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

Abstract

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? 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 (1Edwards J.S. Palsson B.O. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 5528-5533Crossref PubMed Scopus (746) Google Scholar, 2Förster J. Famili I. Fu P. Palsson B.O. Nielsen J. Genome Res. 2003; 13: 244-253Crossref PubMed Scopus (867) Google Scholar), but only 11 intermediates of central carbon metabolism and the cofactors ATP, NADH, and NADPH constitute the core of this intricate biochemical network (3Neidhardt F.C. Ingraham J.L. Schaechter M. Physiology of the Bacterial Cell: a Molecular Approach. Sinauer Associates, Inc., Sunderland, Mass1990Google Scholar, 4Gottschalk G. Bacterial Metabolism. Springer-Verlag, New York, NY1986Crossref Google Scholar). 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 (5Ouzonis C.A. Karp P.D. Genome Res. 2000; 10: 568-576Crossref PubMed Scopus (83) Google Scholar). The electrons of the main respiratory cofactor NADH are transferred primarily to oxygen, thereby driving oxidative phosphorylation of ADP to ATP (3Neidhardt F.C. Ingraham J.L. Schaechter M. Physiology of the Bacterial Cell: a Molecular Approach. Sinauer Associates, Inc., Sunderland, Mass1990Google Scholar, 4Gottschalk G. Bacterial Metabolism. Springer-Verlag, New York, NY1986Crossref Google Scholar, 6Russell J.B. Cook G.M. Microbiol. Rev. 1995; 59: 48-62Crossref PubMed Google Scholar). 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) 1The abbreviations used are: PP pathway, pentose phosphate pathway; TCA cycle, tricarboxylic acid cycle; GC-MS, gas chromatography-mass spectrometry; ED pathway, Entner-Doudoroff pathway; PEP, phosphoenolpyruvate; Pgi, phosphoglucose isomerase; UdhA, energy-independent, soluble transhydrogenase; PntAB, energy-dependent, membrane-bound transhydrogenase; Zwf, glucose-6P dehydrogenase. pathway and the NADPH-dependent isocitrate dehydrogenase in the TCA cycle (Fig. 1; Refs. 4Gottschalk G. Bacterial Metabolism. Springer-Verlag, New York, NY1986Crossref Google Scholar, 7Fraenkel D.G. Neidhardt F.C. Curtiss III, R. Ingraham J.L. Lin E.C.C. Low K.B. Magasanik B. Reznikoff W.S. Riley M. Schaechter M. Umbarger H.E. Escherichia coli and Salmonella: Cellular and Molecular Biology. 2nd Ed. ASM Press, Washington, D. C.1996: 189-198Google Scholar, 8Csonka L.N. Fraenkel D.G. J. Biol. Chem. 1977; 252: 3382-3391Abstract Full Text PDF PubMed Google Scholar). Additionally, nicotinamide nucleotide transhydrogenases may be involved, but ever since their discovery, their physiological role has been a source of speculation and often a matter of controversy (9Hoek J.B. Rydström J. Biochem. J. 1988; 254: 1-10Crossref PubMed Scopus (296) Google Scholar, 10Harold F. The Vital Force: A Study of Bioenergetics. Freeman and Company, New York, NY1986Google Scholar, 11Jackson J.B. Quirk P.G. Cotton N.P. Venning J.D. Gupta S. Bizouarn T. Peake S.J. Thomas C.M. Biochim. Biophys. Acta. 1998; 1365: 79-86Crossref PubMed Scopus (27) Google Scholar). The transhydrogenase reaction [NADPH] + [NAD+] + [H+in] 〈-〉 [NADP+] + [NADH] + [H+out] may be catalyzed by either a membrane-bound, proton-translocating or a soluble, energy-independent isoform (9Hoek J.B. Rydström J. Biochem. J. 1988; 254: 1-10Crossref PubMed Scopus (296) Google Scholar, 12Bizouarn T. Althage M. Pedersen A. Tigerstrom A. Karlsson J. Johansson C. Rydström J. Biochim. Biophys. Acta. 2002; 1555: 122-127Crossref PubMed Scopus (20) Google Scholar). Microbes often contain one isoform or none, with the Enterobacteriaceae as the only known exception that contain both isoforms encoded by the pntAB (13Clarke D.M. Loo T.W. Gillam S. Bragg P.D. Eur. J. Biochem. 1986; 158: 647-653Crossref PubMed Scopus (95) Google Scholar) and udhA (14Boonstra B. French C.E. Wainwright I. Bruce N.C. J. Bacteriol. 1999; 181: 1030-1034Crossref PubMed Google Scholar) genes, respectively. In eukaryotes, the proton-translocating transhydrogenase seems to have a flexible function as a buffer against dissipation of either the mitochondrial redox potential or energy supply (9Hoek J.B. Rydström J. Biochem. J. 1988; 254: 1-10Crossref PubMed Scopus (296) Google Scholar). In prokaryotes, however, the physiological role of the two isoforms remains an exciting matter of debate, and several potential functions were put forward (8Csonka L.N. Fraenkel D.G. J. Biol. Chem. 1977; 252: 3382-3391Abstract Full Text PDF PubMed Google Scholar, 9Hoek J.B. Rydström J. Biochem. J. 1988; 254: 1-10Crossref PubMed Scopus (296) Google Scholar, 15Voordouw G. van der Vies S.M. Themmen A.P.N. Eur. J. Biochem. 1983; 131: 527-533Crossref PubMed Scopus (38) Google Scholar, 16Gerolimatos B. Hanson R.L. J. Bacteriol. 