A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal...

Adenylation domains are critical enzymes that dictate the identity of the amino acid building blocks to be incorporated during nonribosomal peptide (NRP) biosynthesis. NRPs display a wide range of biological activities and are some of the most important drugs currently used in clinics. Traditionally, activity of adenylation domains has been measured by radioactive ATP-[32P]pyrophosphate (PP(i)) exchange assays. To identify adenylation domains for future combinatorial production of novel NRPs as potential drugs, we report a convenient high-throughput nonradioactive method to measure activity of these enzymes. In our assay, malachite green is used to measure orthophosphate (P(i)) concentrations after degradation by inorganic pyrophosphatase of the PP(i) released during aminoacyl-AMP formation by action of the adenylation domains. The assay is quantitative, accurate, and robust, and it can be performed in 96- and 384-well plate formats. The performance of our assay was tested by using NcpB-A(4), one of the seven adenylation domains involved in nostocyclopeptide biosynthesis. The kinetics of pyrophosphate release monitored by this method are much slower than those measured by a traditional ATP-[32P]PP(i) exchange assay. This observation indicates that the formation of the adenylated amino acid and its release are the rate-limiting steps during the catalytic turnover.