An optimized ATP/PP_i‐exchange assay in 96‐well format for screening of adenylation domains for applications in combinatorial biosynthesis

Abstract

We report a new format for measuring ATP/[(32)P]pyrophosphate exchange in a higher throughput assay of adenylation domains (A-domains) of non-ribosomal peptide synthetases. These enzymes are key specificity determinants in the assembly line biosynthesis of non-ribosomal peptides, an important class of natural products with an activity spectrum ranging from antibiotic to antitumor activities. Our assay in 96-well format allows the rapid measurement of approximately 1000 data points per week as a basis for precise assessment of the kinetics of A-domains. The assay also allows quantitative high-throughput screening of the substrate specificity of A-domains identifying alternative, promiscuous substrates. We show that our assay is able to give high quality data for the T278A mutant of the A-domain of the tyrocidine synthetase module TycA with a 330-fold lower k(cat)/K(M). The large dynamic range of this assay will be useful for the screening of libraries of mutant A-domains. Finally we describe and evaluate a procedure for the high-throughput purification of A-domains in 96-well format for the latter purpose. Our approach will be of utility for mechanistic analysis, substrate profiling and directed evolution of the A-domains, to ultimately enable the combinatorial biosynthesis of non-natural analogues of non-ribosomal peptides that may have potential as alternative drug candidates.

A Subdomain Swap Strategy for Reengineering Nonribosomal Peptides

“…We measured adenylation activities of all nine constructs using a pyrophosphate exchange assay (Otten et al, 2007). In an initial screen, sdV-GrsA, sdL-GrsA, sdF2-GrsA, and sdL2-GrsA showed significant pyrophosphate exchange activity stimulated by the respective target amino acid at 1 mM ( Figure 2).…”

“…With sdV-GrsA additionally purified by anion exchange chromatography, PP i exchange increases to 18%. Michaelis-Menten parameters were measured for the adenylation partial reaction in a pyrophosphate exchange assay (Otten et al, 2007). Errors are given as the SD of at least four independent measurements with different batches of protein.…”

Adenylate‐Forming Enzymes of Rubradirin Biosynthesis: RubC1 Is a Bifunctional Enzyme with Aminocoumarin Acyl Ligase and Tyrosine‐Activating Domains

“…This is a well-established assay commonly used for the investigation of aminoacyl-adenylating enzymes. [28,29] It measures the incorporation of radioactive pyrophosphate into ATP. We used the same reaction conditions as employed previously for the investigation of NovH.…”

Alkyne‐Functionalized Chemical Probes for Assaying the Substrate Specificities of the Adenylation Domains in Nonribosomal Peptide Synthetases

“…Cell lysate screening has greatly facilitated enzyme engineering through the expression of mutant enzymes in E. coli cells and the direct assay of the enzymatic activities in the cell lysates. [22,[49][50][51] We thus envision that the chemical conjugation method reported here could be used to screen libraries of A or AT domains and their mutants for altered substrate specificity with alkyne-functionalized substrate analogues.…”

“…[6][7][8][9] To diversify the structures of these natural products further for drug development, the substrate specificities of A and AT domains need to be screened for their ability to incorporate non-native building block molecules into the NRPS and PKS assembly lines for the biosynthesis of natural product analogues. [10][11][12][13][14][15][16][17][18][19][20][21] Current methods for screening the substrate spectra of A or AT domains rely on radioactive assays by measurement of ATP/pyrophosphate (PPi) exchange at the active sites of the A domains [22,23] or by detection of the formation of covalent intermediates between radiolabeled substrates and the NRPS or PKS enzymes. [24] Recently, significant progress has been made in using mass spectrometry to characterize substrate loading in these enzymes.…”