Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics

Abstract

Pyridoxal phosphate (PLP) is an enzyme cofactor required for the chemical transformation of biological amines in many central cellular processes. PLP-dependent enzymes (PLP-DEs) are ubiquitous and evolutionarily diverse, making their classification based on sequence homology challenging. Here we present a chemical proteomic method for reporting on PLP-DEs using functionalized cofactor probes. We synthesized pyridoxal analogues modified at the 2′-position, which are taken up by cells and metabolized in situ. These pyridoxal analogues are phosphorylated to functional cofactor surrogates by cellular pyridoxal kinases and bind to PLP-DEs via an aldimine bond which can be rendered irreversible by NaBH4 reduction. Conjugation to a reporter tag enables the subsequent identification of PLP-DEs using quantitative, label-free mass spectrometry. Using these probes we accessed a significant portion of the Staphylococcus aureus PLP-DE proteome (73%) and annotate uncharacterized proteins as novel PLP-DEs. We also show that this approach can be used to study structural tolerance within PLP-DE active sites and to screen for off-targets of the PLP-DE inhibitor d-cycloserine.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

Fig. 1: Design and synthesis of PL probes.
Fig. 2: Biochemical characterization of PL probes.
Fig. 3: Proteomic profiling with PL2 shows significant enrichment of diverse PLP-DEs.
Fig. 4: Growth medium supplementation with PL1 and PL3 probes enables broad PLPome coverage with proteomic analysis.
Fig. 5: Validation of known and uncharacterized PLP-DEs by UV–vis, MS and gel-based analysis.
Fig. 6: Applications of the PLP labelling method for profiling structural accommodation within PLP-binding sites and identifying PLP-DE off-targets of d-cycloserine.

Data availability

Supplementary information, chemical characterization and proteomic data analysis are available in the online version of the paper. Crystallographic data of alanine racemase structures have been deposited in the Protein Data Bank68 (www.rcsb.org) under PDB codes 6G56, 6G58 and 6G59. The proteomic MS data (raw data and MaxQuant output tables for protein groups and peptides) have been deposited in the ProteomeXchange Consortium69 (http://proteomecentral.proteomexchange.org) via the PRIDE70 partner repository (data set identifier: PXD006483).

References

  1. 1.

    Amadasi, A. et al. Pyridoxal 5′-phosphate enzymes as targets for therapeutic agents. Curr. Med. Chem. 14, 1291–1324 (2007).

    CAS  Article  Google Scholar 

  2. 2.

    Salvo, Di, M. L, Budisa, N. & Contestabile, R. Beilstein Bozen Symposium on Molecular Engineering and Control. (Beilstein Institut: Frankfurt am Main, 2013) 27–66.

  3. 3.

    Eliot, A. C. & Kirsch, J. F. Pyridoxal phosphate enzymes: mechanistic, structural, and evolutionary considerations. Annu. Rev. Biochem. 73, 383–415 (2004).

    CAS  Article  Google Scholar 

  4. 4.

    Toney, M. D. Reaction specificity in pyridoxal phosphate enzymes. Arch. Biochem. Biophys. 433, 279–287 (2005).

    CAS  Article  Google Scholar 

  5. 5.

    Percudani, R. & Peracchi, A. The B6 database: a tool for the description and classification of vitamin B6-dependent enzymatic activities and of the corresponding protein families. BMC Bioinformatics 10, 273 (2009).

    Article  Google Scholar 

  6. 6.

    Percudani, R. & Peracchi, A. A genomic overview of pyridoxal-phosphate-dependent enzymes. EMBO Rep. 4, 850–854 (2003).

    CAS  Article  Google Scholar 

  7. 7.

    Kappes, B., Tews, I., Binter, A. & Macheroux, P. PLP-dependent enzymes as potential drug targets for protozoan diseases. Biochim. Biophys. Acta 1814, 1567–1576 (2011).

    CAS  Article  Google Scholar 

  8. 8.

