An optimized ATP/PP(i)-exchange assay in 96-well format for screening of adenylation domains for applications in combinatorial biosynthesis.
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.
Otten
Linda G
LG
Department of Biochemistry, University of Cambridge, Cambridge, UK.
Schaffer
Michelle L
ML
Villiers
Benoit R M
BR
Stachelhaus
Torsten
T
Hollfelder
Florian
F
D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D001709:Biotechnology / Q000379:methods; D020134:Catalytic Domain; D020650:Combinatorial Chemistry Techniques / Q000379:methods; D011756:Diphosphates / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D008956:Models, Chemical; D015394:Molecular Structure; D010453:Peptide Synthases / Q000378:metabolism*; D015203:Reproducibility of Results; D013379:Substrate Specificity; D014440:Tyrocidine / Q000737:chemistry / Q000378:metabolism
D011756:Diphosphates; D014440:Tyrocidine; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C035180:tyrocidine synthetase