The b-type cytochrome LcpK30 is a latex clearing protein (Lcp), which acts as an endotype dioxygenase to catalyze the extracellular cleavage of the chemically inert aliphatic polymer poly(cis-1,4-isoprene), producing oligo-isoprenoids with different terminal carbonyl groups (aldehyde and ketone, −CH2–CHO and −CH2–COCH3). On the basis of the fact that the muteins of E148A, E148Q, and E148H have substantially reduced reactivity, and the E148-initiated reaction mechanism has been previously proposed, in which a cyclic dioxetane intermediate or an epoxide intermediate may be involved, however, open questions still remain. In this paper, on the basis of the crystal structure of LcpK30, the enzyme–substrate reactant model was constructed, and the cleavage mechanism of the central double bond of poly(cis-1,4-isoprene) was elucidated by performing quantum mechanics/molecular mechanics calculations. Our calculation results revealed that the oxidative cleavage reaction is triggered by the addition of the heme-bound dioxygen to the double bond of the polymer, and E148 does not act as the catalytic base to extract the allylic proton to assist the reaction as previously suggested. Of the two considered pathways, the pathway that involves the dioxetane intermediate was calculated to be more favorable. During the catalysis, the distal oxygen first adds to the double bond of the substrate to form a radical intermediate, and then the Fe–O1 (proximal oxygen) bond cleaves to generate the dioxetane intermediate, which can easily collapse affording the final ketone and aldehyde products. In general, the cleavage mechanism of double C–C bond catalyzed by LcpK30 is similar to those of indoleamine 2,3-dioxygenase, tryptophan 2,3-dioxygenase, and the nonheme stilbene cleavage oxygenase NOV1 that all depend on the iron-bound dioxygen to initiate the cleavage reaction.
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 Fe1 II −Fe2 III −superoxo and Fe1 II − Fe2 IV 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 Fe1 II − Fe2 III −superoxo to install a hydroxyl group into the substrate, generating the high-reactive Fe1 II −Fe2 IV O complex. In the secondround reaction, the Fe1 II −Fe2 IV 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 Fe1 II −Fe2 IV O compared to Fe1 II −Fe2 III −superoxo, besides the C5-hydroxylation, the C3-or C18hydroxylation was also calculated to be feasible.
In this study, we report the discovery of unexpected mechanistic intricacies of Baeyer–Villiger monooxygenases (BVMOs) and provide insights that promise to help in extending their applications in synthetic organic chemistry and biotechnology. The basic mechanism of BVMOs as catalysts in the oxidation of unsymmetrical ketones R1–(CO)–R2 is well known, which involves the intermediacy of short-lived Criegee intermediates. The tendency of R1 or R2 to migrate preferentially in the breakdown of the Criegee intermediate follows the traditional requirement of an antiperiplanar conformation with maximum stabilization of the incipient positive charge. The challenge of inverting the regioselectivity of group migration with the formation of abnormal products was recently met by the semi-rational directed evolution of TmCHMO with the generation of a quadruple mutant. Although a reasonable model explaining the mutational effect was suggested, the theoretical analysis did not include the calculation of both enantiomeric forms of the fleeting chiral Criegee intermediate in transition states and focused only on the wild-type enzyme and the quadruple mutant. The present investigation utilizes complete mutational deconvolution with the experimental construction of a fitness-pathway landscape comprising 4! = 24 upward climbs. We were confronted by the discovery that the absolute configuration of the Criegee intermediate switches from (R) to (S), depending upon the stage of the evolutionary process. On the basis of X-ray structural data, the physical basis of this phenomenon was illuminated by quantum chemical analyses performed on the enzymes at all evolutionary steps of a selected pathway. The hitherto unexplored role of fleeting chiral intermediates in the mechanism of other enzyme types deserves increased attention.
QM/MM calculations reveal that the fatty acid decarboxylase UndA employs the FeIII–OO˙− complex to initiate the β-H abstraction with the monodentate coordination mode. The iron center accepts the extra electron of the substrate radical.
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