背景: 支鏈型胺基酸合成有三種不同的酵素參與。酮醇酸還原異構酶是支鏈型胺基酸合成路徑中的第二個酵素，其總反應依序為: 與鎂離子有關的烷基轉移及NAD(P)H 進行酮基還原。因其合成不存在於人體中，對於開發新型的除草劑與抗生素，酮醇酸還原異構酶是一個很好的標的。動機: 酮醇酸還原異構酶的反應機制及其催化基本原則依然不清楚，一般相信反應是先由基質上三級醇的去質子化開始，但需強鹼，然而，在活性中心附近鹼的來源則不清楚。此外，鎂離子在反應中是扮演催化的功能或只扮演螯合的角色也不清楚。先前QM/MM 計算 (Field et al. JACS, 2000)顯示過高的異構化活化能(40.67 kcak/mol vs. 15 kcal/mol (實驗))。初步結果：藉由DFT計算錯合物模型，我們發現一些可能是掌控催化機制的因素及基本原則: (1). Glu可容易抽取基質上的質子，其能量是由生成Mg-O bond補充; (2).異構化需質子源，質子化的Glu可提供質子源，但需質子穿梭過程; (3). Glu在鎂離子的配位可降低異構化的活化能。方法: QM/MM 計算將首先用於決定反應機制及最低能量反應路徑。搭配umbrella sampling， potential of mean force及WHAM 方法，QM/MM 分子動態模擬將計算反應路徑的自由能。目標: (1).描繪酮醇酸還原異構酶催化的位能曲線，(2).決定基元反應中過渡態的能量及結構，(3). 決定調控酮醇酸還原異構酶催化的基本原理。 ;Background: 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 an 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. Motivations: The KARI reaction mechanism as well as its underlying catalysis principles is remained unsolved. It is believed that the reaction is initialized by the deprotonation of tertiary alcohol of substrate. However, deprotonation of the tertiary alcohol requires a strong base; moreover, the base source around the active site is unclear. In addition, whether the Mg2+ ion pair plays roles in the catalysis rather than chelating substrates is unknown More particularly, previous QM/MM calculation (Field et al. JACS, 2000) shows the calculated activation energy for the alkyl migration is too high (40.67 kcak/mol) when compared to the experimentally-measured value (15 kcal/mol).Preliminary Results: Based on complex model and DFT calculations, we have identified several factors, which might govern the underlying principles of KARI reaction: (i). Glu residue can easily abstract the proton from the tertiary hydroxyl group of substrate, which the required energy is complemented by the formation of Mg-O bond; (ii). Isomerization of substrate needs a proton source near the O=C3 of substrate. Protonated Glu can provide the proton source; (iii). A proton shuttle process is needed to transfer the proton source (GluH) on-O-C2 to O=C3; and (iv). Coordination of Glu residues on Mg(II) reduces the isomerization Ea by weakening the Mg-O bond.Methodology: QM/MM calculations will first be employed to determine the reaction mechanism and the minimum energy reaction pathways. The QM/MM MD simulations in conjunction of umbrella sampling, potential of mean force, and WHAM methods will be employed to determine the free energy of reactions. Objectives: (i). Delineating energy profile of KARI reaction; (ii). Determining the energy and structure of transition state in the elementary reactions in the reaction mechanism and (iii). Determining the underlying principles governing the reaction mechanism. Broader Impact: This project will serve as an interface for integrating research and fundamental education. The computational methods used and developed in this project will be incorporated into the graduate-level courses. The broader impact of this project includes the training of interdisciplinary undergraduates and graduate students for studying biomolecular simulations. Moreover, understanding of the KARI biosynthetic pathways is significantly important in the pharmaceutical and biofuel industries.
