Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases
Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases
复制标题
DOI：
10.1007/978-1-4939-3375-4_5
复制
发表时间：
2016-01-01
期刊：
影响因子：
--
通讯作者：
Hamano, Yoshimitsu
中科院分区：
文献类型：
--
作者：
Maruyama, Chitose;Niikura, Haruka;Hamano, Yoshimitsu
非核糖体肽合成酶（NRPS）是由催化结构域组成的多功能酶。腺苷酸化（A）结构域的底物特异性决定了非核糖体肽生物合成过程中要掺入的氨基酸结构单元。A结构域介导氨基酸底物的ATP依赖性活化，如氨酰-O-AMP和焦磷酸盐（PPi）释放。传统上，A结构域的酶活性通过放射性ATP-[P-32]-PPi交换试验测量，并检测P-32标记的ATP。最近，我们开发了一种直接检测PPi作为黄色18-溴焦磷酸阴离子（[（P2 O 7）Mo 18 O 54]（4-））的比色法。[（P_2O_7）Mo_（18）O_（54）]（4-）被抗坏血酸进一步还原，得到更易分辨的蓝色显色。在这里，我们展示了实验室协议的比色测定PPi释放的A-域反应。
Nonribosomal peptide synthetases (NRPSs) are multifunctional enzymes consisting of catalytic domains. The substrate specificities of adenylation (A) domains determine the amino-acid building blocks to be incorporated during nonribosomal peptide biosynthesis. The A-domains mediate ATP-dependent activation of amino-acid substrates as aminoacyl-O-AMP with pyrophosphate (PPi) release. Traditionally, the enzymatic activity of the A-domains has been measured by radioactive ATP-[P-32]-PPi exchange assays with the detection of P-32-labeled ATP. Recently, we developed a colorimetric assay for the direct detection of PPi as a yellow 18-molybdopyrophosphate anion ([(P2O7)Mo18O54](4-)). [(P2O7)Mo18O54](4-) was further reduced by ascorbic acid to give a more readily distinguishable blue coloration. Here we demonstrate the lab protocols for the colorimetric assay of PPi released in A-domain reactions.