Abstract
Adenylation is a crucial enzymatic process in the biosynthesis of nonribosomal peptide synthetase (NRPS) derived natural products. Adenylation domains are considered the gatekeepers of NRPSs since they select, activate, and load the carboxylic acid substrate onto a downstream peptidyl carrier protein (PCP) domain of the NRPS. We describe a coupled continuous kinetic assay for NRPS adenylation domains that substitutes the PCP domain with hydroxylamine as the acceptor molecule. The pyrophosphate released from the first-half reaction is then measured using a two-enzyme coupling system, which detects conversion of the chromogenic substrate 7-methylthioguanosine (MesG) to 7-methylthioguanine. From profiling substrate specificity of unknown or engineered adenylation domains to studying chemical inhibition of adenylating enzymes, this robust assay will be of widespread utility in the broad field NRPS enzymology.
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References
Schmelz S, Naismith JH (2009) Adenylate-forming enzymes. Curr Opin Struct Biol 19:666–671
Gulick AM (2009) Conformational dynamics in the acyl-CoA synthetases, adenylation domains of non-ribosomal peptide synthetases, and firefly luciferase. ACS Chem Biol 4:811–827
Hur GH, Vickery CR, Burkart MD (2012) Explorations of catalytic domains in non-ribosomal peptide synthetase enzymology. Nat Prod Rep 29:1074–1098
Phelan VV, Du Y, McLean JA, Bachmann BO (2009) Adenylation enzyme characterization using γ-18O4-ATP pyrophosphate exchange. Chem Biol 16:473–478
Kadi N, Challis GL (2009) Chapter 17 siderophore biosynthesis: a substrate specificity assay for nonribosomal peptide synthetase-independent siderophore synthetases involving trapping of acyl-adenylate intermediates with hydroxylamine. Methods Enzymol 458:431–457
Wilson DJ, Aldrich CC (2010) A continuous kinetic assay for adenylation enzyme activity and inhibition. Anal Biochem 404:56–63
Ehmann DE, Shaw-Reid CA, Losey HC et al (2000) The EntF and EntE adenylation domains of Escherichia coli enterobactin synthetase: sequestration and selectivity in acyl-AMP transfers to thiolation domain cosubstrates. Proc Natl Acad Sci U S A 97:2509–2514
McQuade TJ, Shallop AD, Sheoran A et al (2009) A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis. Anal Biochem 386:244–250
Webb MR (1992) A continuous spectrophotometric assay for inorganic phosphate and for measuring phosphate release kinetics in biological systems. Proc Natl Acad Sci U S A 89:4884–4887
Upson RH, Huagland RP, Malekzadeh MN et al (1996) A spectrophotometric method to measure enzymatic activity in reactions that generate inorganic pyrophosphate. Anal Biochem 243:41–45
Stachelhaus T, Marahiel M (1995) Modular structure of peptide synthetases revealed by dissection of the multifunctional enzyme GrsA. J Biol Chem 270:6163–6169
Acknowledgement
C.C.A. acknowledges membership in and support from the Region V “Great Lakes” Regional Center of Excellence in Biodefense and Emerging Infectious Diseases Consortium (National Institutes of Health award 1-U54-AI-057153) and support from R01-AI-070219 for this work.
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Duckworth, B.P., Wilson, D.J., Aldrich, C.C. (2016). Measurement of Nonribosomal Peptide Synthetase Adenylation Domain Activity Using a Continuous Hydroxylamine Release Assay. In: Evans, B. (eds) Nonribosomal Peptide and Polyketide Biosynthesis. Methods in Molecular Biology, vol 1401. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-3375-4_3
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DOI: https://doi.org/10.1007/978-1-4939-3375-4_3
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Publisher Name: Humana Press, New York, NY
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