酮醇酸還原異構酶 (ketol-acid reductoisomerase) 是支鏈型胺基酸合成路徑 (branched chain amino acids biosynthetic pathway)中的第二個酵素，它催化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)。酮醇酸還原異構酶的催化反應可分為兩步驟，第一步為與鎂離子有關的烷基轉移;第二步為利用菸鹼醯胺腺嘌呤二核苷酸或菸鹼醯胺腺嘌呤二核苷酸磷酸(NAD(P)H) 進行酮基還原。研究此種酵素的催化機制有助於開發新型的除草劑及抗生素，因為它們只存在於植物、細菌及真菌，而不存在於動物中。然而，對於酮醇酸還原異構酶的反應機制仍尚不明確，尤其是驅動烷基轉移的起始劑來源是不清楚的。在本篇研究中，我們的研究對象為Sso-ilvC2蛋白，它是硫化葉菌 (Sulfolobus solfataricus) 的酮醇酸還原異構酶，我們利用分子動態柔性擬合 (molecular dynamic flexible fitting) 技術與低溫電子顯微鏡 (cryo-EM) 影像 (解析度 = 3.38 Å) 來建立Sso-ilvC2 蛋白的三維結構，並根據所建立的結構提出三種可能的反應機制。我們利用密度泛函理論、量子力學/分子動力學 (quantum mechanics-molecular mechanics)，以及傘狀抽樣結合量子力學/分子動力學的分子動態模擬 (umbrella sampling quantum mechanics-molecular mechanics molecular dynamic simulations)，研究酮醇酸還原異構酶的催化機制。在研究中我們發現，酵素中的谷胺酸233(Glu233) 扮演著重要的角色。谷胺酸233不直接抽取反應物羥基上的質子，而是藉由抽取鎂離子上配位水的質子，並經過一連串的質子轉移過程，促進了甲基轉移的發生。經由計算得到的反應活化能可以符合實驗的結果。此外，我們所提出的反應機制，其產物與酵素及鎂離子的結合方式，與X光繞射得到的晶體結構也相似，谷胺酸233與產物的羥基形成連結，而不與鎂離子形成配位，實驗的活化能及X光繞射晶體結構都支持我們的反應機制，且此機制有助於研究開發新型的除草劑及抗生素。

The conversion of 2-acetolactate (2-aceto-2-hydroxybutyrate) to 2,3-dihydroxy-isovalerate (2,3-dihydroxy-3-ethylbutyrate) is the second step in the biosynthesis of branched chain amino acids (BCAA) catalyzed by ketol-acid reductoisomerase (KARI). KARIs catalyze an alkyl migration of 2-acetolactate (2-aceto-2-hydroxybutyrate), followed by a ketol-acid reduction in terms of NAD(P)H. BCAA biosynthetic pathway is excellent target for the development of novel antibiotic and herbicide resistance as they are only present in bacteria, plants, and fungi; but absent in animals. However, the KARI reaction mechanism is remained unsolved; in particular, how the alkyl-migration catalyzed is still unknown. Here, we obtain the 3D structure of Sso-ilvC2 protein, a KARI from Sulfolobus solfataricus, from the cryo-EM (resolution = 3.38 Å) optimized by the iterative molecular dynamic flexible fitting (MDFF)-Rosetta technique. Based on the 3D structure of Sso-ilvC2 protein we obtained, we elucidate the catalytic mechanism of KARI reaction with density functional theory, hybrid quantum mechanical/molecular mechanical (QM/MM) theoretical calculations, and umbrella sampling QM/MM MD simulations. We observed that Glu233 plays a critical role in the catalytic reaction. Glu233 initiates the reaction in terms of proton abstraction on the coordinated water on the Mg2+ rather than on the hydroxyl of substrate directly. Proton shuttle by protonated Glu233 activated the methyl migration. The overall activation energy is good agreement with experimentally-measured value. In addition, the product in our mechanism has same structural features of active site of X-ray determined structure, where the Glu233 is bonded with the terminal oxygen of product and does not coordinated with Mg2+. Experimental activation energy and X-ray determined structure supports our catalytic mechanism. Our mechanism provides clues for rational design of de novo antibiotic and herbicide resistances.

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