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

Abstract

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.

Thebaine is Selectively Demethylated by Thebaine 6-O-Demethylase and Codeine-3-O-demethylase at Distinct Binding Sites: A Computational Study

“…The Fe­(IV) = O species in the active site of the nonheme 2OG-dependent enzymes can exist either as a trigonal bipyramidal coordinate (5C) ,− configuration or octahedral-coordinate (6C) configuration. − The QM/MM study of AsqJ and EFE showed that 6C Fe­(IV) = O resembles closely to the experimentally trapped Fe­(IV) = O species obtained in prolyl-4-hydroxylase and taurine dioxygenase (TauD) enzymes. , However, it is not known whether the 5C or 6C is the preferred configuration in T6ODM.…”

“…To understand the oxygen activation by Fe­(II), QM/MM calculations were performed on S = 0, S = 1, S = 2, and S = 3 spin state surfaces for the three representative structures obtained from the MD simulations of Fe­(III) super-oxo complex. The quintet state was suggested to be most favorable in the context of 2OG-dependent nonheme iron enzymes from previous experimental electron paramagnetic resonance (EPR) Mössbauer spectroscopy studies and the QM/MM studies; ,,,− , therefore, it was chosen as the reference to compute the relative energies of other spin states for T6ODM.…”

Enzyme Multifunctionality by Control of Substrate Positioning Within the Catalytic Cycle—A QM/MM Study of Clavaminic Acid Synthase

“…Fe IV is coordinated in a distorted octahedral geometry with the succinate bound in a bidentate fashion. Such an arrangement of the active site was proposed in other studies of non‐haem iron dioxygenases, [56–59] however, depending on the surroundings of the active site, Fe IV can also be in a trigonal bipyramidal arrangement [55, 60–62] or a water molecule can bind to Fe IV as an equatorial ligand [63, 64] . As expected for this complex, the electronic configuration of S is π* xy 1 π* xz 1 π* yz 1 σ* 1 (the occupied orbitals are shown in Figure S2 in the Supporting Information), the σ* orbital is virtual and oriented along the O−Fe−N(His‐279) axis and thus able to accept an electron from the substrate during the initial HAT.…”

“…Such an arrangement of the active site was proposed in other studies of non-haem iron dioxygenases, [56][57][58][59] however,d epending on the surroundings of the actives ite, Fe IV can also be in a trigonal bipyramidal arrangement [55,[60][61][62] or aw ater molecule can bind to Fe IV as an equatorial ligand. [63,64] As expected for this complex, the electronic configurationo fS is p* xy…”

Computational Study of the C5-Hydroxylation Mechanism Catalyzed by the Diiron Monooxygenase PtmU3 as Part of the Platensimycin Biosynthesis

“…The QM/MM method has been applied in the studies of mononuclear iron-containing enzymes. − In our calculations, the whole system was divided into QM and MM regions. All QM/MM calculations were conducted by employing the ChemShell package, which combines TURBOMOLE and DL_POLY programs for treating the QM and MM regions, respectively.…”