Mechanism of Uncoupled Carbocyclization and Epimerization Catalyzed by Two Non-Heme Iron/α-Ketoglutarate Dependent Enzymes

The non-heme iron/α-ketoglutarate dependent enzymes SnoK and SnoN from Streptomyces nogalater are involved in the biosynthesis of anthracycline nogalamycin. Although they have similar active sites, SnoK is responsible for carbocyclization whereas SnoN solely catalyzes the hydroxyl epimerization. Herein, we performed docking, molecular simulations, and a series of combined quantum mechanics and molecular mechanics (QM/MM) calculations to illuminate the mechanisms of two enzymes. The catalytic reactions of two enzymes occur on the quintet state surface. For SnoK, the whole reaction includes two separated hydrogen-abstraction steps and one radical addition, and the latter step is calculated to be rate limiting with an energy barrier of 21.7 kcal/mol. Residue D106 is confirmed to participate in the construction of the hydrogen bond network, which plays a crucial role in positioning the bulky substrate in a specific orientation. Moreover, it is found that SnoN is only responsible for the hydrogen abstraction of the intermediate, and no residue was suggested to be suitable for donating a hydrogen atom to the substrate radical, which further confirms the suggestion based on experiments that either a cellular reductant or another enzyme protein could donate a hydrogen atom to the substrate. Our docking results coincide with the previous structural study that the different roles of two enzymes are achieved by minor changes in the alignment of the substrates in front of the reactive ferryl-oxo species. This work highlights the reaction mechanisms catalyzed by SnoK and SnoN, which is helpful for engineering the enzymes for the biosynthesis of anthracycline nogalamycin.