Abstract
Coenzymes are vital for cellular metabolism and act on the full spectrum of enzymatic reactions. Intrinsic chemical reactivity, enzyme promiscuity and high flux through their catalytic cycles make coenzymes prone to damage. To counteract such compromising factors and ensure stable levels of functional coenzymes, cells use a complex interplay between de novo synthesis, salvage, repair and degradation. However, the relative contribution of these factors is currently unknown, as is the overall stability of coenzymes in the cell. Here, we use dynamic 13C-labelling experiments to determine the half-life of major coenzymes of Escherichia coli. We find that coenzymes such as pyridoxal 5-phosphate, flavins, nicotinamide adenine dinucleotide (phosphate) and coenzyme A are remarkably stable in vivo and allow biosynthesis close to the minimal necessary rate. In consequence, they are essentially produced to compensate for dilution by growth and passed on over generations of cells. Exceptions are antioxidants, which are short-lived, suggesting an inherent requirement for increased renewal. Although the growth-driven turnover of stable coenzymes is apparently subject to highly efficient end-product homeostasis, we exemplify that coenzyme pools are propagated in excess in relation to actual growth requirements. Additional testing of Bacillus subtilis and Saccharomyces cerevisiae suggests that coenzyme longevity is a conserved feature in biology.
This is a preview of subscription content, access via your institution
Access options
Access Nature and 54 other Nature Portfolio journals
Get Nature+, our best-value online-access subscription
$29.99 / 30 days
cancel any time
Subscribe to this journal
Receive 12 digital issues and online access to articles
$119.00 per year
only $9.92 per issue
Rent or buy this article
Prices vary by article type
from$1.95
to$39.95
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Fischer, J. D., Holliday, G. L., Rahman, S. A. & Thornton, J. M. The structures and physicochemical properties of organic cofactors in biocatalysis. J. Mol. Biol. 403, 803–824 (2010).
Eyschen, J. et al. Engineered glycolytic glyceraldehyde-3-phosphate dehydrogenase binds the anti conformation of NAD+ nicotinamide but does not experience A-specific hydride transfer. Arch. Biochem. Biophys. 364, 219–227 (1999).
Lu, P. et al. Absolute protein expression profiling estimates the relative contributions of transcriptional and translational regulation. Nat. Biotechnol. 25, 117–124 (2007).
Bennett, B. D. et al. Absolute metabolite concentrations and implied enzyme active site occupancy in Escherichia coli. Nat. Chem. Biol. 5, 593–599 (2009).
Linster, C. L., Van Schaftingen, E. & Hanson, A. D. Metabolite damage and its repair or pre-emption. Nat. Chem. Biol. 9, 72–80 (2013).
Khersonsky, O. & Tawfik, D. S. Enzyme promiscuity: a mechanistic and evolutionary perspective. Annu. Rev. Biochem. 79, 471–505 (2010).
Oppenheimer, N. J. & Kaplan, N. O. Glyceraldehyde-3-phosphate dehydrogenase catalyzed hydration of the 5-6 double bond of reduced β-nicotinamide adenine dinucleotide (βNADH). Formation of β-6-hydroxy-1,4,5,6-tetrahydronicotinamide adenine dinucleotide. Biochemistry 13, 4685–4694 (1974).
Lerma-Ortiz, C. et al. ‘Nothing of chemistry disappears in biology’: the top 30 damage-prone endogenous metabolites. Biochem. Soc. Trans. 44, 961–971 (2016).
Keller, M. A., Piedrafita, G. & Ralser, M. The widespread role of non-enzymatic reactions in cellular metabolism. Curr. Opin. Biotechnol. 34, 153–161 (2015).
Golubev, A. G. The other side of metabolism: a review. Biochemistry 61, 2018–2039 (1996).
Linster, C. L. et al. Ethylmalonyl-CoA decarboxylase, a new enzyme involved in metabolite proofreading. J. Biol. Chem. 286, 42992–43003 (2011).
