Extended Data Figure 4: The thiazolidine moiety of lugdunin and its formation by peptide cyclisation.

a, Blue rhombus marks the b2-ion from fragmentation route I (Extended Data Fig. 2). The ion was selected from an in-source collision-induced decay (isCID) experiment and was further fragmented in order to find thiazolidine-specific fragment ions. High-resolution MS data are shown with annotated sum formulas and respective deviations from calculated masses (errors in p.p.m.). Shown fragment ions reveal the b1-ion at m/z = 170.0635 Da. Intensity is shown in arbitrary units. b, Photometric detection of thiazolidines at 670 nm. A positive colour reaction with 3-methyl-2-benzothiazolone hydrazone hydrochloride (Sawicki reagent) gives rise to detectable absorbance at 670 nm. Thiazolidine-4-carboxylic acid (thiazolidine) was chosen as positive control, whereas 2-methyl-2-thiazoline (thiazoline) and DMSO acted as negative controls. c, Observable colours for the thiazolidine detection reaction. Thiazolidine-4-carboxlic acid (blue), lugdunin (green) and negative controls (yellow). d, The terminal reductase of LugC is proposed to initiate cleavage of the thioester-bound peptide chain with the aid of an NAD(P)H cofactor. The mature heptapeptide is liberated reductively from the NRPS multienzyme complex and cyclises via the N-terminal amine (l-Cys) and C-terminal aldehyde (l-Val) to form a macrocyclic imine/Schiff base. Subsequent nucleophilic attack of the cysteine thiol group generates the five-membered thiazolidine heterocycle. Nucleophilic attack by the l-cysteine sulfhydryl group may occur either at the re or si face of the imine thus leading to a diastereomeric mixture of two structural populations (depicted with wavy bond). LugC is shown as truncated gene, as indicated by the two lines (||).