Fig. 3: Iterative loading of Gly residues by the A2 domain and chain-length control by the C3 domain. | Nature Communications

Fig. 3: Iterative loading of Gly residues by the A2 domain and chain-length control by the C3 domain.

From: Elucidating the molecular programming of a nonlinear non-ribosomal peptide synthetase responsible for fungal siderophore biosynthesis

Fig. 3: Iterative loading of Gly residues by the A2 domain and chain-length control by the C3 domain.The alternative text for this image may have been generated using AI.

a HPLC traces monitored at 420 nm for the following: (i). production of 3 via heterologous expression of sidC (ΔA1T1), sidA and sidL in S. cerevisiae JHY686; ii - iv). in vitro enzymatic reaction of SidC (ΔA1T1) in the presence of Gly, +/− l-Ser and +/− l-AHO; (v)–(vi). authentic standards of 2 and 3. Experiments were performed in triplicate and representative spectra are shown. b Stacked deconvoluted intact protein mass spectra of holo-SidC C2A2T2 in the presence of Gly, ATP and Mg2+. Assays conducted with excess Gly/ATP are shown in spectra (ii.) and (iii.) at 10 min and 60 min time intervals, and with limited concentrations of Gly/ATP in spectra (iv.) and (v.) after a 10 min incubation. Mass shifts corresponding to mono-/poly-Gly species are highlighted with red arrows, and proposed intermediates are displayed. c Stacked deconvoluted intact protein mass spectra of l-AHO-SidC C3A3T3 following incubation with holo-SidC C2A2T2, Gly, ATP and Mg2+ following 10 min and 60 min incubation periods. Spectrum (i.) shows l-AHO SidC C3A3T3 alone; spectra (ii.) and (iii.) show increasing production of the condensed product, l-AHO-Gly3-SidC C3A3T3 over time. Only the Gly3 condensed product is observed, not Gly1 or Gly2, suggesting that this is not a stepwise process. Instead, Gly3 must be formed on SidC C2A2T2 before the SidC C3 domain will catalyse the condensation reaction. Spectrum iv. shows an experiment where a 60 min pre-incubation of holo-SidC-C2A2T2 with Gly, ATP and Mg2+ was conducted to allow formation of Gly3/Gly5-SidC C2A2T2 (see Fig. 3b, spectrum (iii.), before addition of l-AHO-SidC C3A3T3. Only the Gly3 condensed product is observed, not Gly5, indicating that the SidC C3 domain selectively condenses the Gly3-SidC C2A2T2 species with l-AHO only. Mass shifts corresponding to biosynthetic species are highlighted with red arrows, and proposed intermediates are displayed. Exact measured and observed masses are detailed in Supplementary Tables 2 and 3. Experiments were performed in duplicate and representative spectra are shown.

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