Fig. 5.

Ind6 (Ind56) as a gatekeeper determining the ratio of 2 and 5. (A) The involvement of Ind6 in the Ind3-catalyzed oxazolinone ring formation. Incubation of 6 (in a mixture with 4) and 3 with various combinations of enzymes, in the presence of Mg²⁺and ATP. (B) Identification of the possible leaving group in the Ind3+Ind6 reaction by LC-MS. The ratio of compound (m/z 114.2) production in the Ind3-6 versus Ind3-alone reaction is calculated based on the peak area. (C) Determination of the ratio of 2 in the reaction mixtures of Ind3+Ind6 and Ind3-alone reaction by LC-MS. The ratio of 2 production in the Ind3-6 versus Ind3-alone reaction is calculated based on the peak area at 225 nm. (D) Incubation of 6 (in a mixture with 4) and 3 with various ratios of Ind3/Ind56. (E) Coexpression and purification of Ind5–Ind6 and Ind5–Ind6–Ind3 in Rhodococcus sp. RHA1 (S, soluble fraction; E, elution from nickel–nitrilotriacetic acid column), showing that Ind3 does not form a protein complex with Ind56. (F) In vitro enzymatic total synthesis of indolmycin. EICs for 1 standard compound (i) and ethyl acetate extract from one-pot enzymatic total synthesis of indolmycin (ii).