4-TOLUENE SULFONATE METHYL-MONOOXYGENASE FROM COMAMONAS-TESTOSTERONI T-2 - PURIFICATION AND SOME PROPERTIES OF THE OXYGENASE COMPONENT
4-TOLUENE SULFONATE METHYL-MONOOXYGENASE FROM COMAMONAS-TESTOSTERONI T-2 - PURIFICATION AND SOME PROPERTIES OF THE OXYGENASE COMPONENT
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DOI：
10.1128/jb.173.12.3741-3748.1991
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发表时间：
1991-06-01
影响因子：
3.2
通讯作者：
COOK, AM
中科院分区：
文献类型：
--
作者：
LOCHER, HH;LEISINGER, T;COOK, AM
睾酮单胞菌T-2在TS-盐培养基中合成了一种诱导酶系统，可将4-甲苯磺酸（TS）氧化为4-亚砜醇。我们纯化了这个TS甲基-单加氧酶体系（TSMOS），发现它由两个组分组成。一种单体铁硫黄蛋白（成分B），已被证明在这种生物的4-亚砜苯甲酸酯双加氧酶系统中起还原酶的作用（H. H. Locher， T. Leisinger, and A. M. Cook, Biochem）。[j] . 21 (3): 1 - 2；该加氧酶具有铁硫蛋白的紫外可见光谱特征。天然加氧酶的M(r)s约为152,000，变性条件下的M(r)s约为43,000，表明其为同三聚体或同四聚体酶，其n端氨基酸和氨基酸组成被确定。通过添加Fe2+，纯化酶的活性提高了约5倍。在O2和NADH存在下，组分B和M共同催化TS或p-甲苯磺酸转化为相应的醇。通过观察羧基苄基醇中（O2）O-18的氧原子，证实了该反应为甲基的氧合反应。TSMOS的底物范围包括羧基化的TS类似物（对甲苯、间甲苯和4-苯甲酸乙酯），而对二甲苯、甲苯和对甲酚不是底物。TSMOS还能催化去甲基化；4-甲氧基苯甲酸酯转化为4-羟基苯甲酸酯和甲醛。
Comamonas testosteroni T-2 synthesizes an inducible enzyme system that oxygenates 4-toluene sulfonate (TS) to 4-sulfobenzyl alcohol when grown in TS-salts medium. We purified this TS methyl-monooxygenase system (TSMOS) and found it to consist of two components. A monomeric, iron-sulfur flavoprotein (component B), which has been shown to act as a reductase in the 4-sulfobenzoate dioxygenase system of this organism (H. H. Locher, T. Leisinger, and A. M. Cook, Biochem. J. 274:833-842, 1991), carried electrons from NADH to component M, an oxygenase. This oxygenase had the UV-visible spectral characteristics of an iron-sulfur protein. M(r)s of about 152,000 for the native oxygenase and of 43,000 under denaturing conditions indicated a homotri- or homotetrameric enzyme, whose N-terminal amino acids and amino acid composition were determined. The activity of the purified enzyme was enhanced about fivefold by the addition of Fe2+. In the presence of O2 and NADH, components B and M together catalyzed the stoichiometric transformation of TS or p-toluate to the corresponding alcohol. The reaction was confirmed as oxygenation of the methyl group by observation of an oxygen atom from (O2)O-18 in carboxybenzyl alcohol. The substrate range of TSMOS included carboxylated analogs of TS (p- and m-toluates and 4-ethylbenzoate), whereas p-xylene, toluene, and p-cresol were not substrates. TSMOS also catalyzed demethylation; 4-methoxybenzoate was transformed to 4-hydroxybenzoate and formaldehyde.