Markus A. Keller

The return of metabolism: biochemistry and physiology of the pentose phosphate pathway

The pentose phosphate pathway (PPP) is a fundamental component of cellular metabolism. The PPP is important to maintain carbon homoeostasis, to provide precursors for nucleotide and amino acid biosynthesis, to provide reducing molecules for anabolism, and to defeat oxidative stress. The PPP shares reactions with the Entner–Doudoroff pathway and Calvin cycle and divides into an oxidative and non‐oxidative branch. The oxidative branch is highly active in most eukaryotes and converts glucose 6‐phosphate into carbon dioxide, ribulose 5‐phosphate and NADPH. The latter function is critical to maintain redox balance under stress situations, when cells proliferate rapidly, in ageing, and for the ‘Warburg effect’ of cancer cells. The non‐oxidative branch instead is virtually ubiquitous, and metabolizes the glycolytic intermediates fructose 6‐phosphate and glyceraldehyde 3‐phosphate as well as sedoheptulose sugars, yielding ribose 5‐phosphate for the synthesis of nucleic acids and sugar phosphate precursors for the synthesis of amino acids. Whereas the oxidative PPP is considered unidirectional, the non‐oxidative branch can supply glycolysis with intermediates derived from ribose 5‐phosphate and vice versa, depending on the biochemical demand. These functions require dynamic regulation of the PPP pathway that is achieved through hierarchical interactions between transcriptome, proteome and metabolome. Consequently, the biochemistry and regulation of this pathway, while still unresolved in many cases, are archetypal for the dynamics of the metabolic network of the cell. In this comprehensive article we review seminal work that led to the discovery and description of the pathway that date back now for 80 years, and address recent results about genetic and metabolic mechanisms that regulate its activity. These biochemical principles are discussed in the context of PPP deficiencies causing metabolic disease and the role of this pathway in biotechnology, bacterial and parasite infections, neurons, stem cell potency and cancer metabolism.

Non-enzymatic glycolysis and pentose phosphate pathway-like reactions in a plausible Archean ocean

The reaction sequences of central metabolism, glycolysis and the pentose phosphate pathway provide essential precursors for nucleic acids, amino acids and lipids. However, their evolutionary origins are not yet understood. Here, we provide evidence that their structure could have been fundamentally shaped by the general chemical environments in earths earliest oceans. We reconstructed potential scenarios for oceans of the prebiotic Archean based on the composition of early sediments. We report that the resultant reaction milieu catalyses the interconversion of metabolites that in modern organisms constitute glycolysis and the pentose phosphate pathway. The 29 observed reactions include the formation and/or interconversion of glucose, pyruvate, the nucleic acid precursor ribose‐5‐phosphate and the amino acid precursor erythrose‐4‐phosphate, antedating reactions sequences similar to that used by the metabolic pathways. Moreover, the Archean ocean mimetic increased the stability of the phosphorylated intermediates and accelerated the rate of intermediate reactions and pyruvate production. The catalytic capacity of the reconstructed ocean milieu was attributable to its metal content. The reactions were particularly sensitive to ferrous iron Fe(II), which is understood to have had high concentrations in the Archean oceans. These observations reveal that reaction sequences that constitute central carbon metabolism could have been constrained by the iron‐rich oceanic environment of the early Archean. The origin of metabolism could thus date back to the prebiotic world.

The widespread role of non-enzymatic reactions in cellular metabolism

Graphical abstract

Inhibition of triosephosphate isomerase by phosphoenolpyruvate in the feedback-regulation of glycolysis

The inhibition of triosephosphate isomerase (TPI) in glycolysis by the pyruvate kinase (PK) substrate phosphoenolpyruvate (PEP) results in a newly discovered feedback loop that counters oxidative stress in cancer and actively respiring cells. The mechanism underlying this inhibition is illuminated by the co-crystal structure of TPI with bound PEP at 1.6 Å resolution, and by mutational studies guided by the crystallographic results. PEP is bound to the catalytic pocket of TPI and occludes substrate, which accounts for the observation that PEP competitively inhibits the interconversion of glyceraldehyde-3-phosphate and dihydroxyacetone phosphate. Replacing an isoleucine residue located in the catalytic pocket of TPI with valine or threonine altered binding of substrates and PEP, reducing TPI activity in vitro and in vivo. Confirming a TPI-mediated activation of the pentose phosphate pathway (PPP), transgenic yeast cells expressing these TPI mutations accumulate greater levels of PPP intermediates and have altered stress resistance, mimicking the activation of the PK–TPI feedback loop. These results support a model in which glycolytic regulation requires direct catalytic inhibition of TPI by the pyruvate kinase substrate PEP, mediating a protective metabolic self-reconfiguration of central metabolism under conditions of oxidative stress.

