Structural analysis of ketol-acid reductoisomerase from Sulfolobus solfataricus

• Main Authors: Chih-lin Wang, 汪致霖
• Other Authors: Chin-Yu Chen
• Format: Others
• Language: en_US
• Published: 2015
• Online Access: http://ndltd.ncl.edu.tw/handle/6r6dux

碩士 === 國立中央大學 === 生命科學系 === 103 === Ketol-acid reductoisomerase (KARI) enzyme which is found in plants, fungi and bacteria but not in animals has attracted much interest for production of amino acids, biofuels and development of antimicrobial agents. The KARI is the second enzyme in the branched-chain amino acid biosynthesis pathway, which conversion of 2-acetolactate into 2,3-dihydroxyisovalerate. The conversion involves an Mg2+-dependent alkyl migration followed by a Nicotinamide adenine dinucleotide phosphate (NADPH)-dependent reduction of the 2-keto group. We have found that there are three homologs, IlvC1, IlvC2 and IlvC3 from sulfolobus solfataricus. The amino acid sequences of these three KARIs possess conserved regions despite they are not highly similar to one another and their function in vivo is not clear. In my study, ilvc3 were overexpressed in E. coli BL21 (DE3) and purified by FPLC. The ilvc3 was a dodecamer in solution with molecular weight ~450 kDa. We successfully found the condition of crystallize, however the diffraction of ilvc3 crystal was poor. Therefore, we built a 3D map of Ilvc3 by single particle 3D reconstruction atomic model, which could help us to determine the quaternary structure. It was discovered that the modeling structure of ilvc3 was similar to the other KARI in different species. In future, we attempt to study the function by the modeling atomic structure.