Extended Data Fig. 4: Investigations into the catalytically necessary residues in TvaE.
From: Pseudokinases can catalyse peptide cyclization through thioether crosslinking
(a) Examination of the binding affinities by ITC. TvaE variant was titrated with LP1–52, and obtained KD values are shown. (b) Conversions of 1. Samples including the mixtures of 1 with TvaF (i), TvaEF (ii), TvaEArg220AlaF (iii), TvaEGln224AlaF (iv) and TvaEHis273AlaF (v), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1, 2 or 3, 3a and 3b (left), the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+; and for EICs in HPLC-HR-MS analysis (right), the ESI m/z [M + 6H]6+ mode for both 2 and 3 is 1489.88. (c) Analysis of the production of thioamitide in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2 is 1305.5. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaEArg220Ala (ii), TvaEGln224Ala (iii), or TvaEHis273Ala (iv), respectively. HPLC peaks corresponding to the different products are highlighted by color: green for 2, and light blue for TVA-YJ-2. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).
