StnK3及其突變株於路易斯酸依賴性表異構化反應與逆醛醇反應之蛋白結構解析及反應機轉

Abstract

生物體中的活性物質，通常是由小分子化合物作為起始物，在酵素的催化之下，經由一系列化學反應組裝而成。β-甲基胺基酸就是一個例子，其為增加天然物多樣性的重要組成單元，而其中立體選擇性扮演著重要的角色。酵素StnK3掌理合成過程中的立體選擇性，可催化(3R)-β-MeInPy翻轉成另一個掌性對稱的異構物(3S)-β-MeInPy。 在此篇論文發現StnK3是一個鐵離子依賴型的異構酶，催化反應進行時，鐵離子會被拱活化位的中心點，和3-His-1-Glu四聯體以及β-MeInPy上的α-酮酸形成六配位雙四角錐體，這種特殊的幾何形狀，可以一邊辨識基質(3R)-β-MeInPy，一邊啟動路易士酸導入烯醇化(enolization)，催動異構化發生。StnK3催化的異構化反應是透過前所未有的隱藏版雙鹼基機制達成的，經由活化位的配位轉換，中間體的構造從雙四角錐體變成雙三角體，讓其中一邊脫去的質子，可以從另一側逆向重新接上質子，產生翻轉的異構物(3S)-β-MeInPy。這個質子化的逆向反應，是由質子的內部回歸達成的。 此外，StnK3表現出了一種意料之外的事前校正活性，它會將上游未成熟的基質indolepyruvate，經由逆醛醇縮合(retro-aldol reaction)反應，分裂成吲哚醛(indole aldehyde)和乙醇酸(glycolic acid)，從而避免無謂的循環，將反應效率投注在完成最終產物。在此基礎上，突變體H27A將StnK3從異構酶完全轉化成全功能的逆醛縮酶，可在催化範圍內將β-MeInPy的C-C鍵結打斷形成3-acetylindole。 在這次的研究中，新反應的發現以及機制的闡明不僅有助於破解立體和鏡像選擇性的實驗操作方式，而且可以幫助設計用於生物合成或藥用化學的新生物催化劑。透過催化劑的設計與活性調控，可為新藥開發提供有潛力的先導化合物。

β-Amino acids have long been recognized as important components in a lot of clinically important natural products and contribute to the diversity of small molecules. StnK3 is the key enzyme which control the stereoselectivity of β-methyltryptophan, mediating the chirality-inversion from (2S,3R) to (2S,3S) β-methyltryptophan. In this research, we indicate that StnK3 is a new type of ferric iron dependent epimerase, in which four important residues His64, His66, Glu70, and His109 form a 3-His-1-Glu tetrad motif, providing a scaffold to connect Fe3+ in active site. The 3-His-1-Glu tetrad and an α-keto acid bidentate of β-methylindolepyruvate form a six-coordinate octahedral complex. This particular coordination increases the β-carbon acidity of β-methylindolepyruvate, promoting its enolization, then epimerization is accomplished through an unusual hidden two base internal return mechanism. Furthermore, StnK3 displays an extra proofreading activity to cleavage the immature upstream substrate β-indolepyruvate into indole aldehyde and glycolic acid by retro aldol cleavage reaction. This proofreading activity avoids excess futile cycles but ensures fidelity of the reaction to produce the mature final product. Interestingly, mutant H27A becomes a full-duty retro-aldolase and completely loses its epimerization activity, cleaving the putatively unfavorable C-C bond of (3R)-β-methylindolepyruvate to 3-acetylindole and glycolic acid. Finally, the discovery of a novel reaction and catalytic mechanism of stnk3 not only helps to decipher the code for the stereoselectivity of biosynthesis of (2S,3S)-β-methyltryptophan from L-tryptophan, but also provides the foundation to help design new biocatalysts for the synthesis of industrial or medicinal chemicals.