Fig. 2
Characterization of a two-component cyclopropanase system. a–h HPLC analysis of the relevant metabolites (UV at 374 nm). a Standard of 1.b Standard of 6. c Wild type Streptomyces zelensis NRRL 11183. d The Δc10P mutant. e The Δc10Q mutant. f The Δc10Q mutant complementated with the c10Q gene. g The Δc10P mutant complementated with the Swoo_2002 gene. h The Δc10P mutant complementated with the c10P gene. i Structures of 6 and 7. j–q HPLC analysis of enzymatic products. j–m are the control reactions without C10P, C10Q, Na2S2O4, and SAM, respectively. n Standard of 1. o The complete reaction for C10P and C10Q. p The complete reaction for Swoo_2002 and C10Q. q The reaction for Swoo_2002 and C10Q H138A. All the in vitro enzymatic activity assays were performed in an anaerobic glove box with less than 1 ppm of O2. Reactions were conducted in Tris•HCl buffer (Tris 50 mM, NaCl 100 mM, glycerol 10%, pH 8.0) with the following composition: 1 μM reconstituted Swoo_2002, 2 μM C10Q, 10 μM substrate 6, 1 mM SAM, 5 mM DTT, 5 mM MgCl2, 5 mM Na2S2O4 and 7% DMSO. The reactions were incubated at 28 °C for 12 h