Marbaix, A. Y. et al. Extremely conserved ATP- or ADP-dependent enzymatic system for nicotinamide nucleotide repair. J. Biol. Chem. 286, 41246–41252 (2011).
Vinci, C. R. & Clarke, S. G. Homocysteine methyltransferases Mht1 and Sam4 prevent the accumulation of age-damaged (R,S)-AdoMet in the yeast Saccharomyces cerevisiae. J. Biol. Chem. 285, 20526–20531 (2010).
Huang, L. et al. A family of metal-dependent phosphatases implicated in metabolite damage-control. Nat. Chem. Biol. 12, 621–627 (2016).
Kremer, L. S. et al. NAXE mutations disrupt the cellular NAD(P)HX repair system and cause a lethal neurometabolic disorder of early childhood. Am. J. Hum. Genet. 99, 894–902 (2016).
Mills, P. B. et al. Mutations in antiquitin in individuals with pyridoxine-dependent seizures. Nat. Med. 12, 307–309 (2006).
Coburn, S. P. Location and turnover of vitamin B6 pools and vitamin B6 requirements of humans. Ann. NY Acad. Sci. 585, 76–85 (1990).
Gregory, J. F. III & Quinlivan, E. P. In vivo kinetics of folate metabolism. Annu. Rev. Nutr. 22, 199–220 (2002).
Buescher, J. M. et al. A roadmap for interpreting 13C metabolite labeling patterns from cells. Curr. Opin. Biotechnol. 34, 189–201 (2015).
Kiefer, P. et al. Dynamet: a fully automated pipeline for dynamic LC-MS data. Anal. Chem. 87, 9679 (2015).
Shamir, M., Bar-On, Y., Phillips, R. & Milo, R. Snapshot: timescales in cell biology. Cell 164, 1302–1302 (2016).
Bertoldi, M. & Voltattorni, C. B. Dopa decarboxylase exhibits low pH half-transaminase and high pH oxidative deaminase activities toward serotonin (5-hydroxytryptamine). Prot. Sci. 10, 1178–1186 (2001).
Mewies, M., McIntire, W. S. & Scrutton, N. S. Covalent attachment of flavin adenine dinucleotide (FAD) and flavin mononucleotide (FMN) to enzymes: the current state of affairs. Protein Sci. 7, 7–20 (1998).
Thomas, J. & Cronan, J. E. The enigmatic acyl carrier protein phosphodiesterase of Escherichia coli: genetic and enzymological characterization. J. Biol. Chem. 280, 34675–34683 (2005).
Sorci, L., Ruggieri, S. & Raffaelli, N. NAD homeostasis in the bacterial response to DNA/RNA damage. DNA Repair 23, 17–26 (2014).
Manlapaz-Fernandez, P. & Olivera, B. M. Pyridine nucleotide metabolism in Escherichia coli. IV. Turnover. J. Biol. Chem. 248, 5150–5155 (1973).
Hillyard, D. et al. The pyridine nucleotide cycle. Studies in Escherichia coli and the human cell line D98/AH2. J. Biol. Chem. 256, 8491–8497 (1981).
Baudouin-Cornu, P. et al. Glutathione degradation is a key determinant of glutathione homeostasis. J. Biol. Chem. 287, 4552–4561 (2012).
Hanson, A. D. & Gregory, J. F. III Folate biosynthesis, turnover, and transport in plants. Annu. Rev. Plant Biol. 62, 105–125 (2011).
Thiaville, J. J. et al. Experimental and metabolic modeling evidence for a folate-cleaving side-activity of ketopantoate hydroxymethyltransferase (PanB). Front. Microbiol. 7, 431 (2016).
Jeanguenin, L. et al. Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase. J. Biol. Chem. 285, 41557–41566 (2010).
Ferone, R., Hanlon, M. H., Singer, S. C. & Hunt, D. F. α-Carboxyl-linked glutamates in the folylpolyglutamates of Escherichia coli. J. Biol. Chem. 261, 16356–16362 (1986).
