探討去氧鬼臼毒素合酶催化立體專一性環化反應之結構機轉

Structural Insights into the Stereospecific Cyclization Reaction Catalyzed by Deoxypodophyllotoxin Synthase

吳旻昊(Min-Hao Wu)

指導教授 ： 詹迺立

國立臺灣大學/醫學院/生物化學暨分子生物學研究所/博士(2022年)

https://doi.org/10.6342/NTU202200567

摘要

鬼臼毒素是(podophyllotoxin)一種醫學上重要的芳基四氫萘木脂素，可作為臨床使用的抗腫瘤藥物依託泊苷(etoposide)和替尼泊苷(teniposide)的前驅物。由於對鬼臼毒素的需求持續增長，結合化學與酵素(chemoenzymatic)的合成方法被認為比純化學合成的方式來得更加有效，且能克服鬼臼毒素生成上所遇到的立體化學問題。其中，去氧鬼臼毒素合成酶(deoxypodophyllotoxin synthase)的酵素活性對發展化學酵素合成法相當關鍵。去氧鬼臼毒素合酶能催化亞太因(yatein)的碳6 (C6)及碳7′ (C7′)之間形成碳–碳單鍵，進而生成擁有四環核心的去氧鬼臼毒素(deoxypodophyllotoxin)。此酵素為非血基質鐵及2-側氧戊二酸依賴型加氧酶(non-heme iron- and 2-oxoglutarate-dependent oxygenase)超家族(superfamily)中的一員，該家族成員共通的特徵包括：由雙層β-螺旋(double-stranded β-helix)所構成的催化核心結構，內含一個以二組胺酸一羧酸模體(2-His-1-carboxylate motif)與鐵離子配位所形成的活性中心。先前探討催化機制的研究指出，去氧鬼臼毒素合酶所催化的環化作用(cyclization)可能是經由一個苯甲基自由基(benzylic radical)或是碳陽離子(carbocation)所介導的途徑去完成的。然而，依舊缺乏去氧鬼臼毒素合酶在蛋白結構上的相關資訊。此外，近期的研究指出兩種亞太因的鏡像異構物(enantiomer)皆可做為去氧鬼臼毒素合酶的受質，但羥基化副產物生成與否則截然不同，引發受質立體化學結構如何影響催化途徑的疑問。這些研究也揭露了去氧鬼臼毒素合酶可以主導產物中新生成之掌性中心(chiral center)的立體化學結構。為了闡釋去氧鬼臼毒素合酶催化環化反應中的結構基礎，我們針對去氧鬼臼毒素合酶與鐵離子和消旋亞太因(racemic yatein)複合體進行晶體結構解析。有趣的是，亞太因的鏡像異構物因為與去氧鬼臼毒素合酶結合，而呈現U字型的構形，使其碳7′靠近催化中心的鐵離子，讓氫原子的轉移(hydrogen atom transfer)得以發生。此外，活性中心的空間障礙規範了產物去氧鬼臼毒素在碳7′位置的掌性。我們的實驗結果為去氧鬼臼毒素合酶所催化的合環反應提供了重要的結構觀點，並為去氧鬼臼毒素合酶於化學酵素合成法的應用奠基。

關鍵字

去氧鬼臼毒素合酶 ； 二價鐵及2-側氧戊二酸依賴型加氧酶 ； 環化作用

並列摘要

Podophyllotoxin (PPT) is a medically important aryltetralin lignan that serves as a precursor for the synthesis of clinically used antineoplastic drugs etoposide and teniposide. Owing to the growing demand for PPT, chemoenzymatic approaches are considered more efficient than chemistry-based methods to overcome stereochemical issues arising during PTT synthesis. In particular, the activity of deoxypodophyllotoxin synthase (DPS), which catalyzes the formation of a C–C bond between C6 and C7′ of (−)-yatein to generate the tetracyclic core of (−)-deoxypodophyllotoxin, is crucial for the development of a chemoenzymatic synthesis scheme. DPS belongs to the superfamily of non-heme iron- and 2-oxoglutarate (2OG)-dependent oxygenases (2OGXs), whose members share a conserved catalytic core featuring a double-stranded β-helix (DSBH) fold and an active site harboring a 2-His-1-carboxylate motif for iron coordination. Previous mechanistic studies suggested that DPS-catalyzed cyclization may be achieved through either a benzylic radical or carbocation pathway. However, no structural information on DPS is available. In addition, recent studies have indicated that DPS catalyzes both yatein enantiomers, raising a question about how DPS conducts the cyclization without regard to the stereochemistry of yatein. Meanwhile, these studies also revealed that DPS governs the stereoconfiguration of the newly formed chiral center in the cyclized product. To elucidate the structural basis of DPS-catalyzed cyclization, we have determined DPS crystal structures in complex with iron and racemic yatein. Interestingly, both yatein enantiomers undergo binding-induced conformational changes to adopt a U-shaped conformation, which brings their C7′ close to the iron, presumably allowing hydrogen atom transfer to occur. Moreover, steric restriction imposed by the active site appears to define the chirality of the product (−)-deoxypodophyllotoxin at C7′. Our results provide key structural insights into the DPS-catalyzed ring closure, which may pave a way for utilizing DPS in synthesizing podophyllotoxin-derived drugs.

並列關鍵字

deoxypodophyllotoxin synthase ； Fe(II)- and 2-oxoglutarate-dependent oxygenase ； cyclization

參考文獻

Abad, A., Lopez-Perez, J.L., del Olmo, E., Garcia-Fernandez, L.F., Francesch, A., Trigili, C., Barasoain, I., Andreu, J.M., Diaz, J.F., and San Feliciano, A. (2012) Synthesis and antimitotic and tubulin interaction profiles of novel pinacol derivatives of podophyllotoxins. J. Med. Chem. 55, 6724–6737.

Afonine, P.V., Grosse-Kunstleve, R.W., Echols, N., Headd, J.J., Moriarty, N.W., Mustyakimov, M., Terwilliger, T.C., Urzhumtsev, A., Zwart, P.H., and Adams, P.D. (2012) Towards automated crystallographic structure refinement with phenix.refine. Acta Crystallogr. D Biol. Crystallogr. 68, 352–367.

Aik, W., McDonough, M.A., Thalhammer, A., Chowdhury, R., and Schofield, C.J. (2012) Role of the jelly-roll fold in substrate binding by 2-oxoglutarate oxygenases. Curr. Opin. Struct. Biol. 22, 691–700.

Aik, W.S., Chowdhury, R., Clifton, I.J., Hopkinson, R.J., Leissing, T., McDonough, M.A., Nowak, R., Schofield, C.J., and Walport, L.J. (2015). Introduction to structural studies on 2-oxoglutarate-dependent oxygenases and related enzymes. In 2-Oxoglutarate-dependent oxygenases, (The Royal Society of Chemistry), 59-94.

Basak, A., Salowe, S.P., and Townsend, C.A. (1990) Stereochemical course of the key ring-forming reactions in clavulanic acid biosynthesis. J. Am. Chem. Soc. 112, 1654–1656.