title: Colorimetric Detection of the Adenylation Activity in Nonribosomal Peptide Synthetases.
abstract: 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-[(32)P]-PPi exchange assays with the detection of (32)P-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.
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan.
authors.forename: Chitose
authors.initials: C
authors.lastname: Maruyama
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan.
authors.forename: Haruka
authors.initials: H
authors.lastname: Niikura
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan.
authors.forename: Masahiro
authors.initials: M
authors.lastname: Takakuwa
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan.
authors.forename: Hajime
authors.initials: H
authors.lastname: Katano
authors.affiliation: Department of Bioscience, Fukui Prefectural University, 4-1-1 Matsuoka-Kenjojima, Eiheiji-cho, Yoshida-gun, Fukui, 910-1195, Japan. hamano@fpu.ac.jp.
authors.forename: Yoshimitsu
authors.initials: Y
authors.lastname: Hamano
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000378:metabolism; D000596:Amino Acids / Q000378:metabolism; D001419:Bacteria / Q000737:chemistry / Q000201:enzymology*; D020134:Catalytic Domain; D003124:Colorimetry / Q000379:methods*; D011756:Diphosphates / Q000032:analysis / Q000378:metabolism*; D057075:Enzyme Assays / Q000379:methods*; D010453:Peptide Synthases / Q000737:chemistry / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D000596:Amino Acids; D011756:Diphosphates; D000249:Adenosine Monophosphate; C107241:diphosphoric acid; D000255:Adenosine Triphosphate; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: ATP–[32P]-PPi exchange assay; Adenylation domain; Colorimetric assay; Nonribosomal peptide synthetase; Poly anion