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.

We describe here a novel methodology for rapid diagnosis of metabolic changes, which is based on probabilistic equations that relate GC-MS-derived mass distributions in proteinogenic amino acids to in vivo enzyme activities. This metabolic flux ratio analysis by GC-MS provides a comprehensive perspective on central metabolism by quantifying 14 ratios of fluxes through converging pathways and reactions from [1-13 C] and [U-13 C]glucose experiments. Reliability and accuracy of this method were experimentally verified by successfully capturing expected flux responses of Escherichia coli to environmental modifications and seven knockout mutations in all major pathways of central metabolism. Furthermore, several mutants exhibited additional, unexpected flux responses that provide new insights into the behavior of the metabolic network in its entirety. Most prominently, the low in vivo activity of the EntnerDoudoroff pathway in wild-type E. coli increased up to a contribution of 30% to glucose catabolism in mutants of glycolysis and TCA cycle. Moreover, glucose 6-phosphate dehydrogenase mutants catabolized glucose not exclusively via glycolysis, suggesting a yet unidentified bypass of this reaction. Although strongly affected by environmental conditions, a stable balance between anaplerotic and TCA cycle flux was maintained by all mutants in the upper part of metabolism. Overall, our results provide quantitative insight into flux changes that bring about the resilience of metabolic networks to disruption.Keywords: Entner-Doudoroff pathway; flux analysis; fluxome; METAFoR analysis; pentose phosphate pathway.Comprehensive and quantitative understanding of biochemical reaction networks requires not only knowledge about their constituting components, but also information about the behavior of the network in its entirety. Toward this end, systems-oriented methodologies were developed that simultaneously access the level of reaction intermediates [1] or rates of reactions [2][3][4][5], also referred to as the metabolome [6] and the fluxome [7], respectively. The most important property of biochemical networks are the per se nonmeasurable in vivo reaction rates, which may be estimated by so-called metabolic flux analysis that provides a holistic perspective on metabolism.In its simplest form, metabolic flux analysis relies on flux balancing of extracellular consumption and secretion rates within a stoichiometric reaction model [5]. To increase reliability and resolution of such flux balancing analyses, additional information may be derived from 13 C-labeling experiments. In this approach, 13 C-labeled substrates are administered and 13 C-labeled products of metabolism are analyzed by methods that distinguish between different isotope labeling patterns, in particular NMR and MS [2,3,8]. In the most advanced methodology, a comprehensive isotope isomer (isotopomer) model of metabolism is used to map metabolic fluxes in an iterative fitting procedure on the isotopomer pattern of network metabolites that are deduced from NMR or M...

The structurally conserved and ubiquitous pathways of central carbon metabolism provide building blocks and cofactors for the biosynthesis of cellular macromolecules. The relative uses of pathways and reactions, however, vary widely among species and depend upon conditions, and some are not used at all. Here we identify the network topology of glucose metabolism and its in vivo operation by quantification of intracellular carbon fluxes from 13 C tracer experiments. Specifically, we investigated Agrobacterium tumefaciens, two pseudomonads, Sinorhizobium meliloti, Rhodobacter sphaeroides, Zymomonas mobilis, and Paracoccus versutus, which grow on glucose as the sole carbon source, represent fundamentally different metabolic lifestyles (aerobic, anaerobic, photoheterotrophic, and chemoheterotrophic), and are phylogenetically distinct (firmicutes, ␥-proteobacteria, and ␣-proteobacteria). Compared to those of the model bacteria Escherichia coli and Bacillus subtilis, metabolisms of the investigated species differed significantly in several respects: (i) the Entner-Doudoroff pathway was the almost exclusive catabolic route; (ii) the pentose phosphate pathway exhibited exclusively biosynthetic functions, in many cases also requiring flux through the nonoxidative branch; (iii) all aerobes exhibited fully respiratory metabolism without significant overflow metabolism; and (iv) all aerobes used the pyruvate bypass of the malate dehydrogenase reaction to a significant extent. Exclusively, Pseudomonas fluorescens converted most glucose extracellularly to gluconate and 2-ketogluconate. Overall, the results suggest that metabolic data from model species with extensive industrial and laboratory history are not representative of microbial metabolism, at least not quantitatively. Based on13 C tracer experiments, metabolic-flux analysis emerged as a key methodology to identify the network topology of active reactions and to quantify the in vivo distribution of molecular fluxes throughout metabolism (38, 47). In contrast to global protein, mRNA, or metabolite concentration analyses that assess network composition, flux methods directly assess the operation of metabolic networks by quantifying in vivo reaction velocities. The general principle is based on mass spectrometry (MS) or nuclear magnetic resonance detection of 13 C patterns in products of metabolism. Often, protein-bound amino acids that preserve the carbon backbone of eight metabolic key intermediates are used. The detected 13 C isotope patterns then reflect the activity of intracellular pathways and reactions, whose fluxes can be quantified from the isotope data by using mathematical models with various levels of complexity. In the simplest approach, algebraic equations are used to determine strictly local ratios of converging fluxes analytically by so-called metabolic-flux ratio (METAFoR) analysis (3,16,41,44). Absolute intracellular fluxes in millimoles per gram of biomass per hour may be estimated indirectly by combining such 13 C data with quantitative physiological ...

