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?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.

Expression of the Escherichia coli pntAB genes encoding a membrane-bound transhydrogenase in Corynebacterium glutamicum improves l-lysine formation

“…In C. glutamicum, the proton-motive force is generated mainly by the cytochrome bc 1 -aa 3 supercomplex of the respiratory chain or less efficiently by cytochrome bd oxidase Niebisch 2003, 2005). The transhydrogenase activity of 0.7 μmol min −1 (mg membrane protein) −1 measured in C. glutamicum carrying the pEKEx2-pntAB plasmid was in the same range as that determined for Saccharomyces cerevisiae expressing the E. coli pntAB genes (Anderlund et al 1999) and about 40-fold higher than the intrinsic PntAB-catalyzed transhydrogenase activity of E. coli (Sauer et al 2004).…”

“…The E. coli transhydrogenase PntAB is composed of two membrane-spanning subunits, α (50 kDa) and β (47 kDa), arranged as an α 2 β 2 heterotetramer (Clarke and Bragg 1985;for review, see Jackson 2003). Sauer et al (2004) reported that during aerobic batch cultivation of E. coli in minimal medium, glucose catabolism alone generated less NADPH than was required for biosynthesis and that the membrane-bound transhydrogenase contributed 35-45% of the total anabolic demand for NADPH under these conditions, although it was not essential for growth on glucose.…”

The OxyR Regulon in Nontypeable Haemophilus influenzae

“…NTHI1802 and NTHI1801 are homologues of pntA and pntB which encode the ␣ and ␤ subunits of NAD(P) transhydrogenase, respectively. In E. coli, PntAB has a role in the generation of NADPH; treatment of E. coli with H 2 O 2 induces both this activity and an increase in NADPH-dependent oxidase and peroxidase activity (11,73). Both the thioredoxin and glutaredoxin systems utilize the reducing power of NADPH to reduce disulfide bonds, so this process will likely be important during periods of oxidative stress.…”

NADH Availability Limits Asymmetric Biocatalytic Epoxidation in a Growing Recombinant Escherichia coli Strain

“…Fluxes into biomass were calculated from the known metabolite requirements for macromolecular compounds and the growth rate-dependent RNA and protein contents (16). The stoichiometric matrix of 20 linear equations and 22 metabolites included free interchange between the NADH and NADPH pools by the action of the dissolved and membrane-bound transhydrogenases UdhA and PntAB, respectively (60 We assumed that 55% of glucose-6-phosphate is catabolized through glycolysis as determined by Emmerling et al (16) for E. coli JM101 growing at a dilution rate of 0.09 h Ϫ1 . Moreover, we assumed phosphoenolpyruvate to oxaloacetate as the only anaplerotic reaction.…”