Hydroxamate-based colorimetric assay to assess amide bond formation by adenylation domain of nonribosomal peptide synthetases

Ryotaro Hara, Ryohei Suzuki, Kuniki Kino

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    8 Citations (Scopus)

    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<sup>3+</sup> 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.

    Original languageEnglish
    Pages (from-to)89-91
    Number of pages3
    JournalAnalytical Biochemistry
    Volume477
    DOIs
    Publication statusPublished - 2015 May 15

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    Keywords

    • Adenylation domain
    • Amide bond
    • Aminoacyl-AMP
    • Colorimetric assay
    • Hydroxylamine
    • Nonribosomal peptide synthetase

    ASJC Scopus subject areas

    • Biochemistry
    • Biophysics
    • Molecular Biology
    • Cell Biology

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