Hara Ryotaro, Suzuki Ryohei, Kino Kuniki
Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
Anal Biochem. 2015 May 15;477:89-91. doi: 10.1016/j.ab.2015.01.006. Epub 2015 Jan 20.
We demonstrated the usefulness of a hydroxamate-based colorimetric assay for predicting amide bond formation (through an aminoacyl-AMP intermediate) by the adenylation domain of nonribosomal peptide synthetases. By using a typical adenylation domain of tyrocidine synthetase (involved in tyrocidine biosynthesis), we confirmed the correlation between the absorbance at 490 nm of the l-Trp-hydroxamate-Fe(3+) complex and the formation of l-Trp-l-Pro, where l-Pro was used instead of hydroxylamine. Furthermore, this assay was adapted to the adenylation domains of surfactin synthetase (involved in surfactin biosynthesis) and bacitracin synthetase (involved in bacitracin biosynthesis). Consequently, the formation of various aminoacyl l-Pro formations was observed.
我们证明了基于异羟肟酸的比色测定法在预测非核糖体肽合成酶的腺苷化结构域形成酰胺键（通过氨酰 - AMP中间体）方面的实用性。通过使用酪菌素合成酶（参与酪菌素生物合成）的典型腺苷化结构域，我们证实了l-色氨酸 - 异羟肟酸 - Fe(3+)复合物在490 nm处的吸光度与l-色氨酸 - l-脯氨酸的形成之间的相关性，其中使用l-脯氨酸代替羟胺。此外，该测定法适用于表面活性素合成酶（参与表面活性素生物合成）和杆菌肽合成酶（参与杆菌肽生物合成）的腺苷化结构域。因此，观察到了各种氨酰基l-脯氨酸的形成。
