Fig. 1: Characterization of the PA biosynthesis.
From: Azetidine amino acid biosynthesis by non-haem iron-dependent enzymes
a, LC–MS analysis of the fermentation broths of S. cacaoi and its mutants. Shown are the EICs for polyoxin A (EIC = 617.2053) and the EIC for polyoxin D (EIC = 522.1317). b, Transformation of l-Ile to PA by PolF. HPLC UV chromatograms (325 nm) of PolF assays containing 300 μM l-Ile, 30 μM apo-PolF, 100 μM Fe(II), 1 mM ascorbate and ~0.5 mM O2 (i). Also shown are control assays performed under the same conditions but without ascorbate (ii), with boiled PolF (iii), without Fe (iv) and without O2 (v). c, Transformation of l-Val to MAA by PolF. HPLC UV chromatograms (325 nm) of a PolF assay with 300 μM l-Val, 30 μM apo-PolF, 100 μM Fe(II), 1 mM ascorbate and ~0.5 mM O2 (i), and a control assay with boiled PolF (ii). See Supplementary Fig. 4 for the HPLC chromatograms of the entire retention time range for b and c. d,e,g, PolF reactions with l-Val (3,4-dh-Val) under multiple turnover (d) and single turnover conditions (e), and with 3,4-dh-Val under single turnover conditions (g). f, Reactions catalysed by PolF with l-Val as substrate. The rate constant of each step is calculated from the kinetic fittings of the data in e and g. See Supplementary Table 3 for details. h,i, PolF reactions with l-Ile under single turnover (h) and multiple turnover conditions (i). The products of the l-Ile products were characterized by LC–MS and a comparison with the l-Val reaction. The solid lines in e,g,h are a nonlinear curve fit of equation (1) (Methods). All the kinetic parameters are summarized in Supplementary Table 3. The kinetic experiments were performed in triplicate. Error bars represent one standard deviation calculated from the three replicates. Source data for a–e, g–i are provided.
