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

• Published in: Biotechnology journal 2007-02, Vol.2 (2), p.232-240
• Main Authors: Otten, Linda G., Schaffer, Michelle L., Villiers, Benoit R.M., Stachelhaus, Torsten, Hollfelder, Florian
• Format: Article
• Language: English
• ISSN: 1860-6768; 1860-7314
• DOI: 10.1002/biot.200600220

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.