Water-Mediated, Metal-Catalyzed, and Base-Induced (WMMCBI) Mechanism of a novel Tertiary -ketol Rearrangement in Ketol–Acid Reductoisomerase

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題名:	Water-Mediated, Metal-Catalyzed, and Base-Induced (WMMCBI) Mechanism of a novel Tertiary -ketol Rearrangement in Ketol–Acid Reductoisomerase
作者:	莊宜銓;Zhuang, Yi-Chuan
貢獻者:	化學學系
關鍵詞:	酮醇酸還原異構;KARI;QM/MM MD;Umbrella Sampling
日期:	2021-08-10
上傳時間:	2021-12-07 11:21:14 (UTC+8)
出版者:	國立中央大學
摘要:	支鏈型胺基酸合成路徑 (branched chain amino acids biosynthetic pathway)中的第二個酵素—酮醇酸還原異構? (ketol-acid reductoisomerase, KARI)，可催化2-羥-2-甲基-3-氧代丁酸 (2-乙醯-2-羥基丁酸) 轉換為2,3-二羥-3-甲基丁酸 (2,3-二羥-3-甲基戊酸)。由於支鏈型胺基酸合成路徑只存在於細菌、植物與菌類中，但不存在於動物中，因此可以做為設計新世代抗生素的研發目標。然而，酮醇酸還原異構?的反應機制仍未完全清楚；尤其，2-羥-2-甲基-3-氧代丁酸中三級羥基的去質子化反應。在此，我們利用 molecular dynamic flexible fitting技術與低溫電子顯微鏡 (cryo-EM) 影像來建立Sso-KARI 蛋白的三維水溶液結構。根據此蛋白質三維結構，我們利用量子力學/分子力學的分子動態模擬結合傘狀抽樣(QM/MM MD/US) 計算出羥基酮重排反應的自由能曲線，對此酵素的催化機制進行研究。我們發現，以水分子為媒介，金屬催化，鹼誘導(WMMCBI)機制有利於2-羥-2-甲基-3-氧代丁酸中三級羥基的去質子化反應及接下來的質子傳導。在此機制中包括基質上三級羥基、鎂上的配位水以及谷氨酸233。此去質子化反應只需4.87千卡/莫耳的活化能，其反應機制涉及到以下兩步驟：谷氨酸233作為鹼抽取鎂上配位水的質子並生成氫氧根離子中間物;以及氫氧根離子中間物做為強鹼對基質的三級羥基進行去質子化反應。相較而言，藉由谷氨酸233作為鹼對基質進行直接去質子化是動力學禁止的反應。WMMCBI機制研究揭示了三級羥基的去質子化反應並提供設計抗生素的線索。;The ketol-acid reductoisomerase (KARI) catalyzes conversion of (S)-2-acetolactate or (S)-2-aceto-2-hydroxybutyrate to 2,3-dihydroxy-3-alkylbutyrate is the second step in the biosynthesis of branched chain amino acids (BCAA). As BCAA biosynthetic pathway is only present in bacteria, plants, and fungi; but absent in animals, thus it is an excellent target for the development of new-generation antibiotics and herbicides. However, the KARI reaction mechanism is yet to be fully solved; in particular, the source of “strong” base needed to first deprotonate the tertiary hydroxyl group of (S)-2-acetolactate is remained unknown. Here, we first optimized the 3D solution structure of archaea ketol-acid reductoisomerase (Sso-KARI) from Sulfolobus solfataricus (Sso) from the cryo-EM maps by iterative molecular dynamic flexible fitting-Rosetta techniques. On the basis of the structure of Sso-KARI:2Mg2+:NADH:(S)-2-acetolactate complex, we decipher the catalytic mechanism of KARI reaction with hybrid quantum mechanics/molecular mechanics (QM/MM) molecular dynamics (MD) simulations in conjunction with umbrella sampling (US). We have determined the entire free energy profile of the -ketol rearrangement and provided detailed mechanistic insights. Our results show that the Water-Mediated, Metal-Catalyzed, and Base-Induced (WMMCBI) mechanism is preferred for the deprotonation of tertiary hydroxyl group of (S)-2-acetolactate and the following proton shuttle. In the WMMCBI mechanism, water coordinated on Mg2+, hydroxyl group of substrate, and Glu233 forms a proton wire route having deprotonation activation energy of 4.87 kcal/mol only. The WMMCBI mechanism has two steps involving am hydroxide intermediate: first, the Glu233 serves as a general base to cleavage the Mg2+bound water and a hydroxide intermediate coordinated on Mg2+ is formed. Second, the hydroxide acts as a strong base to deprotonate the ternary hydroxyl group of substrate. In contrast, direct deprotonation of substrate by Glu233 is kinetically forbidden. WMMCBI mechanism opens a novel way for the design of catalysts for the tertiary -ketol rearrangement and provides clues for the development of new-generation antibiotics.
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