摘要
Clostridium sticklandii此菌株內的鳥胺酸異構酶（D-ornithine aminomutase ）可將D-ornithine催化成(2R, 4S)-2, 4- diaminopentanoic acid。鳥胺酸異位酶由兩個次單元所組成，分別為OraS與OraE。OraS由121個胺基酸組成，分子量為12800 Da；OraE由753個胺基酸所組成，分子量為82900 Da。 OraSE在催化的過程中，需要兩個輔酶AdoCbl與PLP的參與。本論文的結果顯示，E. coil內蛋白質摺疊的途徑，與在試管內的途徑並不相同的，前者並不需要輔酶B6或B12的參與。此外，鳥胺酸異位酶與PLP結合係數經測定為224 ± 41 nM。最後，我們還成功的自Samonella typhimurium ATCC 19585選殖、定序、表現出cobU基因，CobU蛋白可應用於合成AdoCbl的類似物AdoCbi-GDP，而我們也成功的利用酵素化學法，大量合成出AdoCbi-GDP，以供日後研究所需。
並列摘要
D-Ornithine aminomutase from Clostridium sticklandii catalyzes the 1,2- rearrangement of D-ornithine to (2R,4S)-2,4-diaminopentanoic acid. It comprises two strongly associating subunits. The oraS gene, which encodes a protein of 121 amino acid residues with Mr 12,800, and oraE gene, which encodes a protein of 753 amino acid residues with Mr 82,900. Two different coenzymes, pyridoxal phosphate (PLP) and adenosylcobalamin (Ado-Cbl), are involved in this enzymatic reaction. In this study, The apparent Kd for coenzyme B6 binding to D-ornithine aminomutase is 224 ± 41 nM as measured by equilibrium dialysis. OraSE–K629M, is catalytically inactive and unable to bind PLP. Because no coenzyme is involved in protein folding during in vivo translation of OraSE–K629M in E. coli, in vitro refolding of the enzyme employs a different folding mechanism. In both cases, the association of the S and E subunit is important for D-ornithine aminomutase to maintain an active conformation. The cloning, sequencing, and expression of the cobU gene from Salmonella typhimurium ATCC 19585 was also reported. A new single-step method based on hydrophobic interaction chromatography developed to facilitate the purification work of CobU protein. With the use of purified CobU protein, the coenezyme B12 analog, AdoCbi-GDP, was synthesized for futwe studies.