Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase
Expression, purification, and characterization of Mycobacterium tuberculosis mycothione reductase
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DOI：
10.1021/bi991025h
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发表时间：
1999-09-07
期刊：
影响因子：
2.9
通讯作者：
Blanchard, JS
中科院分区：
文献类型：
--
作者：
Patel, MP;Blanchard, JS
来自人类病原体结核分枝杆菌的菌硫酮还原酶已被克隆，在耻垢分枝杆菌中表达，并以 43% 的产率纯化 145 倍，达到同质性。菌硫酮还原酶与功能上同源的谷胱甘肽和锥硫酮还原酶的氨基酸序列比对表明，催化重要的氧化还原活性二硫键、组氨酸-谷氨酸离子对以及参与结合 FAD 辅因子和底物 NADPH 的区域的保守性。均质的 50 kDa 亚基酶以同二聚体形式存在，并且是 NADPH 依赖性的，并且对结构独特的低分子量二硫化物、菌硫酮具有高度特异性，NADPH 和菌硫酮的米氏常数分别为 8 和 73 μM。 HPLC 分析表明每个单体存在 1 mol 结合的 FAD 作为辅因子，在 462 nm 处呈现出 lambda(max) 吸收光谱，消光系数为 11 300 M-1 cm(-1)。用 NADH 对酶进行还原滴定表明存在假定的催化硫醇盐之一的电荷转移复合物，FAD 在约 100 nm 处吸收。 530 纳米。与连续截短的菌硫酮和其他二硫化物和吡啶核苷酸类似物的反应表明，菌硫酮的葡萄糖胺部分需要严格的最小二硫键底物要求。该酶对二硫化物和醌底物表现出双双乒乓动力学。使用 NADH 和硫代 NADP(+) 观察转氢酶活性，证实了其动力学机制。我们建议麦硫酮还原酶作为氧化还原酶的 I 类黄素蛋白二硫化物还原酶家族的最新成员。
Mycothione reductase from the human pathogen Mycobacterium tuberculosis has been cloned, expressed in Mycobacterium smegmatis, and purified 145-fold to homogeneity in 43% yield. Amino acid sequence alignment of mycothione reductase with the functionally homologous glutathione and trypanothione reductase indicates conservation of the catalytically important redox-active disulfide, histidine-glutamate ion pair, and regions involved in binding both the FAD cofactor and the substrate NADPH. The homogeneous 50 kDa subunit enzyme exists as a homodimer and is NADPH-dependent and highly specific for the structurally unique low-molecular mass disulfide, mycothione, exhibiting Michaelis constants of 8 and 73 mu M for NADPH and mycothione, respectively. HPLC analysis indicated the presence of 1 mol of bound FAD per monomer as the cofactor exhibiting an absorption spectrum with a lambda(max) at 462 nm with an extinction coefficient of 11 300 M-1 cm(-1), The reductive titration of the enzyme with NADH indicates the presence of a charge-transfer complex of one of the presumptive catalytic thiolates and FAD absorbing at ca. 530 nm. Reaction with serially truncated mycothione and other disulfides and pyridine nucleotide analogues indicates a strict minimal disulfide substrate requirement for the glucosamine moiety of mycothione. The enzyme exhibits bi-bi ping-pong kinetics with both disulfide and quinone substrates. Transhydrogenase activity is observed using NADH and thio-NADP(+), confirming the kinetic mechanism. We suggest mycothione reductase as the newest member of the class I flavoprotein disulfide reductase family of oxidoreductases.