Structural insight into the mechanism of AsqJ-catalyzed consecutive oxidation reactions

• Main Authors: Hsuan-Jen Liao, 廖宣任
• Other Authors: Nei-Li Chan
• Format: Others
• Language: zh-TW
• Published: 2019
• Online Access: http://ndltd.ncl.edu.tw/handle/kkagv6

博士 === 國立臺灣大學 === 生物化學暨分子生物學研究所 === 107 === AsqJ is a non-heme FeII/2-oxoglutarate-dependent dioxygenase recently discovered in Aspergillus nidulans, which catalyzes the sequential desaturation and epoxidation on the substrate cyclopeptin to produce cyclopenin. Each AsqJ-mediated oxidation step is accompanied by the breakdown of 2-oxoglutarate (2OG) into succinate and CO2, which results in the formation of a high-valent FeⅣ-oxo species as a key intermediate. Crystal structures of the AsqJ in its NiII-substituted state and in complexes with cyclopeptin and its analogs have already been determined. However, a more complete and detailed mechanistic picture of the AsqJ-catalyzed reactions has remained to be further explored. Here, we report the high-resolution crystal structures of iron-bound AsqJ with cyclopeptin and its C3-epimer (D-cyclopeptin), respectively. An XANES spectrum collected from an AsqJ•Fe•2OG•cyclopeptin crystal unambiguously shows the presence of iron in the crystal. This structure provides the first visualization of AsqJ in its native, Fe-bound form. More importantly, different from the binding configuration of cyclopeptin where the C3-H and one of the C10-Hs are poised toward the iron center, in the structure of AsqJ•Fe•2OG•D-cyclopeptin, the C3-H points away from the iron center. These findings suggest that a pathway involving hydrogen atom abstraction at the C10 position of the substrate by a short-lived Fe(IV)-oxo species and the subsequent formation of a carbocation or a hydroxylated intermediate more likely accounts for AsqJ-catalyzed desaturation. We have also determined several structures on AsqJ that may correspond to intermediate states occurred during the epoxidation step, two different binding conformations of 2-CF3 (an analog of cyclopeptin) in complex with AsqJ, and structures resulted from in-crystal reaction. The two intermediate structures of AsqJ and a product-bound AsqJ complex suggest that the AsqJ-catalyzed epoxidation may go through ferric-oxo formation followed by O-C10 bond formation and epoxide production. Interestingly, superimposition analysis shows that the distance from the oxo group to C10 of substrate in conformation 1 of bound 2-CF3 analog (2.6 Å) is longer than the 2.0 Å seen in the intermediate showing O-C10 bond formation. This result may explain why 2-CF3 is less reactive toward AsqJ-catalyzed epoxidation. Finally, crystal structures of two O2 –bound AsqJ complexes with cyclopeptin and dehydrocyclopeptin, the bicyclic Fe(Ⅳ)-peroxyhemiketal intermediate, the Fe(Ⅳ)-oxo intermediate mimicked by VOSO4 replacement, and the Fe(Ⅳ)-oxo intermediate via rearrangement mimicked by soaking succinate are determined. Together, these results not only depict the mechanism of AsqJ-catalyzed desaturation and epoxidation, but also provide more detailed information on dioxygen activation of 2OG-dependent dioxygenase superfamily.