The widespread role of non-enzymatic reactions in cellular metabolism.

Enzymes shape cellular metabolism, are regulated, fast, and for most cases specific. Enzymes do not however prevent the parallel occurrence of non-enzymatic reactions. Non-enzymatic reactions were important for the evolution of metabolic pathways, but are retained as part of the modern metabolic network. They divide into unspecific chemical reactivity and specific reactions that occur either exclusively non-enzymatically as part of the metabolic network, or in parallel to existing enzyme functions. Non-enzymatic reactions resemble catalytic mechanisms as found in all major enzyme classes and occur spontaneously, small molecule (e.g. metal-) catalyzed or light-induced. The frequent occurrence of non-enzymatic reactions impacts on stability and metabolic network structure, and has thus to be considered in the context of metabolic disease, network modeling, biotechnology and drug design.We acknowledge funding from the Wellcome Trust (RG 093735/Z/10/Z), the ERC (starting Grant 260809). Markus A Keller is supported by the Austrian Science Funds by an Erwin Schroeder postdoctoral fellowship (FWF, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This paper was originally published in Current Opinion in Biotechnology (Keller MA, Piedrafita G, Ralser M, Current Opinion in Biotechnology 2015, 34, 153–161, doi:10.1016/j.copbio.2014.12.020)

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.Work in the Ralser lab is funded from the Wellcome Trust (RG 093735/Z/10/Z), the ERC (Starting grant 260809). Markus A. Keller is supported by the Austrian Science Funds by an Erwin Schrödinger postdoctoral fellowship (FWF, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This is the final version of the article. It first appeared from MDPI via http://dx.doi.org/10.3390/biom503210

A haploproficient interaction of the transaldolase paralogue NQM1 with the transcription factor VHR1 affects stationary phase survival and oxidative stress resistance.

BACKGROUND: Studying the survival of yeast in stationary phase, known as chronological lifespan, led to the identification of molecular ageing factors conserved from yeast to higher organisms. To identify functional interactions among yeast chronological ageing genes, we conducted a haploproficiency screen on the basis of previously identified long-living mutants. For this, we created a library of heterozygous Saccharomyces cerevisiae double deletion strains and aged them in a competitive manner. RESULTS: Stationary phase survival was prolonged in a double heterozygous mutant of the metabolic enzyme non-quiescent mutant 1 (NQM1), a paralogue to the pentose phosphate pathway enzyme transaldolase (TAL1), and the transcription factor vitamin H response transcription factor 1 (VHR1). We find that cells deleted for the two genes possess increased clonogenicity at late stages of stationary phase survival, but find no indication that the mutations delay initial mortality upon reaching stationary phase, canonically defined as an extension of chronological lifespan. We show that both genes influence the concentration of metabolites of glycolysis and the pentose phosphate pathway, central metabolic players in the ageing process, and affect osmolality of growth media in stationary phase cultures. Moreover, NQM1 is glucose repressed and induced in a VHR1 dependent manner upon caloric restriction, on non-fermentable carbon sources, as well as under osmotic and oxidative stress. Finally, deletion of NQM1 is shown to confer resistance to oxidizing substances. CONCLUSIONS: The transaldolase paralogue NQM1 and the transcription factor VHR1 interact haploproficiently and affect yeast stationary phase survival. The glucose repressed NQM1 gene is induced under various stress conditions, affects stress resistance and this process is dependent on VHR1. While NQM1 appears not to function in the pentose phosphate pathway, the interplay of NQM1 with VHR1 influences the yeast metabolic homeostasis and stress tolerance during stationary phase, processes associated with yeast ageing.We thank the Max Planck Society, Wellcome Trust (RG 093735/Z/10/Z), the ERC (Starting grant 260809), and the Isaac Newton Trust for funding. Markus A Keller is supported by an Erwin Schroedinger postdoctoral fellowship (FWF, Austria, J 3341). Markus Ralser is a Wellcome Trust Research Career Development and Wellcome-Beit Prize fellow.This is the final version of the article. It first appeared from BMC via http://dx.doi.org/10.1186/s12863-015-0171-

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

Screening for PTSD and functional impairment in trauma-exposed young children: evaluation of alternative CBCL-PTSD subscales

The Child Behavior Checklist (CBCL 1.5–5 years) posttraumatic stress disorder (PTSD) subscale by Dehon & Scheeringa (2006) as a screener for PTSD in trauma-exposed young children has yielded inconsistent results so far. Therefore, the aim of this study was to create and examine the validity of alternative CBCL-PTSD subscales and compare them to the existing CBCL-PTSD subscale based on the DSM-5 PTSD diagnostic criteria for children 6 years and younger. Further, the CBCL-PTSD subscales were examined regarding their usefulness in screening for posttraumatic stress-related functional impairment. The sample comprised 116 trauma-exposed young children (M$_{age}$ = 3.42 years, SD$_{age}$ = 1.21 years, female = 49.1%). The psychometric properties of the existing CBCL-PTSD subscale as well as the alternative subscales based on expert rating (CBCL-PTSD-17) and based on variable importance (CBCL-PTSD-6) were evaluated by means of receiver operating characteristic curves, sensitivity, specificity, positive predictive values, and negative predictive values. Area under the curves for all three investigated CBCL-PTSD subscales were good to excellent for PTSD and functional impairment. Further, all three CBCL-PTSD subscales showed high sensitivity for PTSD and functional impairment. Considering the length and the performance of the three investigated subscales, the CBCL-PTSD-6 appears to be a promising and clinically useful CBCL-PTSD subscale as a screener for PTSD and functional impairment due to the easiest and most practicable application. For purposes of discriminant validation of the CBCL-PTSD-6, young children without a history of trauma should be compared to young children with trauma history