The Impact of Non-Enzymatic Reactions and Enzyme Promiscuity on Cellular Metabolism during (Oxidative) Stress Conditions

Cellular metabolism assembles in a structurally highly conserved, but functionally dynamic system, known as the metabolic network. This network involves highly active, enzyme-catalyzed metabolic pathways that provide the building blocks for cell growth. In parallel, however, chemical reactivity of metabolites and unspecific enzyme function give rise to a number of side products that are not part of canonical metabolic pathways. It is increasingly acknowledged that these molecules are important for the evolution of metabolism, affect metabolic efficiency, and that they play a potential role in human disease—age-related disorders and cancer in particular. In this review we discuss the impact of oxidative and other cellular stressors on the formation of metabolic side products, which originate as a consequence of: (i) chemical reactivity or modification of regular metabolites; (ii) through modifications in substrate specificity of damaged enzymes; and (iii) through altered metabolic flux that protects cells in stress conditions. In particular, oxidative and heat stress conditions are causative of metabolite and enzymatic damage and thus promote the non-canonical metabolic activity of the cells through an increased repertoire of side products. On the basis of selected examples, we discuss the consequences of non-canonical metabolic reactivity on evolution, function and repair of the metabolic network.

Unbiased Metabolomic Investigation of Alzheimer’s Disease Brain Points to Dysregulation of Mitochondrial Aspartate Metabolism

Alzheimers disease (AD) is the most common cause of adult dementia. Yet the complete set of molecular changes accompanying this inexorable, neurodegenerative disease remains elusive. Here we adopted an unbiased lipidomics and metabolomics approach to surveying frozen frontal cortex samples from clinically characterized AD patients (n = 21) and age-matched controls (n = 19), revealing marked molecular differences between them. Then, by means of metabolomic pathway analysis, we incorporated the novel molecular information into the known biochemical pathways and compared it with the results of a metabolomics meta-analysis of previously published AD research. We found six metabolic pathways of the central metabolism as well as glycerophospholipid metabolism predominantly altered in AD brains. Using targeted metabolomics approaches and MS imaging, we confirmed a marked dysregulation of mitochondrial aspartate metabolism. The altered metabolic pathways were further integrated with clinical data, showing various degrees of correlation with parameters of dementia and AD pathology. Our study highlights specific, altered biochemical pathways in the brains of individuals with AD compared with those of control subjects, emphasizing dysregulation of mitochondrial aspartate metabolism and supporting future venues of investigation.

Regulation of ribosomal DNA amplification by the TOR pathway

Significance We tend to think of our genome as an unchanging store of information; however, recent evidence suggests that genomes vary between different cells in the same organism. How these differences arise and what effects they have remain unknown, but clearly our genome can change. In a single-celled organism, genome changes occur at random, and advantageous changes slowly propagate by natural selection. However, it is known that the DNA encoding ribosomes can change simultaneously in a whole population. Here we show that signaling pathways that sense environmental nutrients control genome change at the ribosomal DNA. This demonstrates that not all genome changes occur at random and that cells possess specific mechanisms to optimize their genome in response to the environment. Repeated regions are widespread in eukaryotic genomes, and key functional elements such as the ribosomal DNA tend to be formed of high copy repeated sequences organized in tandem arrays. In general, high copy repeats are remarkably stable, but a number of organisms display rapid ribosomal DNA amplification at specific times or under specific conditions. Here we demonstrate that target of rapamycin (TOR) signaling stimulates ribosomal DNA amplification in budding yeast, linking external nutrient availability to ribosomal DNA copy number. We show that ribosomal DNA amplification is regulated by three histone deacetylases: Sir2, Hst3, and Hst4. These enzymes control homologous recombination-dependent and nonhomologous recombination-dependent amplification pathways that act in concert to mediate rapid, directional ribosomal DNA copy number change. Amplification is completely repressed by rapamycin, an inhibitor of the nutrient-responsive TOR pathway; this effect is separable from growth rate and is mediated directly through Sir2, Hst3, and Hst4. Caloric restriction is known to up-regulate expression of nicotinamidase Pnc1, an enzyme that enhances Sir2, Hst3, and Hst4 activity. In contrast, normal glucose concentrations stretch the ribosome synthesis capacity of cells with low ribosomal DNA copy number, and we find that these cells show a previously unrecognized transcriptional response to caloric excess by reducing PNC1 expression. PNC1 down-regulation forms a key element in the control of ribosomal DNA amplification as overexpression of PNC1 substantially reduces ribosomal DNA amplification rate. Our results reveal how a signaling pathway can orchestrate specific genome changes and demonstrate that the copy number of repetitive DNA can be altered to suit environmental conditions.

