Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex
Expression and purification of the recombinant subunits of toluene/o-xylene monooxygenase and reconstitution of the active complex
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DOI：
10.1046/j.1432-1033.2002.03281.x
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发表时间：
2002-11-01
期刊：
影响因子：
--
通讯作者：
Di Donato, A
中科院分区：
文献类型：
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作者：
Cafaro, V;Scognamiglio, R;Di Donato, A
本文报道了斯氏假单胞菌OX 1甲苯/邻二甲苯单加氧酶复合物各组分编码基因的克隆、表达、纯化和鉴定。此外，重组亚基的活性复合物的重建已经获得，并确定了每个组件在电子供体到分子氧的电子转移中的功能作用。亚基B，E和A的共表达导致形成一个具有四级结构（X1）的亚复合物，命名为H（2），具有羟化酶活性。Tomo F组件是一种NADH氧化还原酶。纯化的酶每摩尔蛋白质中含有约1 mol的FAD、2 mol的铁和2 mol的酸不稳定的硫化物，如预期的那样存在一个[2Fe-2S]簇，并具有典型的黄素氧还蛋白吸收光谱。我们已经证明，这取决于我们已经纠正的基因序列中的微小错误。C组分是Rieske型铁氧还蛋白，其铁和酸不稳定硫化物含量与一个[2Fe-2S]簇的存在一致。该簇是非常敏感的氧damage.Mixtures的亚复合物H和亚基F，C和D能够氧化对甲酚成4-甲基cathecol，从而证明了完整的功能性的重组亚基作为purified.Finally，实验证据报告，这有力地支持了电子转移的模型。亚基F是电子传递链的第一个成员，它将电子从NADH转移到C，C将它们隧穿到H亚复合物，并最终到达分子氧。
This paper describes the cloning of the genes coding for each component of the complex of toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1, their expression, purification and characterization. Moreover, the reconstitution of the active complex from the recombinant subunits has been obtained, and the functional role of each component in the electron transfer from the electron donor to molecular oxygen has been determined.The coexpression of subunits B, E and A leads to the formation of a subcomplex, named H, with a quaternary structure (BEA)(2), endowed with hydroxylase activity.Tomo F component is an NADH oxidoreductase. The purified enzyme contains about 1 mol of FAD, 2 mol of iron, and 2 mol of acid labile sulfide per mol of protein, as expected for the presence of one [2Fe-2S] cluster, and exhibits a typical flavodoxin absorption spectrum.Interestingly, the sequence of the protein does not correspond to that previously predicted on the basis of DNA sequence. We have shown that this depends on minor errors in the gene sequence that we have corrected.C component is a Rieske-type ferredoxin, whose iron and acid labile sulfide content is in agreement with the presence of one [2Fe-2S] cluster. The cluster is very sensitive to oxygen damage.Mixtures of the subcomplex H and of the subunits F, C and D are able to oxidize p-cresol into 4-methylcathecol, thus demonstrating the full functionality of the recombinant subunits as purified.Finally, experimental evidence is reported which strongly support a model for the electron transfer. Subunit F is the first member of an electron transport chain which transfers electrons from NADH to C, which tunnels them to H sub-complex, and eventually to molecular oxygen.