title: Online Pyrophosphate Assay for Analyzing Adenylation Domains of Nonribosomal Peptide Synthetases.
abstract: Nonribosomal peptide synthetases (NRPSs) produce many important and structurally complex natural products. Because of their architectures, reprogramming NRPSs has long been attempted to access new bioactive compounds. However, detailed characterization of NRPS catalysis and substrate selectivity by adenylation (A) domains is needed to support such efforts. We present a simple coupled NADH/pyrophosphate (PPi  ) detection assay for analyzing A domain catalysis in vitro. PPi formation is coupled to the consumption of NADH by four enzymatic steps and is detected spectroscopically (λ=340 nm) for simple analysis. We demonstrate the effectiveness of this assay with several adenylation domains, including a stand-alone A domain (DltA, cell wall biosynthesis) and an embedded A domain (Tcp10, teicoplanin biosynthesis). Substrate acceptance of the Tcp10 A domain was explored for the first time, thus demonstrating the applicability of the assay for complex, multi-domain NRPSs.
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Tiia
authors.initials: T
authors.lastname: Kittilä
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany.
authors.forename: Melanie
authors.initials: M
authors.lastname: Schoppet
authors.affiliation: Department of Biomolecular Mechanisms, Max Planck Institute for Medical Research, Jahnstrasse 29, 69120 Heidelberg, Germany. max.cryle@monash.edu.|EMBL Australia, Monash University, Clayton, Victoria, 3800, Australia. max.cryle@monash.edu.|The Department of Biochemistry and Molecular Biology and, ARC Centre of Excellence in Advanced Molecular Imaging, Monash University, 15 Innovation Walk, Clayton, Victoria, 3800, Australia. max.cryle@monash.edu.
authors.forename: Max J
authors.identifier: http://orcid.org/0000-0002-9739-6157
authors.initials: MJ
authors.lastname: Cryle
mesh_terms: D000249:Adenosine Monophosphate / Q000032:analysis / Q000737:chemistry; D020134:Catalytic Domain; D011756:Diphosphates / Q000737:chemistry; D057075:Enzyme Assays / Q000379:methods*; D007700:Kinetics; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011506:Proteins / Q000737:chemistry; D017334:Teicoplanin / Q000737:chemistry
chemical_list: D011756:Diphosphates; D011506:Proteins; C533392:TCP10L3 protein, human; D000249:Adenosine Monophosphate; C107241:diphosphoric acid; D017334:Teicoplanin; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: adenylation domain; antibiotics; enzymes; non-ribosomal peptide synthetase; pyrophosphate detection; teicoplanin