Brandsch R, Bichler V
FEBS Lett. 1985 Nov 18;192(2):204-8. doi: 10.1016/0014-5793(85)80108-3.
The 6-hydroxy-D-nicotine oxidase gene of Arthrobacter oxidans was cloned into E.coli with the aid of the expression vector pKK223-3. This enzyme, as well as the E.coli enzymes succinate dehydrogenase and fumarate reductase, bears the cofactor FAD covalently attached to the polypeptide through a His-N3-8 alpha-linkage. The amino acid sequence surrounding the histidine residue involved in FAD binding in 6-hydroxy-D-nicotine oxidase and the two E.coli enzymes, however, show no homology. Nevertheless, 6-hydroxy-D-nicotine oxidase is expressed in E.coli in vivo and in an E.coli-derived coupled transcription-translation system as a covalently flavinylated, enzymatically active polypeptide.
借助表达载体pKK223 - 3，将氧化节杆菌的6 - 羟基 - D - 尼古丁氧化酶基因克隆到大肠杆菌中。这种酶以及大肠杆菌的琥珀酸脱氢酶和延胡索酸还原酶都带有通过His - N3 - 8α连接与多肽共价连接的辅因子FAD。然而，参与6 - 羟基 - D - 尼古丁氧化酶以及两种大肠杆菌酶中FAD结合的组氨酸残基周围的氨基酸序列没有同源性。尽管如此，6 - 羟基 - D - 尼古丁氧化酶在大肠杆菌体内以及大肠杆菌来源的偶联转录 - 翻译系统中表达为共价黄素化的、具有酶活性的多肽。