    Mehta, P. K. & Christen, P. The molecular evolution of pyridoxal-5′-phosphate-dependent enzymes. Adv. Enzymol. Relat. Areas Mol. Biol. 74, 129–184 (2000).

    CAS  PubMed  Google Scholar 

  9. 9.

    Catazaro, J., Caprez, A., Guru, A., Swanson, D. & Powers, R. Functional evolution of PLP-dependent enzymes based on active-site structural similarities. Proteins 82, 2597–2608 (2014).

    CAS  Article  Google Scholar 

  10. 10.

    Denessiouk, K. A., Denesyuk, A. I., Lehtonen, J. V., Korpela, T. & Johnson, M. S. Common structural elements in the architecture of the cofactor-binding domains in unrelated families of pyridoxal phosphate-dependent enzymes. Proteins 35, 250–261 (1999).

    CAS  Article  Google Scholar 

  11. 11.

    Fleischman, N. M. et al. Molecular characterization of novel pyridoxal-5′-phosphate-dependent enzymes from the human microbiome. Protein Sci. 23, 1060–1076 (2014).

    CAS  Article  Google Scholar 

  12. 12.

    Whittaker, M. M., Penmatsa, A. & Whittaker, J. W. The Mtm1p carrier and pyridoxal 5′-phosphate cofactor trafficking in yeast mitochondria. Arch. Biochem. Biophys. 568, 64–70 (2015).

    CAS  Article  Google Scholar 

  13. 13.

    Simon, E. S. & Allison, J. Determination of pyridoxal-5′-phosphate (PLP)-bonding sites in proteins: a peptide mass fingerprinting approach based on diagnostic tandem mass spectral features of PLP-modified peptides. Rapid Commun. Mass Spectrom. 23, 3401–3408 (2009).

    CAS  Article  Google Scholar 

  14. 14.

    Wu, Y Chen, J., Liu, Z. & Wang, F. Identification of pyridoxal phosphate modified proteins using mass spectrometry. Rapid Commun. Mass Spectrom. 32, 195–200 2018).

    CAS  Article  Google Scholar 

  15. 15.

    Schnackerz, K. D. & Cook, P. F. Resolution of pyridoxal 5′-phosphate from O-acetylserine sulfhydrylase from Salmonella typhimurium and reconstitution of apoenzyme with cofactor and cofactor analogues as a probe of the cofactor binding site. Arch. Biochem. Biophys. 324, 71–77 (1995).

    CAS  Article  Google Scholar 

  16. 16.

    Morino, Y. & Snell, E. E. Coenzyme activity of homologues of pyridoxal phosphate. Biochemistry 57, 1692–1699 (1967).

    CAS  Google Scholar 

  17. 17.

    Mechanik, M. L., Torchinsky, Y. M., Florentiev, V. L. & Karpeisky, M. Y. Interaction of the apoenzyme of l-glutamate decarboxylase with pyridoxal phosphate analogues. FEBS Lett. 13, 177–180 (1971).

    Article  Google Scholar 

  18. 18.

    Cravatt, B. F., Wright, A. T. & Kozarich, J. W. Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu. Rev. Biochem. 77, 383–414 (2008).

    CAS  Article  Google Scholar 

  19. 19.

    Korytnyk, W., Srivastava, S. C., Angelino, N., Potti, P. G. & Paul, B. A general method for modifying the 2-methyl group of pyridoxol. Synthesis and biological activity of 2-vinyl- and 2-ethynylpyridoxols and related compounds. J. Med. Chem. 16, 1096–1101 1973).

    CAS  Article  Google Scholar 

  20. 20.

    Kaiser, E. M. et al. Regiointegrity of carbanions derived by selective metalations of dimethylpyridines and -quinolines. J. Organomet. Chem. 213, 405–417 (1981).

    CAS  Article  Google Scholar 

  21. 21.

    Kim, Y.-C. & Jacobson, K. A. Versatile synthesis of 6-alkyl and aryl substituted pyridoxal derivatives. Synthesis 1, 119–122 (2000).

    CAS  Article  Google Scholar 

  22. 22.

