Abstract
4′-Phosphopantetheinylation is an essential posttranslational modification of the primary and secondary metabolic pathways in prokaryotes and eukaryotes. Several peptide-based natural products are biosynthesized by large, multifunctional enzymes known as nonribosomal peptide synthetases (NRPSs), responsible for producing virulence factors and many pharmaceuticals. The thiolation (T) domain serves as a covalent tether for substrates and intermediates in nonribosomal peptide biosynthesis and must be posttranslationally modified with a 4′-phosphopantetheinyl group. To detect 4′-phosphopantetheinylation of NRPS in bacterial proteomes, we developed a 5′-(vinylsulfonylaminodeoxy)adenosine scaffold with a clickable functionality, enabling effective chemical labeling of 4′-phosphopantethylated NRPSs. In this chapter, we describe the design and synthesis of an activity-based protein profiling probe and summarize our work toward developing a series of protocols for the labeling and visualization of 4′-phosphopantetheinylation of endogenous NRPSs in complex proteomes.
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References
Beld J, Sonnenschein EC, Vickery CR et al (2014) The phosphopantetheinyl transferases: catalysis of a post-translational modification crucial for life. Nat Prod Rep 31:61–108
Hur GH, Vickery CR, Burkart MD (2012) Explorations of catalytic domains in non- ribosomal peptide synthetase enzymology. Nat Prod Rep 10:1074–1098
Minandri F, Imperi F, Frangipani E et al (2016) Role of iron systems in Pseudomonas aeruginosa virulenceand airway infection. Infect Immum 84:2324–2335
Leblanc C, Prudhomme T, Tabouret G et al (2012) 4′-Phosphopantetheunyl transferase PptT, a new drug target required for Mycobacterium tuberculosis growth and persistence in vivo. PLoS Pathog 8:e10003097
Kasai S, Ishikawa F, Suzuki T et al (2016) A chemical proteomic probe for detecting native carrier protein motifs in nonribosomal peptide synthetases. Chem Commun 52:14129–14132
Qiao C, Wilson DJ, Bennett EM et al (2007) A mechanism-based aryl carrier protein/thiolation domain affinity probe. J Am Chem Soc 129:6350–6351
Sundlov JA, Shi C, Wilson DJ et al (2012) Structural and functional investigation of the intermolecular interaction between NRPS adenylation and carrier protein domains. Chem Biol 19:188–198
Konno S, Ishikawa F, Suzuki T et al (2015) Active site-directed proteomic probes for adenylation domains in nonribosomal peptide synthetases. Chem Commun 51:2262–2265
Ishikawa F, Konno S, Suzuki T et al (2015) Profiling nonribosomal peptide synthetase activities using chemical proteomic probes for adenylation domains. ACS Chem Biol 10:1989–1997
Ishikawa F, Kasai S, Kakeya H et al (2017) Visualizing the adenylation activities and protein-protein interactions of aryl acid adenylating enzymes. Chembiochem 18:2199–2204
Cravatt BF, Wright AT, Kozarich JW (2008) Activity-based protein profiling: from enzyme chemistry to proteomic chemistry. Annu Rev Biochem 77:383–414
Sanman LE, Bogyo M (2014) Activity-based profiling of proteases. Annu Rev Biochem 83:249–273
Rivero MR, Alonso I, Carretero JC (2004) Vinyl sulfoxides as stereochemical controllers in intermolecular Pauson-Khand reactions: applications to the enantioselective synthesis of natural cyclopentanoids. Chem Eur J 10:5443–5459
Skiles JW, Miao C, Sorcek R et al (1992) Inhibition of human leukocytoelastase by N-substituted peptides containing α,α-difluorostatone residues at P1. J Med Chem 35:4795–4808
Reuter DC, McIntosh JE, Guinn AC et al (2003) Synthesis of vinyl sulfonamides using the Horner reaction. Synthesis 15:2321–2324
Wei Y-H, Wang L-F et al (2004) Optimization iron supplement strategies for enhanced surfactin production with Bacillus subtilis. Biotechnol Prog 20:979–983
Yeh M-S, Wei Y-H, Chang JS (2005) Enhanced production of surfactin from Bacillus subtilis by addition of solid carriers. Biotechnol Prog 21:1329–1334
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72:248–254
Augenstein DC, Thrasher KD, Sinskey AJ et al (1974) Optimization in the recovery of a labile intracellular enzyme. Biotechnol Bioeng 16:1433–1447
Acknowledgments
This work was partly supported by Grants-in-Aid for Research (C) (19 K05722 to F.I.) from JSPS and by grants from the Noda Institute for Scientific Research (F.I.), Research Foundation for Pharmaceutical Sciences (F.I.), Japan Foundation for Applied Enzymology (F.I.), and Institute Foundation for Science, Osaka (IFO) (F.I.). We are also thankful for the financial support provided by the Antiaging Project for Private Universities.
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Ishikawa, F., Tanabe, G. (2023). Chemical Labeling of Protein 4′-Phosphopantetheinylation in Surfactin-Producing Nonribosomal Peptide Synthetases. In: Burkart, M., Ishikawa, F. (eds) Non-Ribosomal Peptide Biosynthesis and Engineering. Methods in Molecular Biology, vol 2670. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3214-7_15
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DOI: https://doi.org/10.1007/978-1-0716-3214-7_15
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