A novel [2Fe-2S] ferredoxin from Pseudomonas putida mt2 promotes the reductive reactivation of catechol 2,3-dioxygenase
A novel [2Fe-2S] ferredoxin from Pseudomonas putida mt2 promotes the reductive reactivation of catechol 2,3-dioxygenase
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DOI：
10.1074/jbc.273.16.9622
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发表时间：
1998-04-17
影响因子：
4.8
通讯作者：
Jouanneau, Y
中科院分区：
文献类型：
--
作者：
Hugo, N;Armengaud, J;Jouanneau, Y
邻苯二酚α，3-双加氧酶（XS LE）是TOL质粒编码的降解甲苯和二甲苯的途径的组分，并催化芳环的双氧分解裂解。纯化的XylE在体外是氧敏感的和不稳定的，特别是在取代的儿茶酚底物存在下，但它在体内被另一种蛋白质XylT稳定，该蛋白质由位于xylE上游的xylT基因编码。在这项研究中，我们从含有高表达xylT基因的重组大肠杆菌菌株中纯化了XylT产物，并将其表征为新型[2Fe-2S]铁氧还蛋白，它是在中性pH下具有净正电荷的可溶性铁氧还蛋白的第一个实例。铁硫簇的EPR信号具有菱形对称性，与植物型铁氧还蛋白的情况一样，但XylT吸收光谱更接近于肾上腺素还蛋白的吸收光谱。在pH 8.5下，中点氧化还原电位测定为-373 +/-6 mV。XylT对于[2Fe-2S]铁氧还蛋白是异常不稳定的，在25 ° C空气中半衰期为69分钟，在37 ° C氩气中半衰期为70分钟。利用光化学还原的5-脱氮黄素来控制还原剂的产生，证明XylT介导纯化的无活性儿茶酚2的快速再活化，3-双加氧酶在体外，XylE由4-甲基儿茶酚失活导致活性位点铁氧化成高自旋三价铁状态，这是可通过EPR检测的，光谱证据表明，XylT通过还原酶的活性位点中的铁原子来重新激活XylE。这是铁氧还蛋白介导的酶再活化的第一例。恶臭假单胞菌mt 2细胞中XylT的表达水平低，并且计算的XylT/XylE摩尔比与XylE再活化涉及催化非化学计量量的XylT的提议一致。
Catechol a,3-dioxygenase (XS LE) is a component of the TOL plasmid-encoded pathway for the degradation of toluene and xylenes and catalyzes the dioxygenolytic cleavage of the aromatic ring. Purified XylE is oxygen-sensitive and unstable in vitro, particularly in the presence of substituted catechol substrates, but it is stabilized in vivo by another protein, XylT, encoded by the xylT gene located just upstream of xylE, In this study, we have purified to homogeneity the XylT product from a recombinant Escherichia coli strain containing a hyper-expressible xylT gene and characterized it as a novel [2Fe-2S] ferredoxin, It is the first example of a soluble ferredoxin with a net positive charge at neutral pH, The EPR signal of the iron sulfur cluster has rhombic symmetry as is the case for plant-type ferredoxins, but the XylT absorbance spectrum resembles more closely that of adrenodoxin, The midpoint redox potential was determined to be -373 +/- 6 mV, at pH 8.5, XylT was unusually unstable for a [2Fe-2S] ferredoxin, with half-lives of 69 min at 25 degrees C in air and 70 min at 37 degrees C in argon, With photochemically reduced 5-deazaflavin for the controlled generation of reductant, it was demonstrated that XylT mediates the rapid reactivation of purified inactive catechol 2,3-dioxygenase in vitro, Inactivation of XylE by 4-methylcatechol resulted in oxidation of the active site iron to a high spin ferric state that was detectable by EPR, Spectroscopic evidence presented here demonstrates that XylT reactivates XylE through reduction of the iron atom in the active site of the enzyme. It is the first instance of a ferredoxin-mediated reactivation of an enzyme. The level of expression of XylT in Pseudomonas putida mt2 cells is low and the calculated XylT/XylE molar ratio is consistent with the proposal that XylE reactivation involves catalytic nonstoichiometric amounts of XylT.