Sulfate radicals enable a non-enzymatic Krebs cycle precursor

The evolutionary origins of the Krebs cycle (tricarboxylic acid cycle) are not currently clear. Despite the existence of a simple non-enzymatic Krebs cycle catalyst being dismissed only a few years ago as ‘an appeal to magic’, citrate and other intermediates have since been discovered on a carbonaceous meteorite and do interconvert non-enzymatically. To identify a metabolism-like non-enzymatic Krebs cycle catalyst, we used combinatorial, quantitative high-throughput metabolomics to systematically screen iron and sulfate compounds in a reaction mixture that orients on the typical components of Archaean sediment. Krebs cycle intermediates were found to be stable in water and in the presence of most molecule species, including simple iron sulfate minerals. However, in the presence of sulfate radicals generated from peroxydisulfate, the intermediates underwent 24 interconversion reactions. These non-enzymatic reactions covered the critical topology of the oxidative Krebs cycle, the glyoxylate shunt and the succinic-semialdehyde pathway. Assembled in a chemical network, the reactions achieved over 90% carbon recovery. Our results show that a non-enzymatic precursor of the Krebs cycle is biologically sensible, efficient, and forms spontaneously in the presence of sulfate radicals.

Tetrahydrobiopterin and alkylglycerol monooxygenase substantially alter the murine macrophage lipidome

Significance We have recently identified the sequence of the ether lipid-cleaving enzyme alkylglycerol monooxygenase. Like nitric oxide synthases and aromatic amino acid hydroxylases, alkylglycerol monooxygenase needs tetrahydrobiopterin as cofactor. Whereas the former enzymes have well-established roles in the cell, the physiology of alkylglycerol monooxygenase is still not clear. Here we show its regulation in murine macrophage differentiation and its dependence on the cofactor tetrahydrobiopterin in live murine macrophage-like RAW264.7 cells. Upon modulation of the activity of alkylglycerol monooxygenase and the key enzyme in tetrahydrobiopterin biosynthesis, we observe extensive changes in various lipid classes ranging from ether lipids to far more complex lipids. These findings point to an important role of tetrahydrobiopterin in cellular lipid homeostasis. Tetrahydrobiopterin is a cofactor synthesized from GTP with well-known roles in enzymatic nitric oxide synthesis and aromatic amino acid hydroxylation. It is used to treat mild forms of phenylketonuria. Less is known about the role of tetrahydrobiopterin in lipid metabolism, although it is essential for irreversible ether lipid cleavage by alkylglycerol monooxygenase. Here we found intracellular alkylglycerol monooxygenase activity to be an important regulator of alkylglycerol metabolism in intact murine RAW264.7 macrophage-like cells. Alkylglycerol monooxygenase was expressed and active also in primary mouse bone marrow-derived monocytes and “alternatively activated” M2 macrophages obtained by interleukin 4 treatment, but almost missing in M1 macrophages obtained by IFN-γ and lipopolysaccharide treatment. The cellular lipidome of RAW264.7 was markedly changed in a parallel way by modulation of alkylglycerol monooxygenase expression and of tetrahydrobiopterin biosynthesis affecting not only various ether lipid species upstream of alkylglycerol monooxygenase but also other more complex lipids including glycosylated ceramides and cardiolipins, which have no direct connection to ether lipid pathways. Alkylglycerol monooxygenase activity manipulation modulated the IFN-γ/lipopolysaccharide–induced expression of inducible nitric oxide synthase, interleukin-1β, and interleukin 1 receptor antagonist but not transforming growth factor β1, suggesting that alkylglycerol monooxygenase activity affects IFN-γ/lipopolysaccharide signaling. Our results demonstrate a central role of tetrahydrobiopterin and alkylglycerol monooxygenase in ether lipid metabolism of murine macrophages and reveal that alteration of alkylglycerol monooxygenase activity has a profound impact on the lipidome also beyond the class of ether lipids.

Hyperpolarized [U-2H, U-13C]Glucose reports on glycolytic and pentose phosphate pathway activity in EL4 tumors and glycolytic activity in yeast cells

A resonance at ∼181 ppm in the 13C spectra of tumors injected with hyperpolarized [U‐2H, U‐13C]glucose was assigned to 6‐phosphogluconate (6PG), as in previous studies in yeast, whereas in breast cancer cells in vitro this resonance was assigned to 3‐phosphoglycerate (3PG). These peak assignments were investigated here using measurements of 6PG and 3PG 13C‐labeling using liquid chromatography tandem mass spectrometry (LC‐MS/MS)