentioning

confidence: 99%

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Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

Yamaguchi

Stout

2003

Journal of Biological Chemistry

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The nicotinamide nucleotide transhydrogenases of mitochondria and bacteria are proton pumps that couple hydride ion transfer between NAD(H) and NADP(H) bound, respectively, to extramembranous domains I and III, to proton translocation by the membrane-intercalated domain II. Previous experiments have established the involvement of three conserved domain II residues in the proton pumping function of the enzyme: His 91 , Ser 139 , and Asn 222 , located on helices 9, 10, and 13, respectively. Eight highly conserved domain II glycines in helices 9, 10, 13, and 14 were mutated to alanine, and the mutant enzymes were assayed for hydride transfer between domains I and III and for proton translocation by domain II. One of the glycines on helix 14, Gly 252 , was further mutated to Cys, Ser, Thr, and Val, expression levels of the mutant enzymes were evaluated, and each was purified and assayed. The results show that Gly 252 is essential for function and support a model for the proton channel composed of helices 9, 10, 13, and 14. Gly Nicotinamide nucleotide transhydrogenases (TH)1 of mitochondria and microorganisms are membrane-intercalated enzymes that couple the transfer of a hydride ion between soluble domains to translocation of a proton through the integral membrane domain.They catalyze the direct and stereospecific transfer of hydride between the 4A position of NAD(H) and the 4B position of NADP(H). The transhydrogenation reaction is coupled to transmembrane proton translocation with a H ϩ /H Ϫ stoichiometry of unity (Reaction 1) (1-3).In bovine mitochondria, the proton motive force accelerates the forward reaction 10 -12-fold and shifts the equilibrium toward product formation. Due to this activity, a function of TH is to produce NADPH for reduction of toxic H 2 O 2 by glutathione reductase and glutathione peroxidase. In the reverse direction, transhydrogenation from NADPH to NAD results in outward proton translocation and creation of a proton motive force. Because there is essentially no difference in the reduction potential of the nicotinamide cofactors, the driving force for the reverse reaction is the difference in binding affinities for substrates (NADPH and NAD) versus products (NADH and NADP). Consequently, TH provides a system in which to study the transformation of substrate binding energy into proton translocation.The amino acid sequences of over 50 TH enzymes are available, but only the enzymes from bovine mitochondria (4), Escherichia coli (5), and Rhodobacter capsulatus (6) have been purified. The bovine enzyme is a homodimer of monomer molecular mass of 109 kDa. The monomer is composed of three domains: an amino-terminal ϳ430-residue-long extramembranous domain I that binds NAD(H), a ϳ400-residue-long central domain II that is composed of 14 transmembrane ␣-helices, and a carboxyl-terminal ϳ200-residue-long extramembranous domain III that binds NADP(H) (1, 4, 7). The extramembranous domains I and III come together in the mitochondrial matrix to form the active site for hydride transfer. Bovine TH lack...

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Section: Nicotinamide Nucleotide Transhydrogenases (Th)supporting

confidence: 56%

Section: Nicotinamide Nucleotide Transhydrogenases (Th)mentioning

confidence: 99%

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

Yamaguchi

Stout

2003

Journal of Biological Chemistry

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“…In many animal mitochondria and bacteria, transhydrogenases couple the reversible hydride transfer between NADPH + H + and NAD + to proton translocation across a membrane [Bizouarn et al, 2002;Cotton et al, 2001]. In G. oxydans the enzyme may fulfill two functions: (i) the oxidation of NADPH derived from intermediary metabolism ( fig.…”

Section: Respiratory Chainmentioning

confidence: 99%

Physiology of Acetic Acid Bacteria in Light of the Genome Sequence of <i>Gluconobacter oxydans</i>

Deppenmeier¹,

Ehrenreich²

2008

Microb Physiol

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Acetic acid bacteria are a distinct group of microorganisms within the family Acetobacteriaceae. They are characterized by their ability to incompletely oxidize a wide range of carbohydrates and alcohols. The great advantage of these reactions is that many substrates are regio- and stereoselectively oxidized. This feature is already exploited in several combined biotechnological-chemical procedures for the synthesis of sugar derivatives. Therefore, it is important to understand the basic concepts of this type of physiology to construct strains for improved or new oxidative fermentations. Based on the genome sequence of Gluconobacteroxydans, we will shed light on the central carbon metabolism, the composition of the respiratory chain and the analysis of uncharacterized oxidoreductases. In this context, the role of membrane-bound and -soluble dehydrogenases are of major importance in the process of incomplete oxidation. Other topics deal with the question of how these organisms generate energy and assimilate carbon. Furthermore, we will discuss how acetic acid bacteria thrive in their nutrient-rich environment and how they outcompete other microorganisms.

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“…out ] may be catalyzed by either a membranebound, proton-translocating or a soluble, energy-independent isoform (9,12). Microbes often contain one isoform or none, with the Enterobacteriaceae as the only known exception that contain both isoforms encoded by the pntAB (13) and udhA (14) genes, respectively.…”

mentioning

confidence: 99%

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

Sauer

Canonaco

Heri

et al. 2004

Journal of Biological Chemistry

523

526

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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?About 1,000 anabolic reactions synthesize the macromolecular components that make up functional cells (1, 2), but only 11 intermediates of central carbon metabolism and the cofactors ATP, NADH, and NADPH constitute the core of this intricate biochemical network (3, 4). These intermediates and cofactors must be supplied through the catabolism of different substrates at appropriate rates and stoichiometries for balanced growth; hence, anabolism and catabolism are delicately balanced and regulated to enable growth under fluctuating environmental conditions. Although chemically very similar, the redox cofactors NADH and NADPH serve distinct biochemical functions and participate in more than 100 enzymatic reactions (5). The electrons of the main respiratory cofactor NADH are transferred primarily to oxygen, thereby driving oxidative phosphorylation of ADP to ATP (3,4,6). NADPH, in contrast, exclusively drives anabolic reduction reactions. Despite the important role in linking the fundamental processes of catabolism and anabolism, however, even a qualitative understanding of NADPH metabolism is still missing for most organisms.The primary NADPH-generating reactions are considered to be the oxidative pentose phosphate (PP) 1 pathway and the NADPH-dependent isocitrate dehydrogenase in the TCA cycle (Fig.

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The organization of the membrane domain and its interaction with the NADP(H)-binding site in proton-translocating transhydrogenase from E. coli

Cited by 22 publications

References 31 publications

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

Essential Glycine in the Proton Channel of Escherichia coli Transhydrogenase

Physiology of Acetic Acid Bacteria in Light of the Genome Sequence of <i>Gluconobacter oxydans</i>

The Soluble and Membrane-bound Transhydrogenases UdhA and PntAB Have Divergent Functions in NADPH Metabolism of Escherichia coli

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