    Korytnyk, W. & Ahrens, H. 5-homopyridoxals, 5-thiopyridoxal, and related compounds. Synthesis, tautomerism, and biological properties. J. Med. Chem. 14, 947–952 1971).

    CAS  Article  Google Scholar 

  23. 23.

    di Salvo, M. L., Contestabile, R. & Safo, M. K. Vitamin B6 salvage enzymes: mechanism, structure and regulation. Biochim. Biophys. Acta 1814, 1597–1608 (2011).

    Article  Google Scholar 

  24. 24.

    Denesyuk, A. I., Denessiouk, K. A., Korpela, T. & Johnson, M. S. Functional attributes of the phosphate group binding cup of pyridoxal phosphate-dependent enzymes. J. Mol. Biol. 316, 155–172 (2002).

    CAS  Article  Google Scholar 

  25. 25.

    Nodwell, M. B., Koch, M. F., Alte, F., Schneider, S. & Sieber, S. A. A subfamily of bacterial ribokinases utilizes a hemithioacetal for pyridoxal phosphate salvage. J. Am. Chem. Soc. 136, 4992–4999 (2014).

    CAS  Article  Google Scholar 

  26. 26.

    Strominger, J. L., Ito, I. & Threnn, R. H. Competitive inhibition of enzymatic reactions by oxamycin. J. Am. Chem. Soc. 82, 998–999 (1960).

    CAS  Article  Google Scholar 

  27. 27.

    Griswold, W. R. & Toney, M. D. Role of the pyridine nitrogen in pyridoxal 5′-phosphate catalysis: activity of three classes of PLP enzymes reconstituted with deazapyridoxal 5′-phosphate. J. Am. Chem. Soc. 133, 14823–14830 (2011).

    CAS  Article  Google Scholar 

  28. 28.

    Scaletti, E. R., Luckner, S. R. & Krause, K. L. Structural features and kinetic characterization of alanine racemase from Staphylococcus aureus (Mu50). Acta Crystallogr. D 68, 82–92 (2012).

    CAS  Article  Google Scholar 

  29. 29.

    Rostovtsev, V. V., Green, L. G., Fokin, V. V. & Sharpless, K. B. A stepwise huisgen cycloaddition process: copper(I)-catalyzed regioselective ‘ligation’ of azides and terminal alkynes. Angew. Chem. Int. Ed. 41, 2596–2599 (2002).

    CAS  Article  Google Scholar 

  30. 30.

    Speers, A. E. & Cravatt, B. F. Profiling enzyme activities in vivo using click chemistry methods. Chem. Biol. 11, 535–546 (2004).

    CAS  Article  Google Scholar 

  31. 31.

    Tornoe, C. W., Christensen, C. & Meldal, M. Peptidotriazoles on solid phase: [1,2,3]-triazoles by regiospecific copper(I)-catalyzed 1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org. Chem. 67, 3057–3064 (2002).

    CAS  Article  Google Scholar 

  32. 32.

    Mukherjee, T., Hanes, J., Tews, I., Ealick, S. E. & Begley, T. P. Pyridoxal phosphate: biosynthesis and catabolism. Biochim. Biophys. Acta 1814, 1585–1596 (2011).

    CAS  Article  Google Scholar 

  33. 33.

    Fey, P. D.et al. A genetic resource for rapid and comprehensive phenotype screening of nonessential Staphylococcus aureus genes. mBio 4, e00537-12 (2013).

    Article  Google Scholar 

  34. 34.

    Cox, J. et al. Accurate proteome-wide label-free quantification by delayed normalization and maximal peptide ratio extraction, termed MaxLFQ. Mol. Cell. Proteomics. 13, 2513–2526 (2014).

    CAS  Article  Google Scholar 

  35. 35.

    Saxon, E. & Bertozzi, C. R. Cell surface engineering by a modified Staudinger reaction. Science 287, 2007–2010 (2000).

    CAS  Article  Google Scholar 

  36. 36.

