Computational Study of the Catalytic Mechanism of Ketol–Acid Reductoisomerase of Sso-ilvC2 Protein

• Main Authors: Sheng-Uei Weng, 翁聖崴
• Other Authors: Hui-Hsu Tsai
• Format: Others
• Language: en_US
• Published: 2018
• Online Access: http://ndltd.ncl.edu.tw/handle/6cbsa7

碩士 === 國立中央大學 === 化學學系 === 106 === The ketol-acid reductoisomerase (KARI) is an enzyme necessary for the branched chain amino acids (BCAA) biosynthetic pathway. KARIs catalyze an alkyl-migration of substrate followed by a ketol-acid reduction in terms of NAD(P)H. However, the KARI reaction mechanism is remained unclear; in particular, the roles of Mg2+ ion pair in the catalysis and how the KARI reaction is initialized are still unknown. In this study, we obtain the 3D structure of Sso-ilvC2 protein, a KARI from Sulfolobus solfataricus, from the cryo-EM (resolution = 3.4 Å) in terms of the molecular dynamic flexible fitting (MDFF) technique. We employ the first principle DFT calculations to investigate the activation energy of methyl migration under different catalysis conditions of substrate based on the active site structure of Sso-ilvC2 protein obtained from the cryo-EM structure refined by MD simulations. We observed that the substrate when its ketone group is protonated has lowest active energy (21.5 kcal/mol) for the methyl migration.