First insights into structure-function relationships of alkylglycerol monooxygenase

Alkylglycerol monooxygenase is a tetrahydrobiopterin-dependent enzyme that cleaves the O-alkyl-bond of alkylglycerols. It is an exceptionally unstable, hydrophobic membrane protein which has never been purified in active form. Recently, we were able to identify the sequence of alkylglycerol monooxygenase. TMEM195, the gene coding for alkylglycerol monooxygenase, belongs to the fatty acid hydroxylases, a family of integral membrane enzymes which have an 8-histidine motif crucial for catalysis. Mutation of each of these residues resulted in a complete loss of activity. We now extended the mutational analysis to another 25 residues and identified three further residues conserved throughout all members of the fatty acid hydroxylases which are essential for alkylglycerol monooxygenase activity. Furthermore, mutation of a specific glutamate resulted in an 18-fold decreased affinity of the protein to tetrahydrobiopterin, strongly indicating a potential important role in cofactor interaction. A glutamate residue in a comparable amino acid surrounding had already been shown to be responsible for tetrahydrobiopterin binding in the aromatic amino acid hydroxylases. Ab initio modelling of the enzyme yielded a structural model for the central part of alkylglycerol monooxygenase where all essential residues identified by mutational analysis are in close spatial vicinity, thereby defining the potential catalytic site of this enzym

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.We acknowledge funding from the European Commission (Brussels) Role ofMitochondria in Conserved Mechanisms of Aging (MIMAGE) Project (Contract 512020, to M.B.), the Cancer Research Programme Grant (C197/A3514 to K.M.B.), Cancer Research UK and ERC Grants 322842-METABOp53 (supporting E.C.), the Wellcome Trust (RG 093735/Z/10/Z to M.R.), the ERC (Starting grant 260809 to M.R.), the German Research Foundation DFG (PR 1527/1-1 to A.P.), and the Austrian Science Fund (FWF) S9302-B05 (to M.B.). V.O.-S. is supported by Consejo Nacional de Ciencia y Tecnologia (CONACyT) Mexico postdoctoral fellowship 203450, M.A.K. by the FWF (Austria) by an Erwin Schroedinger postdoctoral fellowship (J 3341). M.R. is a Wellcome-Trust Research career development and Wellcome-Beit prize fellow.This is the final published version. It is also available from Wiley at http://onlinelibrary.wiley.com/doi/10.1111/brv.12140/abstract

Drug interactions and pharmacogenetic factors contribute to variation in apixaban concentration in atrial fibrillation patients in routine care

Factor Xa-inhibitor apixaban is an oral anticoagulant prescribed in atrial fibrillation (AF) for stroke prevention. Its pharmacokinetic profile is known to be affected by cytochrome P450 (CYP)3A metabolism, while it is also a substrate of the efflux transporters ATP-binding cassette (ABC)B1 (P-glycoprotein) and ABCG2 (breast cancer resistance protein, BCRP). In this study, we assessed the impact of interacting medication and pharmacogenetic variation to better explain apixaban concentration differences among 358 Caucasian AF patients. Genotyping (ABCG2, ABCB1, CYP3A4*22, CYP3A5*3) was performed by TaqMan assays, and apixaban quantified by mass spectrometry. The typical patient was on average 77.2 years old, 85.5 kg, and had a serum creatinine of 103.1 µmol/L. Concomitant amiodarone, an antiarrhythmic agent and moderate CYP3A/ABCB1 inhibitor, the impaired-function variant ABCG2 c.421C \u3e A, and sex predicted higher apixaban concentrations when controlling for age, weight and serum creatinine (multivariate regression; R2 = 0.34). Our findings suggest that amiodarone and ABCG2 genotype contribute to interpatient apixaban variability beyond known clinical factors

Re-structuring of marine communities exposed to environmental change

Species richness is the most commonly used but controversial biodiversity metric in studies on aspects of community stability such as structural composition or productivity. The apparent ambiguity of theoretical and experimental findings may in part be due to experimental shortcomings and/or heterogeneity of scales and methods in earlier studies. This has led to an urgent call for improved and more realistic experiments. In a series of experiments replicated at a global scale we translocated several hundred marine hard bottom communities to new environments simulating a rapid but moderate environmental change. Subsequently, we measured their rate of compositional change (re-structuring) which in the great majority of cases represented a compositional convergence towards local communities. Re-structuring is driven by mortality of community components (original species) and establishment of new species in the changed environmental context. The rate of this re-structuring was then related to various system properties. We show that availability of free substratum relates negatively while taxon richness relates positively to structural persistence (i.e., no or slow re-structuring). Thus, when faced with environmental change, taxon-rich communities retain their original composition longer than taxon-poor communities. The effect of taxon richness, however, interacts with another aspect of diversity, functional richness. Indeed, taxon richness relates positively to persistence in functionally depauperate communities, but not in functionally diverse communities. The interaction between taxonomic and functional diversity with regard to the behaviour of communities exposed to environmental stress may help understand some of the seemingly contrasting findings of past research