Mechanistic Insights into the Oxidative Rearrangement Catalyzed by the Unprecedented Dioxygenase ChaP Involved in Chartreusin Biosynthesis

Abstract

ChaP is a non-heme iron-dependent dioxygenase belonging to the vicinal oxygen chelate (VOC) enzyme superfamily that catalyzes the final α-pyrone ring formation in the biosynthesis of chartreusin. In contrast to other common dioxygenases, for example, 2,3-catechol dioxygenase which uses the dioxygen molecule as the oxidant, ChaP requires the flavinactivated oxygen (O 2 2− ) as the equivalent. Previous experiments showed that the ChaP-catalyzed ring rearrangement contains two successive C−C bond cleavages and one lactonization; however, the detailed reaction mechanism is unknown. In this work, on the basis of the recently obtained crystal structure of ChaP, the computational model was constructed and the catalytic mechanism of ChaP was explored by performing quantum mechanical/molecular mechanical (QM/MM) calculations. Our calculation results reveal that ChaP uses the proximal oxygen in iron-coordinated HOO − to attack the carbonyl carbon of the substrate, whereas the previous proposal that Asp49 acts as a base to deprotonate the iron-coordinated HOO − to generate O 2 2− is unlikely. In the first stage reaction, owing to the coordination of the substrate with iron, the substrate is activated by accepting an electron from iron and the resulting oxy-radical intermediate formed by O−O cleavage can easily trigger the ring rearrangement. In the final decarboxylation, the phenolic anion of the substrate cooperatively accepts the proton of iron-coordinated HOO − to facilitate the attack of the distal oxygen, and the proton-coupled electron transfer (PCET) from the substrate to the Fe IV O plays a key role for the decarboxylation. These findings may provide useful information for understanding the ChaP-catalyzed oxidative rearrangement and other flavindependent non-heme dioxygenases.

Cluster Model Study into the Catalytic Mechanism of α-Ketoglutarate Biodegradation by the Ethylene-Forming Enzyme Reveals Structural Differences with Nonheme Iron Hydroxylases

“…We started the work by calculating the iron­(III)-superoxo reactant using models A , B , and C in various low-energy spin states and compared the work with previous studies and available crystal structure coordinates. In agreement with several analogous studies on enzymatic nonheme iron­(III)-superoxo species, the ground state is the quintet spin state, while the triplet and singlet states are well higher in energy. − The optimized geometries of 5 Re B and 5 Re C are shown in Figure . Geometrically, they are very similar, and an overlay of the two structures put most protein chains in virtually the same positions.…”

Computational Study of Aromatic Hydroxylation Catalyzed by the Iron-Dependent Hydroxylase PqqB Involved in the Biosynthesis of Redox Cofactor Pyrroloquinoline Quinone

“…Starting from 7 IM2′, the system energy keeps continuously increasing with the decrease of the O d –C 3 distance, suggesting the septet state to be unreactive because of the two spin-parallel electrons in dioxygen and substrate. This result is similar to those of other nonheme dioxygenases. , Since the quintet state is the reactive state, we only considered the quintet state in the following calculations.…”

“…This electronic structure coincides with the calculation results of other nonheme dioxygenases, in which both the dioxygen and substrate bind to the iron center to generate the substrate radical-Fe II -superoxo species. [21][22][23]39 To further illuminate the electron characteristic of 5 IM2′, we further performed NBO calculations, and three types of orbital interactions with large second-order perturbative (ΔE 2 ) are displayed in Figure S8. In one type with a ΔE (2) value of 9.43 kcal/mol, the donor is the lone pair (LP) p orbital of O P , and the acceptor is the d orbital of iron center.…”

“…This result is similar to those of other nonheme dioxygenases. 23,39 Since the quintet state is the reactive state, we only considered the quintet state in the following calculations. According to our calculations, the attack of oxygen on the noncarbonyl C 3 of the substrate is in concert with the broken of Fe−O p bond, which corresponds to a relatively high barrier of 19.9 kcal/mol (see Figure S10).…”

“…To avoid the collapse of the active site, the Fe and Fe-coordinated atoms were kept frozen during the MD simulation. This strategy is commonly used for treating the metal-containing enzymes. ,,− The calculated RMSDs of the backbone atoms of the protein are displayed in Figure S1b. One can see that the system has basically reached equilibrium after 7 ns.…”