Tokumoto, U. & Takahashi, Y. Genetic analysis of the isc operon in Escherichia coli involved in the biogenesis of cellular iron–sulfur proteins. J. Biochem. 130, 63–71 (2001).
Monod, J. The growth of bacterial cultures. Annu. Rev. Microbiol. 3, 371–394 (1949).
Wilson, A. C. & Pardee, A. B. Regulation of flavin synthesis by Escherichia coli. J. Gen. Microbiol. 28, 283–303 (1962).
Rechsteiner, M., Hillyard, D. & Olivera, B. M. Magnitude and significance of NAD turnover in human cell line D98/AH2. Nature 259, 695–696 (1976).
Gaballa, A. et al. Biosynthesis and functions of bacillithiol, a major low-molecular-weight thiol in Bacilli. Proc. Natl Acad. Sci. USA 107, 6482–6486 (2010).
Mandal, M. et al. Riboswitches control fundamental biochemical pathways in Bacillus subtilis and other bacteria. Cell 113, 577–586 (2003).
Larrabee, K. L., Phillips, J. O., Williams, G. J. & Larrabee, A. R. The relative rates of protein synthesis and degradation in a growing culture of Escherichia coli. J. Biol. Chem. 255, 4125–4130 (1980).
Christiano, R., Nagaraj, N., Frohlich, F. & Walther, T. C. Global proteome turnover analyses of the yeasts S. cerevisiae and S. pombe. Cell Rep. 9, 1959–1965 (2014).
Eden, E. et al. Proteome half-life dynamics in living human cells. Science 331, 764–768 (2011).
Toyama, B. H. et al. Identification of long-lived proteins reveals exceptional stability of essential cellular structures. Cell 154, 971–982 (2013).
Thayer, N. H. et al. Identification of long-lived proteins retained in cells undergoing repeated asymmetric divisions. Proc. Natl Acad. Sci. USA 111, 14019–14026 (2014).
Golubev, A., Hanson, A. D. & Gladyshev, V. N. Non-enzymatic molecular damage as a prototypic driver of aging. J. Biol. Chem. 292, 6029–6038 (2017).
Kun, A., Papp, B. & Szathmary, E. Computational identification of obligatorily autocatalytic replicators embedded in metabolic networks. Genome Biol. 9, R51 (2008).
Schmidt, R. et al. Computing autocatalytic sets to unravel inconsistencies in metabolic network reconstructions. Bioinformatics 31, 373–381 (2015).
Baba, T. et al. Construction of Escherichia coli K-12 in-frame, single-gene knockout mutants: the Keio collection. Mol. Syst. Biol. 2, 2006.0008 (2006).
Mülleder, M. et al. A prototrophic deletion mutant collection for yeast metabolomics and systems biology. Nat. Biotechnol. 30, 1176–1178 (2012).
Tannler, S., Decasper, S. & Sauer, U. Maintenance metabolism and carbon fluxes in Bacillus species. Microb. Cell Fact. 7, 19 (2008).
Baganz, F. et al. Suitability of replacement markers for functional analysis studies in Saccharomyces cerevisiae. Yeast 13, 1563–1573 (1997).
Bolten, C. J. et al. Sampling for metabolome analysis of microorganisms. Anal. Chem. 79, 3843–3849 (2007).
Müller, J. E. et al. Core pathways operating during methylotrophy of Bacillus methanolicus MGA3 and induction of a bacillithiol-dependent detoxification pathway upon formaldehyde stress. Mol. Microbiol. 98, 1089–1100 (2015).
Rabinowitz, J. D. & Kimball, E. Acidic acetonitrile for cellular metabolome extraction from Escherichia coli. Anal. Chem. 79, 6167–6173 (2007).
Campbell, K., Vowinckel, J., Keller, M. A. & Ralser, M. Methionine metabolism alters oxidative stress resistance via the pentose phosphate pathway. Antioxid. Redox Signal. 24, 543–547 (2016).
