Fig. 4.

¹H NMR analysis of FzmN activity. a The canonical glutamine synthetase reaction catalyzed by FzmN. The γ-protons on the Glu substrate (Hγ) and Gln product (Hγ’) are highlighted. b ¹H NMR analysis of a reaction mixture containing FzmN, Glu, NH₄Cl, ATP, and MgCl₂. Peaks corresponding to the γ-protons on Glu and Gln are indicated. c ¹H NMR analysis of the reaction mixture containing Glu, NH₄Cl, ATP, and MgCl₂ without FzmN. d The putative reaction catalyzed by FzmN during the biosynthesis of fosfazinomycin. The γ-protons on the Glu substrate (γ) and the glutamylacetylhydrazine (5) product (γ”) and the methyl protons on the acetylhydrazine substrate (CH₃) and 5 (CH’₃) are indicated. e ¹H NMR analysis of the reaction mixture containing FzmN, Glu, acetylhydrazine, ATP, and MgCl₂. Resonances corresponding to the γ-protons and methyl protons on the substrates and the product are indicated. f ¹H NMR analysis of the reaction mixture containing Glu, acetylhydrazine, ATP, and MgCl₂ without FzmN. The chemical shifts of several of the protons are sensitive to conditions (pH, salt), and hence spiking with authentic materials was used for assignments