    Ito, T. et al. Conserved pyridoxal protein that regulates Ile and Val metabolism. J. Bacteriol. 195, 5439–5449 (2013).

    CAS  Article  Google Scholar 

  37. 37.

    Prunetti, L. et al. Evidence that COG0325 proteins are involved in PLP homeostasis. Microbiology 162, 694–706 (2016).

    CAS  Article  Google Scholar 

  38. 38.

    Mozzarelli, A. & Bettati, S. Exploring the pyridoxal 5′-phosphate-dependent enzymes. Chem. Rec. 6, 275–287 (2006).

    CAS  Article  Google Scholar 

  39. 39.

    Finn, R. D. et al. InterPro in 2017—beyond protein family and domain annotations. Nucleic Acids Res. 45, D190–D199 (2017).

    CAS  Article  Google Scholar 

  40. 40.

    Kuhn, M. L., Majorek, K. A., Minor, W. & Anderson, W. F. Broad-substrate screen as a tool to identify substrates for bacterial Gcn5-related N-acetyltransferases with unknown substrate specificity. Protein Sci. 22, 222–230 (2013).

    CAS  Article  Google Scholar 

  41. 41.

    Mukherjee, J. J. & Dekker, E. E. Purification, properties, and N-terminal amino acid sequence of homogeneous Escherichia coli 2-amino-3-ketobutyrate CoA ligase, a pyridoxal phosphate-dependent enzyme. J. Biol. Chem. 262, 14441–14447 (1987).

    CAS  PubMed  Google Scholar 

  42. 42.

    Ye, Q. Z., Liu, J. & Walsh, C. T. p-Aminobenzoate synthesis in Escherichia coli: purification and characterization of PabB as aminodeoxychorismate synthase and enzyme X as aminodeoxychorismate lyase. Proc. Natl Acad. Sci. USA 87, 9391–9395 (1990).

    CAS  Article  Google Scholar 

  43. 43.

    Soper, T. S. & Manning, J. M. Different modes of action of inhibitors of bacterial d-amino acid transaminase. A target enzyme for the design of new antibacterial agents. J. Biol. Chem. 256, 4263–4268 (1981).

    CAS  PubMed  Google Scholar 

  44. 44.

    Malashkevich, V. N., Strop, P., Keller, J. W., Jansonius, J. N. & Toney, M. D. Crystal structures of dialkylglycine decarboxylase inhibitor complexes. J. Mol. Biol. 294, 193–200 (1999).

    CAS  Article  Google Scholar 

  45. 45.

    Yew, W. W., Wong, C. F., Wong, P. C., Lee, J. & Chau, C. H. Adverse neurological reactions in patients with multidrug-resistant pulmonary tuberculosis after coadministration of cycloserine and ofloxacin. Clin. Infect. Dis. 17, 288–289 (1993).

    CAS  Article  Google Scholar 

  46. 46.

    Caminero, J. A., Sotgiu, G., Zumla, A. & Migliori, G. B. Best drug treatment for multidrug-resistant and extensively drug-resistant tuberculosis. Lancet Infect. Dis. 10, 621–629 (2010).

    CAS  Article  Google Scholar 

  47. 47.

    Neuhaus, F. C. Selective inhibition of enzymes utilizing alanine in the biosynthesis of peptidoglycan. Antimicrob. Agents Chemother. 7, 304–313 (1967).

    CAS  PubMed  Google Scholar 

  48. 48.

    Peisach, D., Chipman, D. M., Van Ophem, P. W., Manning, J. M. & Ringe, D. d-Cycloserine inactivation of d-amino acid aminotransferase leads to a stable noncovalent protein complex with an aromatic cycloserine-PLP derivative. J. Am. Chem. Soc. 120, 2268–2274 (1998).

    CAS  Article  Google Scholar 

  49. 49.

    Fenn, T. D., Stamper, G. F., Morollo, A. A. & Ringe, D. A side reaction of alanine racemase: transamination of cycloserine. Biochemistry 42, 5775–5783 (2003).

    CAS  Article  Google Scholar 

  50. 50.