1978; 134: 394-400Crossref PubMed Google Scholar, 17Liang A. Houghton R.L. FEBS Lett. 1980; 109: 185-188Crossref PubMed Scopus (4) Google Scholar). Based on the isoform distribution in different organisms and the generally more reduced state of the NADP(H) pool compared with the NAD(H) pool (10Harold F. The Vital Force: A Study of Bioenergetics. Freeman and Company, New York, NY1986Google Scholar), it has been hypothesized (15Voordouw G. van der Vies S.M. Themmen A.P.N. Eur. J. Biochem. 1983; 131: 527-533Crossref PubMed Scopus (38) Google Scholar) that the physiological function of membrane-bound and soluble transhydrogenases in microbes might be generation and reoxidation of NADPH, respectively. Indeed, organisms lacking transhydrogenases, such as the yeast Saccharomyces cerevisiae, cannot tolerate imbalances between catabolic NADPH production and anabolic NADPH consumption (18Boles E. Lehnert W. Zimmermann F.K. Eur. J. Biochem. 1993; 217: 469-477Crossref PubMed Scopus (87) Google Scholar, 19Nissen T.L. Anderlund M. Nielsen J. Villadsen J. Kielland-Brandt M.C. Yeast. 2001; 18: 19-32Crossref PubMed Scopus (95) Google Scholar), unless a soluble isoform is expressed (19Nissen T.L. Anderlund M. Nielsen J. Villadsen J. Kielland-Brandt M.C. Yeast. 2001; 18: 19-32Crossref PubMed Scopus (95) Google Scholar, 20Fiaux J. Çakar Z.P. Sonderegger M. Wüthrich K. Szyperski T. 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Press, Scholar). we used the with 100 of and of was to for at and the was by of at at and at the for udhA and for and for the that the and the and were the between and other were with were for the of by that the were on by and the were to the with as an by of in was used to flux E. U. Eur. J. Biochem. 2003; PubMed Scopus Google Scholar, E. U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). from were and in of at for in in a at were at in of and of for The distribution in the was by gas chromatography-mass and the of were for E. U. Eur. J. Biochem. 2003; PubMed Scopus Google Scholar). The were to in metabolic and which of through reactions and to the of from and E. U. Eur. J. Biochem. 2003; PubMed Scopus Google Scholar). carbon were from the physiological with a and in were from the known for macromolecular (3Neidhardt F.C. Ingraham J.L. Schaechter M. Physiology of the Bacterial Cell: a Molecular Approach. 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Lett. 2001; PubMed Google Scholar, E. U. Eur. J. Biochem. 2003; PubMed Scopus Google Scholar, E. U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The flux a metabolic with more than excess NADPH formation and the mutant was the only with an of from and a of from (Fig. as was E. U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar). The flux distribution of the identified cycle E. U. J. Biol. Chem. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar), i.e. flux through the and flux from to (Fig. of the mutant was on glucose E. U. J. Biol. 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C.1996: 189-198Google Scholar), we that transhydrogenases important in NADPH the divergent physiological functions of the two transhydrogenase isoforms in E. coli that catabolic NADPH production from anabolic NADPH the membrane-bound transhydrogenase PntAB the energy-dependent transfer of from NADH to the soluble transhydrogenase the energy-independent In standard aerobic batch glucose catabolism NADPH than was required for the membrane-bound transhydrogenase was a major pathway of NADPH formation that contributed 35–45% of the anabolic for NADPH, which the role of the membrane-bound isoform in of E. coli A. Houghton R.L. FEBS Lett. 1980; 109: 185-188Crossref PubMed Scopus (4) Google Scholar). NADPH formation was essential for batch growth on glucose, because PntAB mutants on this The of NADPH formation was for primarily through increased PP pathway Although and growth on glucose, the soluble transhydrogenase function batch growth on this but the do of both isoforms in a metabolic conditions that to excess NADPH formation, however, the soluble transhydrogenase was essential for conditions were growth on acetate or in phosphoglucose isomerase mutants that catabolized glucose exclusively through the PP pathway, thereby a excess of NADPH, as was for S. J. Çakar Z.P. Sonderegger M. Wüthrich K. Szyperski T. U. 2003; PubMed Scopus Google Scholar). metabolic with excess NADPH formation in E. coli is growth in because respiratory growth with TCA cycle of of the glucose in produced NADPH in the isocitrate dehydrogenase reaction M. M. A. J. M. Szyperski T. Wüthrich K. U. J. Bacteriol. 2002; PubMed Scopus Google Scholar, C. T. 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