A nonradioactive high-throughput assay for screening and characterization of adenylation domains for nonribosomal peptide combinatorial biosynthesis

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• 作者：Thomas J. McQuade ; Abbie D. Shallop ; Anita Sheoran ; James E. DelProposto ; Oleg V. Tsodikov ; Sylvie Garneau-Tsodikova
• 关键词：Adenylation domain ; Colorimetric ; High-throughput screening ; Nonradioactive ; Malachite green ; Inorganic pyrophosphatase ; Combinatorial biosynthesis ; Nostocyclopeptide
• 刊名：Analytical Biochemistry
• 出版年：2009
• 出版时间：15 March 2009
• 年：2009
• 卷：386
• 期：2
• 页码：244-250
• 全文大小：438 K

文摘

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-A₄, 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.