HAMA: A multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes.
(2019) HAMA: A multiplexed LC-MS/MS assay for specificity profiling of adenylate-forming enzymes. Chem Sci 10, 10395-10399.
Adenylation enzymes selecting substrates for ribosomal and nonribosomal protein and peptide biosynthesis have been
popular targets of enzyme engineering. Previous standard assays for adenylation specificity have been cumbersome and
failed to reflect the competition conditions inside a cell because they measure substrates one at a time. We have
developed an adenylation assay based on hydroxamate quenching and LC-MS/MS detection of hydroxamate products
testing dozens of competing amino acid substrates in parallel. Streamlined specificity profiling of adenylation enzymes will
facilitate engineering and directed evolution of ribosomal and nonribosomal peptide synthesis.