Expression of theE. coli nadBGene and Characterization of the Gene Product L-Aspartate Oxidase

生物化学 分子生物学 结构基因 基因产物 等电点 生物 化学 溴化氰 辅因子 半胱氨酸 基因 肽序列 立体化学 基因表达 大肠杆菌
作者
Jana Seifert,N. Kunz,Ralf Flachmann,Albrecht Läufer,Klaus‐Dieter Jany,Hans Günter Gassen
出处
期刊:Biological chemistry Hoppe-Seyler [De Gruyter]
卷期号:371 (1): 239-248 被引量:47
标识
DOI:10.1515/bchm3.1990.371.1.239
摘要

Quinolinic acid is synthesized in E. coli by the enzymes L-aspartate oxidase and quinolinate synthase A, the genes of which are named nadB and nadA. In our previous work we cloned and characterized the two genes (Flachmann, R., Kunz, N., Seifert, J., Gütlich, M., Wientjes, F.J., Läufer, A. & Gassen, H.G. (1988) Eur. J. Biochem. 175, 221-228). Here we report on the expression of the nadB gene under control of the inducible left promoter of the bacteriophage lambda. The yield of the active gene product L-aspartate oxidase was enhanced up to 20% of the soluble cell protein. The enzyme was purified to homogeneity in a three-step procedure and the reading frame of the L-aspartate oxidase gene was confirmed by Edman degradation of five cyanogen bromide peptides. L-Aspartate oxidase shows no classical Michaelis-Menten behaviour but is subject to a substrate inactivation. The apparent Km values were different for substrate concentrations below and above 1mM and were determined to 0.5 mM and 4.1mM, respectively. The active form of the enzyme is a monomer of 60,284 Da and contains one molecule of FAD and nine cysteine residues, four of which built up two disulfide bonds. The isoelectric point of the protein was determined to be at pH 5.6. Chemical modifications of the enzyme showed that at least one tyrosine and one histidine residue are essential for enzyme activity. The coenzyme-binding domain is located in the amino-terminal part of the polypeptide chain as revealed by a sequence comparison to other dinucleotide binding enzymes. Furthermore, there is evidence for a relationship to fumarate reductase and succinate dehydrogenase of E. coli.
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