    Sieradzki, K. & Tomasz, A. Suppression of beta-lactam antibiotic resistance in a methicillin-resistant Staphylococcus aureus through synergic action of early cell wall inhibitors and some other antibiotics. J. Antimicrob. Chemother. 39, 47–51 (1997).

    CAS  Article  Google Scholar 

  51. 51.

    Roze, U. & Strominger, J. L. Alanine racemase from Staphylococcus aureus: conformation of its substrates and its inhibitor, d-cycloserine. Mol. Pharm. 2, 92–94 (1966).

    CAS  Google Scholar 

  52. 52.

    Lambert, M. P. & Neuhaus, F. C. Mechanism of d-cycloserine action: alanine racemase from Escherichia coli W. J. Bacteriol. 110, 978–987 (1972).

    CAS  PubMed  PubMed Central  Google Scholar 

  53. 53.

    Prosser, G. A. & de Carvalho, L. P. Metabolomics reveal d-alanine:d-alanine ligase as the target of d-cycloserine in Mycobacterium tuberculosis. ACS Med. Chem. Lett. 4, 1233–1237 (2013).

    CAS  Article  Google Scholar 

  54. 54.

    Halouska, S. et al. Metabolomics analysis identifies d-alanine-d-lanine ligase as the primary lethal target of d-cycloserine in mycobacteria. J. Proteome Res. 13, 1065–1076 (2014).

    CAS  Article  Google Scholar 

  55. 55.

    Contestabile, R. et al. l-Threonine aldolase, serine hydroxymethyltransferase and fungal alanine racemase. A subgroup of strictly related enzymes specialized for different functions. Eur. J. Biochem. 268, 6508–6525 (2001).

    CAS  Article  Google Scholar 

  56. 56.

    di Salvo, M. L. et al. Alanine racemase from Tolypocladium inflatum: a key PLP-dependent enzyme in cyclosporin biosynthesis and a model of catalytic promiscuity. Arch. Biochem. Biophys. 529, 55–65 (2013).

    Article  Google Scholar 

  57. 57.

    Takenaka, T., Ito, T., Miyahara, I., Hemmi, H. & Yoshimura, T. A new member of MocR/GabR-type PLP-binding regulator of d-alanyl-d-alanine ligase in Brevibacillus brevis. FEBS J. 282, 4201–4217 (2015).

    CAS  Article  Google Scholar 

  58. 58.

    Kleiner, P., Heydenreuter, W., Stahl, M., Korotkov, V. S. & Sieber, S. A. A whole proteome inventory of background photocrosslinker binding. Angew. Chem. Int. Ed. 56, 1396–1401 (2017).

    CAS  Article  Google Scholar 

  59. 59.

    Anderson, L. N. et al. Live cell discovery of microbial vitamin transport and enzyme–cofactor interactions. ACS Chem. Biol. 11, 345–354 (2016).

    CAS  Article  Google Scholar 

  60. 60.

    Broncel, M., Serwa, R. A., Bunney, T. D., Katan, M. & Tate, E. W. Global profiling of Huntingtin-associated protein E (HYPE)-mediated AMPylation through a chemical proteomic approach. Mol. Cell. Proteomics 15, 715–725 (2016).

    CAS  Article  Google Scholar 

  61. 61.

    Grammel, M. & Hang, H. C. Chemical reporters for biological discovery. Nat. Chem. Biol. 9, 475–484 (2013).

    CAS  Article  Google Scholar 

  62. 62.

    Romine, M. F. et al. Elucidation of roles for vitamin B12 in regulation of folate, ubiquinone, and methionine metabolism. Proc. Natl Acad. Sci. USA 114, E1205–E1214 (2017).

    CAS  Article  Google Scholar 

  63. 63.

    Westcott, N. P., Fernandez, J. P., Molina, H. & Hang, H. C. Chemical proteomics reveals ADP-ribosylation of small GTPases during oxidative stress. Nat. Chem. Biol. 13, 302–308 (2017).

