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

Abstract

PtmU3 is a newly identified nonheme diiron monooxygenase, which installs a C-5 β-hydroxyl group into the C-19 CoA-ester intermediate involved in the biosynthesis of unique diterpene-derived scaffolds of platensimycin and platencin. PtmU3 possesses a noncanonical diiron active site architecture of a saturated six-coordinate iron center and lacks the μ-oxo bridge. Although the hydroxylation process is a simple reaction for nonheme mononuclear iron-dependent enzymes, how PtmU3 employs the diiron center to catalyze the H-abstraction and OH-rebound is still unknown. In particular, the electronic characteristic of diiron is also unclear. To understand the catalytic mechanism of PtmU3, we constructed two reactant models in which both the Fe1II-Fe2III-superoxo and Fe1II-Fe2IV═O are considered to trigger the H-abstraction and performed a series of quantum mechanics/molecular mechanics calculations. Our calculation results reveal that PtmU3 is a special monooxygenase, that is, both atoms of the dioxygen molecule can be incorporated into two molecules of the substrate by the successive reactions. In the first-round reaction, PtmU3 uses the Fe1II-Fe2III-superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1II-Fe2IV═O complex. In the second-round reaction, the Fe1II-Fe2IV═O species is responsible for the hydroxylation of another molecule of the substrate. In the diiron center, Fe2 adopts the high spin state (S = 5/2) during the catalysis, whereas for Fe1, in addition to its structural role, it may also play an assistant role for Fe1 catalysis. In the two successive OH-installing steps, the H-abstraction is always the rate-liming step. E241 and D308 not only act as bridging ligands to connect two Fe ions but also take part in the electron reorganization. Owing to the high reactivity of Fe1II-Fe2IV═O compared to Fe1II-Fe2III-superoxo, besides the C5-hydroxylation, the C3- or C18-hydroxylation was also calculated to be feasible.