以結構觀點深入探討AsqJ所催化連續性的氧化反應機制

Abstract

小巢狀麴菌之AsqJ為一非血基質鐵/-酮戊二酸依賴型雙氧化酶，負責催化受質cyclopeptin連續性的脫氫以及環氧化反應以形成產物cyclopenin。 此氧化反應起始於氧分子與二價鐵離子的結合，再藉由氧分子誘發-酮戊二酸之氧化脫羧反應、使其裂解為二氧化碳以及琥珀酸，同時形成具高氧化力之四價鐵與單氧鍵結的重要中間產物(ferryl-oxo intermediate)，進而輔助受質之脫氫或環氧化。目前已解出的AsqJ 晶體結構包括以鎳取代活性中心鐵的原態構形、及其與受質cyclopeptin或受質衍生物形成的複合體。然而，對於AsqJ所催化之反應的詳細機制則仍待進一步闡明。在本篇研究中，我們首先解出了AsqJ分別與受質cyclopeptin及其C3-表異構物(即D-cyclopeptin)結合的兩個高解析度複合體結構。X-光近緣吸收光譜顯示與受質cyclopeptin結合的AsqJ複合體當中有鐵的訊號。這意味著此AsqJ結構確為與鐵結合的活性態構形。比較此二結構中cyclopeptin與D-cyclopeptin的構形可知，cyclopeptin之三號碳的氫原子以及十號碳上其中一個氫原子皆朝向鐵原子中心; 然而D-cyclopeptin只有十號碳上其中一個氫原子朝向鐵原子中心，而三號碳上的氫原子是遠離鐵原子中心的。基於此結果可推論AsqJ所催化的脫氫反應是透過四價鐵與單氧鍵結的過渡狀態先在十號碳的位置進行氫原子轉移，後續的反應會經形成碳正離子或是羥基化過渡狀態完成。其次，我們分別利用結晶後浸換或同時進行晶體內反應以及共結晶的實驗方法，得到多個AsqJ在催化環氧化反應過程中可能出現的反應中間物結構、以及兩種與AsqJ結合時呈現不同構型的2-CF3受質類似物結構。結構分析指出AsqJ所催化的環氧化反應可能是經由形成三價鐵與單氧鍵結的過渡狀態，再由此單氧原子與受質上的十號碳形成單鍵鍵結，最後形成環氧產物。有趣的是，結構疊合分析顯示構型一的2-CF3上的十號碳與單氧原子之間的距離(2.6 Å)較在形成O-C10鍵結(2.0 Å)的反應中間物遠。此結果與2-CF3受質類似物較不易進行環氧化反應的結果一致。我們也解出兩個有氧分子結合且分別與受質cyclopeptin以及dehydrocyclopeptin結合的AsqJ複合體結構、反應中間物 bicyclic Fe(Ⅳ)-peroxyhemiketal intermediate結構、以硫酸氧釩進行鐵取代所模擬的四價鐵與單氧鍵結過渡狀態結構、以及藉由琥珀酸進行置換所獲得模擬單氧原子重組後的四價鐵與單氧鍵結過度狀態的結構。這些實驗結果對於-酮戊二酸依賴型雙氧化酶進行雙氧活化的機制提供更詳細的資訊。總結以上的研究成果，這些先前尚未被發現的晶體結構完整地描繪出AsqJ所催化進行脫氫與環氧化反應的機制。

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.