    CAS  Article  Google Scholar 

  64. 64.

    Wright, M. H. et al. Validation of N-myristoyltransferase as an antimalarial drug target using an integrated chemical biology approach. Nat. Chem. 6, 112–121 (2014).

    CAS  Article  Google Scholar 

  65. 65.

    Eirich, J. et al. Pretubulysin derived probes as novel tools for monitoring the microtubule network via activity-based protein profiling and fluorescence microscopy. Mol. Biosyst. 8, 2067–2075 (2012).

    CAS  Article  Google Scholar 

  66. 66.

    Tyanova, S., Temu, T. & Cox, J. The MaxQuant computational platform for mass spectrometry-based shotgun proteomics. Nat. Protoc. 11, 2301–2319 (2016).

    CAS  Article  Google Scholar 

  67. 67.

    Cox, J. et al. Andromeda: a peptide search engine integrated into the MaxQuant environment. J. Proteome Res. 10, 1794–1805 (2011).

    CAS  Article  Google Scholar 

  68. 68.

    Berman, H. M. et al. The Protein Data Bank. Nucleic Acids Res. 28, 235–242 (2000).

    CAS  Article  Google Scholar 

  69. 69.

    Vizcaino, J. A. et al. ProteomeXchange provides globally coordinated proteomics data submission and dissemination. Nat. Biotechnol. 32, 223–226 (2014).

    CAS  Article  Google Scholar 

  70. 70.

    Vizcaino, J. A. et al. 2016 update of the PRIDE database and its related tools. Nucleic Acids Res. 44, 11033 (2016).

    CAS  Article  Google Scholar 

Download references

Acknowledgements

This project received funding from the European Research Council (ERC) and the European Union’s Horizon 2020 research and innovation programme (grant agreement no. 725085, CHEMMINE, ERC consolidator grant). Further financial support includes doctoral scholarships to A.H. from the Deutscher Akademischer Austausch Dienst (DAAD) and to M.S. from the Studienstiftung des Deutschen Volkes. The authors thank the Network on Antimicrobial Resistance in Staphylococcus aureus (NARSA) for the supply of the Nebraska Transposon Mutant Library (NTML). The authors also thank M. Wolff and K. Bäuml for technical assistance, the Swiss Light Source (SLS) and European Synchrotron Radiation Facility (ESRF) for beam time, and the staff of beamlines PX I (SLS), ID23-2 and ID29 (ESRF) for setting up of the beamlines for data collection. The authors thank M. H. Wright and B. M. Williams for critical proofreading of the manuscript.

Author information

Affiliations

Authors

Contributions

A.H., M.B.N. and S.A.S. conceived and designed the project. A.H., M.B.N. and M.P. synthesized PL probes. A.H., M.B.N. and M.P. conducted biochemical characterization of probes and PLP-DEs, including purification of recombinant proteins, enzyme kinetics assays, UV–vis measurements and in vitro protein labelling experiments (MS and gel-based). S.S. performed crystallization and determined X-ray structures of Alr. A.H. completed cell-based labelling experiments and growth curves. V.C.K. prepared and analysed targeted metabolomic samples and characterized JW8. A.H. designed, executed and analysed proteomic experiments. N.C.B. and M.S. contributed proteomics expertise and analysed PLP binding sites. A.H. and S.A.S. wrote the manuscript.

Corresponding author

Correspondence to Stephan A. Sieber.

Ethics declarations

Competing interests

The authors declare no competing interests.

Additional information

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Supplementary information

Supplementary Information

Supplementary Data, Supplementary Schemes, Supplementary Figures and Supplementary Methods and Protocols

Proteomics tables and binding-site identification

Supplementary proteomics tables and identification of binding-sites

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Hoegl, A., Nodwell, M.B., Kirsch, V.C. et al. Mining the cellular inventory of pyridoxal phosphate-dependent enzymes with functionalized cofactor mimics. Nature Chem 10, 1234–1245 (2018). https://doi.org/10.1038/s41557-018-0144-2

Download citation

Further reading

Search

Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing