Crystallization and Preliminary X-ray Crystallographic Analysis of Imidase from Pig Liver and Agrobacterium radiobacter

• Main Authors: Sheng-kuo Chiang, 江盛國
• Other Authors: Yuh-ju Sun
• Format: Others
• Language: zh-TW
• Published: 2003
• Online Access: http://ndltd.ncl.edu.tw/handle/96900308603730575837

碩士 === 國立清華大學 === 生物技術研究所 === 91 === Imidase also known as dihydropyrimidinase (E.C 3.5.2.2), hydantoinase, dihydropyrimidine hydrase or dihydropyrimidine amidohydrolase, that catalyzes the reversible hydrolysis of 5,6-dihydrouracil to 3-ureidopropionate and many other imides. The degradation of pyrimidine (uracil and thymine) is the only known physiological function of imidase. In vitro data indicate that imidase has a broad substrate specificity and prefer xenobiotics over natural substrates. Substrate specificity, metal content and amino-acid sequence all differ significantly between bacterial and mannalian imide-hydrolyzing enzymes. In our study, a thermophilic imidase were purified from pig liver and Agrobacterium radiobacter and crystallized. In pig liver imidase, two kinds of imidase crystals were grown by the hanging-drop vapor-diffusion method using polyethylene glycol MME 5000 and 2-propanol as precipitants. One belongs to the triclinic P1 space group,with unit-cell parameters a = 96.35Å , b = 96.87Å , c = 154.87Å,  = 82.10o,  = 72.54o,  = 77.19o, and the other belongs to the orthorhombic C2221 space group, with unit-cell parameters a = 113.92Å , b =157.22Å , c =156.21Å. In the Agrobacterium radiobacter imidase (A. radio.- D-hydantoinase), two kinds of imidase crystals were grown by the hanging-drop vapor-diffusion method using ammonium sulfate as precipitant. One belongs to cubic F23 (or rhombohedral R3) space group, with unit-cell parameters a = b = c =179.73Å (a = b = 127.09Å , c = 311.29Å ,  = 120o), and the other belongs to tetragonal I4 space group, with unit-cell parameters a = b =129.39Å , c = 173.31Å.