title: Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases.
abstract: 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.
authors.affiliation: Research Institute for Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Ryotaro
authors.initials: R
authors.lastname: Hara
authors.affiliation: Department of Applied Chemistry, Faculty of Science and Engineering, Waseda University, Tokyo 169-8555, Japan.
authors.forename: Ryohei
authors.initials: R
authors.lastname: Suzuki
authors.affiliation: 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. Electronic address: kkino@waseda.jp.
authors.forename: Kuniki
authors.initials: K
authors.lastname: Kino
mesh_terms: D000249:Adenosine Monophosphate / Q000378:metabolism; D000577:Amides / Q000737:chemistry*; D056449:Bacillales / Q000201:enzymology; D003124:Colorimetry / Q000379:methods*; D006877:Hydroxamic Acids / Q000737:chemistry*; D010453:Peptide Synthases / Q000737:chemistry* / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D017434:Protein Structure, Tertiary
chemical_list: D000577:Amides; D006877:Hydroxamic Acids; D000249:Adenosine Monophosphate; D010453:Peptide Synthases; C104385:non-ribosomal peptide synthase
keywords: Adenylation domain; Amide bond; Aminoacyl-AMP; Colorimetric assay; Hydroxylamine; Nonribosomal peptide synthetase