Fig. 2: Enzyme assays with substrate 3 in presence of O2 (shown are the RP-HPLC chromatograms at λ = 254 nm).
From: Enzymatic spiroketal formation via oxidative rearrangement of pentangular polyketides
Incubation times before reaction quenching are indicated to the left. The respective enzyme and cofactor composition is shown to the right. No conversion of 3 was observed in control assays lacking NADPH (trace 1) or GrhO5 (trace 2). Traces 3–8 show time points from the same, discontinuous assay with GrhO5, in which shunt product 9 accumulated aside from intermediate 10. Addition of GrhO1 boosted 10 formation and counteracted 9 formation (trace 9). Addition of GrhO6 (traces 10 and 11) lead to conversion of 10 into 4 (that rapidly forms ring-opened 12). Note that 7 is not observed due to poor separation resulting from polymerization and irreversible binding. The structures of the intermediates are shown above. The proposed enzymatic steps are presented in Fig. 3. All assays were at least conducted three times independently and representative examples are shown (for uncropped chromatograms, see Supplementary Fig. 8).
