Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.
Purification and properties of NADH-ferredoxinTOL reductase. A component of toluene dioxygenase from Pseudomonas putida.
复制标题
NADH-铁氧还蛋白TOL 还原酶的纯化和性质。
DOI：
10.1016/s0021-9258(19)69675-4
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发表时间：
1981
期刊：
影响因子：
--
通讯作者：
D. Gibson
中科院分区：
文献类型：
--
作者：
V. Subramanian;T. Liu;W. Yeh;M. Narro;D. Gibson
恶臭假单胞菌细胞在甲苯作用下生长后，含有一个能将甲苯氧化成(+)-1(S)，2(R)-dihydroxy-3-methyl-cyclohexa-3，5-diene.的多组份的酶系统这些成分中的一个已经被纯化到均一，并被证明是一种黄素蛋白，它含有FAD作为唯一可检测到的修复基团。FAD在纯化过程中被从酶中去除。平衡透析实验表明，该酶可结合一摩尔FAD/摩尔的酶蛋白。在十二烷基硫酸钠和巯基乙醇存在下，经凝胶过滤和聚丙烯酰胺凝胶电泳法测定，该酶的表观分子量为46,000。后一种结果表明存在单一的多肽链。该酶的氨基酸组成表明，疏水氨基酸亮氨酸、异亮氨酸和缬氨酸的含量相对较高，并且在组成上与在某些单加氧酶系统中起作用的黄素蛋白非常相似。纯化的酶催化二氯吲酚、亚硝基四氮唑、铁氰化铁和铁氧还原醇的还原。它还原细胞色素c的能力和在甲苯双加氧酶体系中发挥作用的能力完全依赖于铁氧还蛋白TOL的存在。
Cells of Pseudomonas putida, after growth with toluene, contain a multicomponent enzyme system that oxidizes toluene to (+)-1(S),2(R)-dihydroxy-3-methyl-cyclohexa-3,5-diene. One of these components has been purified to homogeneity and shown to be a flavoprotein that contains FAD as the only detectable prosthetic group. Fad was removed from the enzyme during purification. However, equilibrium dialysis experiments showed that the enzyme can bind one mol of FAD/mol of enzyme protein. The apparent molecular weight of the enzyme is 46,000, as judged by gel filtration and polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and mercaptoethanol. The latter result suggests the presence of a single polypeptide chain. The amino acid composition of the enzyme reveals a relatively high content of the hydrophobic amino acids leucine, isoleucine, and valine and is remarkably similar in composition to the flavoproteins that function in certain monooxygenase enzyme systems. The purified enzyme catalyzes the reduction of dichloroindophenol, nitrobluetetrazolium, ferricyanide, and ferredoxinTOL. Its ability to reduce cytochrome c and to function in the toluene dioxygenase enzyme system is absolutely dependent on the presence of ferredoxinTOL.