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.

Even though transcriptional regulation plays a key role in establishing the metabolic network, the extent to which it actually controls the in vivo distribution of metabolic fluxes through different pathways is essentially unknown. Based on metabolism-wide quantification of intracellular fluxes, we systematically elucidated the relevance of global transcriptional regulation by ArcA, ArcB, Cra, Crp, Cya, Fnr, and Mlc for aerobic glucose catabolism in batch cultures of Escherichia coli. Knockouts of ArcB, Cra, Fnr, and Mlc were phenotypically silent, while deletion of the catabolite repression regulators Crp and Cya resulted in a pronounced slow-growth phenotype but had only a nonspecific effect on the actual flux distribution. Knockout of ArcA-dependent redox regulation, however, increased the aerobic tricarboxylic acid (TCA) cycle activity by over 60%. Like aerobic conditions, anaerobic derepression of TCA cycle enzymes in an ArcA mutant significantly increased the in vivo TCA flux when nitrate was present as an electron acceptor. The in vivo and in vitro data demonstrate that ArcA-dependent transcriptional regulation directly or indirectly controls TCA cycle flux in both aerobic and anaerobic glucose batch cultures of E. coli. This control goes well beyond the previously known ArcA-dependent regulation of the TCA cycle during microaerobiosis.Metabolic networks consist of hundreds of metabolites that are interconnected through a large number of biochemical and regulatory reactions. In principle, metabolites could flow through various reactions, but only few specific pathways are used in reality (20). This distribution of molecular fluxes is regulated by multiple mechanisms at several levels that include gene expression, posttranscriptional control, enzyme kinetics, and allosteric control. While transcriptional regulation is generally considered the main mode of regulation in bacteria, the extent to which it actually controls the distribution of metabolic fluxes through different pathways is mostly unknown. Based on metabolism-wide comparisons of metabolic flux and gene expression during growth on different substrates, the flux through some central metabolic pathways was found to correlate, at least qualitatively, with the expression level, but in many cases there was no apparent correlation (12,24,41,42). In parasitic protists, the glycolytic flux was demonstrated to be rarely completely controlled at the transcriptional level and mostly not even largely controlled at this level (65).Transcriptional regulation itself involves a complex network of global and specific regulators, in which global regulators exhibit pleiotropic phenotypes and regulate several operons that belong to different functional groups (26). In Escherichia coli, only seven global regulators (ArcA, Crp, Fis, Fnr, Ihf, Lrp, and NarL) directly modulate the expression of about one-half of all genes (39). The following three regulators have specific metabolic functions that involve altering expression of genes that are involved in central carbon m...

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