Extended Data Fig. 6: Investigations into the catalytic function of H86 in CP.
From: Pseudokinases can catalyse peptide cyclization through thioether crosslinking
(a) Conversions of 1 variants. Samples including 1H86A (i) and the mixtures of 1H86A with TvaE (ii), TvaF (iii) or TvaEF (iv), and 1H86F (v) and the mixtures of 1H86F with TvaE (vi), TvaF (vii) or TvaEF (viii), respectively, were examined after incubation 30 °C for 2-h. For HR-MS analysis of 1H86A, 3bH86A, 1H86F, and 3bH86F, the dashed lines indicate the monoisotopic peaks of ESI m/z [M + 6H]6+ (calculated 1485.7110, 1475.3814, 1498.3829, and 1488.0533, respectively. For observed details and errors, see Supplementary Figs. 36–39). The mutation sites in the precursor peptide are indicated by asterisks. (b) Analysis of the production of thioamitides in S. coelicolor. For EIC in HPLC-HR-MS analysis, the ESI m/z [M + H]+ mode for TVA-YJ-2, TVA-YJ-2H86A, and TVA-YJ-2H86F are 1305.5. 1195.5, and 1271.5, respectively. Tested strains include the S. coelicolor strains harboring a wild-type tva gene cluster (i) and an engineered cluster to encode mutant TvaAH86A (ii), or TvaAH86F (iii), respectively. The HPLC peak corresponding to the TVA-YJ-2 variants is highlighted by light blue. Related experiments were performed >= 3 times (with >= 2 parallel samples for each).
