酮醇酸還原異構酶是參與支鏈型胺基酸生合成途徑的第二個酵素。此酵素作用的方式分成兩步驟:第一步是需要鎂離子專一性地參與，將受質的烷基異構化(把甲基或是乙基，由受質中心的第二個碳，轉移至第三個碳)；第二步是利用NADPH或是NADH將受質第二個碳上的酮基還原成氫氧基，這一步仍需要鎂離子的參與，但是其他二價離子如錳離子與鈷離子的存在也能催化此反應。此酵素只存在於微生物、真菌、與植物中，動物體內並沒有這個酵素。因此、酮醇酸還原異構酶不僅在微生物基因工程中，用於生質燃料的生產，同時也是研發除草劑與抗生素的標的物。我們由耐熱抗酸的古生菌硫磺礦硫化葉菌，鑑定出三個可能是酮醇酸還原異構酶的基因，並將其轉殖入大腸桿菌，大量表達並純化這三個酵素。由圓二色光譜的結果顯示，這三個酮醇酸還原異構酶都具有熱穩定性與酸耐受性。我們也利用X光單晶繞射技術與新穎的冷凍電鏡，解析出其中Sso-KARI-C2的蛋白質結構。這個蛋白質是以六個雙體為單位來組成十二聚體，形成四面體的結構。在此計畫中，我們預計利用具有互補性的結構生物學工具---X光單晶繞射與冷凍電鏡，將三個Sso-KARI 酵素(全酵素或是缺輔基酵素)的結構解析達近似原子級的解析度。此外、利用酵素動力學實驗來研究酮醇酸還原異構酶在高溫下的酵素活性、輔基的喜好(NADPH or NADH)、二價金屬離子對活性的效應以及酵素抑制劑的作用。我們相信Sso-KARI酮醇酸還原異構酶，在高溫下仍能保有不錯的催化活性，具備在厭氧或惡劣條件下的催化能力，此於代謝工程或工業上具備很高的應用潛力。 ;Ketol-acid reductoisomerase (KARI) is a bifunctional enzyme in the second step of biosynthetic pathways for branched-chain amino acids and catalyzes an unusual two-step reaction which involves a Mg2+-dependent alkyl migration followed by a NAD(P)H-dependent reduction of the 2-keto group. KARI enzyme is present in microorganisms and plants but not in mammals. Not only an important enzyme in the biofuel production, KARI is also an attractive target for the herbicide and antibiotic developments. Here, we characterized three putative KARI enzymes from Sulfolobus solfataricus, namely Sso-KARI-C1, Sso-KARI-C2 and Sso-KARI-C3. We also determined the 3D structure of Sso-KARI C2 by X-ray crystallography (2.5 Å resolution) and cryo-EM (~2.92 Å resolution). The Sso-KARI-C2 forms dodecamer by assembling six dimers as a tetrahedral shape. Circular dichroism analysis reveals that three Sso-KARI enzymes are thermostable and pH tolerant. In this proposal, we are planning to determine the 3D structure of three Sso-KARIs both in apo- and holo-form at high resolution either by X-ray crystallography or by advanced cryo-EM technique. The thermoactivity, cofactor preference, metal ion effect on enzyme activity, and inhibitors of Sso-KARIs will be further investigated by enzyme kinetic assay. The Sso-KARIs could be one of the unique enzymes that display high activity at elevated temperature. They are potential candidates for the usages in metabolic engineering or industrial applications under anaerobic or harsh conditions.