Kiefer, P., Delmotte, N. & Vorholt, J. A. Nanoscale ion-pair reversed-phase HPLC-MS for sensitive metabolome analysis. Anal. Chem. 83, 850–855 (2011).
Mashego, M. R. et al. MIRACLE: mass isotopomer ratio analysis of U-13C-labeled extracts. A new method for accurate quantification of changes in concentrations of intracellular metabolites. Biotechnol. Bioeng. 85, 620–628 (2004).
Kiefer, P., Schmitt, U. & Vorholt, J. A. eMZed: an open source framework in Python for rapid and interactive development of LC/MS data analysis workflows. Bioinformatics 29, 963–964 (2013).
Moseley, H. N. Correcting for the effects of natural abundance in stable isotope resolved metabolomics experiments involving ultra-high resolution mass spectrometry. BMC Bioinformatics 11, 139 (2010).
Haug, K. et al. MetaboLights—an open-access general-purpose repository for metabolomics studies and associated meta-data. Nucleic Acids Res. 41, D781–D786 (2013).
Acknowledgements
The authors thank U. Sauer and J.-C. Portais for discussions and comments on the manuscript and P. Christen for technical support with LC-MS. This work was supported by the Swiss National Science Foundation (grant no. 31003A-173094) and ETH Zurich.
Author information
Authors and Affiliations
Contributions
J.H., P.K., F.M. and J.A.V. planned the project and designed the experiments. J.H. and F.M. performed the experiments. J.H. and P.K. set-up LC-MS methods and data analysis. J.H. performed measurements and data analysis. J.H. and J.A.V. wrote the manuscript with input from all authors.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing financial interests.
Supplementary information
Supplementary Information
Supplementary Figures 1-9, Supplementary Tables 1-6, Supplementary Methods, Supplementary References. (PDF 2291 kb)
Supplementary Data 1
Excel file containing analysed LC-MS and LC-MS/MS data from a long-term dynamic labelling switch experiment (12C to 13C) with Escherichia coli. (XLSX 301 kb)
Supplementary Data 2
Excel file containing analysed LC-MS and LC-MS/MS data from a long-term dynamic labelling switch experiment (13C to 12C) with Escherichia coli. (XLSX 301 kb)
Supplementary Data 3
Excel file containing analysed LC-MS and LC-MS/MS data from a long-term dynamic labelling switch experiment (12C to 13C) with Escherichia coli Δfdx. (XLSX 315 kb)
Supplementary Data 4
Excel file containing analysed LC-MS and LC-MS/MS data from a long-term dynamic labelling switch experiment (12C to 13C) with Escherichia coli. (XLSX 283 kb)
Supplementary Data 5
Excel file containing analysed LC-MS and LC-MS/MS data from a long-term dynamic labelling switch experiment (12C to 13C) with Saccharomyces cerevisiae. (XLSX 199 kb)
Rights and permissions
About this article
Cite this article
Hartl, J., Kiefer, P., Meyer, F. et al. Longevity of major coenzymes allows minimal de novo synthesis in microorganisms. Nat Microbiol 2, 17073 (2017). https://doi.org/10.1038/nmicrobiol.2017.73
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/nmicrobiol.2017.73
This article is cited by
-
Metabolic adaptation to vitamin auxotrophy by leaf-associated bacteria
The ISME Journal (2022)
-
The polar oxy-metabolome reveals the 4-hydroxymandelate CoQ10 synthesis pathway
Nature (2021)
-
Inhibiting Mycobacterium tuberculosis CoaBC by targeting an allosteric site
Nature Communications (2021)
-
Reciprocal growth control by competitive binding of nucleotide second messengers to a metabolic switch in Caulobacter crescentus
Nature Microbiology (2020)
-
Untargeted metabolomics links glutathione to bacterial cell cycle progression
Nature Metabolism (2020)