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Qualitative theoretical approaches such as graph theory and stoichiometric analyses are beginning to uncover the architecture and systemic functions of complex metabolic reaction networks. At present, however, only a few, largely unproven quantitative concepts propose functional design principles of the global flux distribution. As operational units of function, molecular fluxes determine the systemic cell phenotype by linking genes, proteins and metabolites to higher-level biological functions. In sharp contrast to other 'omics' analyses, 'fluxome' analysis remained tedious. By large-scale quantification of in vivo flux responses, we identified a robust flux distribution in 137 null mutants of Bacillus subtilis. On its preferred substrate, B. subtilis has suboptimal metabolism because regulators of developmental programs maintain a 'standby' mode that invests substantial resources in anticipation of changing environmental conditions at the expense of optimal growth. Network rigidity and robustness are probably universal functional design principles, whereas the standby mode may be more specific.

Most chemical techniques used to produce antibody−drug conjugates (ADCs) result in a heterogeneous mixture of species with variable drug-to-antibody ratios (DAR) which will potentially display different pharmacokinetics, stability, and safety profiles. Here we investigated two strategies to obtain homogeneous ADCs based on site-specific modification of deglycosylated antibodies by microbial transglutaminase (MTGase), which forms isopeptidic bonds between Gln and Lys residues. We have previously shown that MTGase solely recognizes Gln295 within the heavy chain of IgGs as a substrate and can therefore be exploited to generate ADCs with an exact DAR of 2. The first strategy included the direct, onestep attachment of the antimitotic toxin monomethyl auristatin E (MMAE) to the antibody via different spacer entities with a primary amine functionality that is recognized as a substrate by MTGase. The second strategy was a chemo-enzymatic, two-step approach whereby a reactive spacer entity comprising a bio-orthogonal thiol or azide function was attached to the antibody by MTGase and subsequently reacted with a suitable MMAE-derivative. To this aim, we investigated two different chemical approaches, namely, thiol-maleimide and strain-promoted azide−alkyne cycloaddition (SPAAC). Direct enzymatic attachment of MMAE-spacer derivatives at an 80 molar excess of drug yielded heterogeneous ADCs with a DAR of between 1.0 to 1.6. In contrast to this, the chemo-enzymatic approach only required a 2.5 molar excess of toxin to yield homogeneous ADCs with a DAR of 2.0 in the case of SPAAC and 1.8 for the thiol-maleimide approach. As a proof-of-concept, trastuzumab (Herceptin) was armed with the MMAE via the chemo-enzymatic approach using SPAAC and tested in vitro. Trastuzumab-MMAE efficiently killed BT-474 and SK-BR-3 cells with an IC 50 of 89.0 pM and 21.7 pM, respectively. Thus, the chemo-enzymatic approach using MTGase is an elegant strategy to form ADCs with a defined DAR of 2. Furthermore, the approach is directly applicable to a broad variety of antibodies as it does not require prior genetic modifications of the antibody sequence.

Complete oxidation of carbohydrates to CO 2 is considered to be the exclusive property of the ubiquitous tricarboxylic acid cycle, the central process in cellular energy metabolism of aerobic organisms. Based on metabolism-wide in vivo quantification of intracellular carbon fluxes, we describe here complete oxidation of carbohydrates via the novel P-enolpyruvate (PEP)-glyoxylate cycle, in which two PEP molecules are oxidized by means of acetyl coenzyme A, citrate, glyoxylate, and oxaloacetate to CO 2 , and one PEP is regenerated. Key reactions are the constituents of the glyoxylate shunt and PEP carboxykinase, whose conjoint operation in this bi-functional catabolic and anabolic cycle is in sharp contrast to their generally recognized functions in anaplerosis and gluconeogenesis, respectively. Parallel operation of the PEP-glyoxylate cycle and the tricarboxylic acid cycle was identified in the bacterium Escherichia coli under conditions of glucose hunger in a slow-growing continuous culture. Because the PEPglyoxylate cycle was also active in glucose excess batch cultures of an NADPH-overproducing phosphoglucose isomerase mutant, one function of this new central pathway may be the decoupling of catabolism from NADPH formation that would otherwise occur in the tricarboxylic acid cycle.Structuring of cellular networks into pathways with distinct functions is pivotal for comprehension of "textbook" biochemistry. The ability of existing model pathways to portray flux through complex metabolic networks, however, is an open question that is just beginning to be addressed theoretically (1-3) and experimentally (4, 5). Microbial growth on the most abundant carbon-source glucose represses transcription of metabolic functions that are required on alternative carbon sources. This universal phenomenon is referred to as catabolite repression and includes a number of mechanistically distinguishable but physiologically related regulation mechanisms (6, 7). Although catabolite repression is strong under conditions of feast with excess glucose, microbes typically thrive under conditions of starvation (absence of nutrients) or hunger (suboptimal supply of nutrients) in their natural environments (8,9). This metabolic state of hunger, between optimal growth and starvation, can be studied in glucose-limited continuous (chemostat) cultures with very low glucose concentrations at a rate of growth that is controlled by the experimenter. Catabolite repression is absent under the severe glucose limitation in slow growing chemostat cultures (9 -11), and, as a consequence, increased in vivo activity of repressed metabolic enzymes is often observed with advanced methods of metabolic flux analyses based on 13 C-labeling experiments (4, 5, 12, 13).Here we elucidate metabolic impacts of severe and absent catabolite repression during growth of Escherichia coli in glucose-excess batch cultures and glucose-limited chemostat cultures. Direct analytical interpretation of 13 C-labeling patterns in proteinogenic amino acids was used to establish the ...

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