A continuous fluorescence assay to measure nicotianamine synthase activity

• DOI: 10.1016/bs.mie.2024.06.013
• Publication Date: 2024-07-08T06:56:07Z
• Authors: Thiago M. Pasin; Kathleen M. Meneely; Deegan M. Ruiz; Audrey L. Lamb
• Keywords: S-Adenosylmethionine; Kinetics; Spectrometry, Fluorescence; Alkyl and Aryl Transferases; Enzyme Assays

S-adenosylmethionine (SAM) is most widely known as the biological methylating agent of methyltransferases and for generation of radicals by the iron-sulfur dependent Radical SAM enzymes. SAM also serves as a substrate in biosynthetic reactions that harvest the aminobutyrate moiety of the methionine, producing methylthioadenosine as a co-product. These reactions are found in the production of polyamines such as spermine, siderophores derived from nicotianamine, and opine metallophores staphylopine and pseudopaline, among others. This procedure defines a highly sensitive, continuous fluorescence assay for the determination of steady state kinetic parameters for enzymes that generate the co-product methylthioadenosine.