A new class of arylamine oxygenases:: Evidence that p-aminobenzoate N-oxygenase (AurF) is a di-iron enzyme and further mechanistic studies
A new class of arylamine oxygenases:: Evidence that p-aminobenzoate N-oxygenase (AurF) is a di-iron enzyme and further mechanistic studies
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DOI：
10.1002/cbic.200600136
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发表时间：
2006-08-01
期刊：
影响因子：
3.2
通讯作者：
Zhao, Huimin
中科院分区：
文献类型：
--
作者：
Simurdiak, Michael;Lee, Jungkul;Zhao, Huimin
芳胺加氧酶是催化芳基胺不寻常的氧化成芳基硝基的酶。虽然芳胺加氧酶似乎广泛存在并应用于多种代谢反应中，但[1,2]氨基硝基氧合酶（PrnD）[2]和对氨基苯甲酸酯加氧酶（AurF）[3]是已知的仅有的两个例子。近年来，人们对PrnD进行了生物化学和力学表征。[2,4]然而，AurF的表征有限，主要是由于难以在体外获得功能性酶。[1,5]此外，先前的数据库搜索（BLAST， PROSITE）未能在AurF achtungtrennung序列中发现任何已知的氧化还原酶辅因子结合位点或基序；这表明AurF可能是一类新的芳胺加氧酶[1,3]。我们使用生物学工作台（http://workbench）对AurF进行了自己的BLAST搜索。sdsc。edu)，并鉴定出4个与AurF序列同源性较低的推定蛋白（同源性为15-17%）。Clustal W进一步测序发现了一些保守的天冬氨酸、谷氨酸和组氨酸残基，以及EX28-37DEXXH基序的两个拷贝。该基序在几种双铁加氧酶中保存良好，[6-13]表明AurF可能是一种双核非血红素铁加氧酶。为了验证这一假设，我们将硫链霉菌（ATCC 12310）的aurF基因克隆到pTrc99A中，并在大肠杆菌BL21 （DE3）中异源表达。如先前链霉菌实验报道的那样，在使用磷酸盐-盐水溶液反应介质时，无法观察到大肠杆菌中AurF的体内活性。[5,14]然而，在反应介质中加入葡萄糖、甘油、醋酸盐或琥珀酸盐等碳源可以恢复大肠杆菌体内的AurF活性；这表明酶的功能需要某种细胞过程。这一结果与在链霉菌实验中观察到的结果不同。在我们的实验室和其他的一样
Arylamine oxygenases are enzymes that catalyze the unusual oxidation of an aryl amine group to an aryl nitro group. Although arylamine oxygenases seem to be widespread and used in diverse metabolic reactions,[1, 2] aminopyrrolnitrin oxygenase (PrnD)[2] and p-aminobenzoate oxygenase (AurF)[3] are the only two known examples. Recently, PrnD was biochemically and mechanistically characterized.[2, 4] AurF, however, has had limited characterization, mainly due to the difficulty of obtaining functional enzymes in vitro.[1, 5] In addition, previous database searches (BLAST, PROSITE) failed to identify any known oxidoreductase cofactor binding sites or motifs in the AurF ACHTUNGTRENNUNGsequence;[1, 3] this indicated that AurF could be a novel class of arylamine oxygenases.We performed our own BLAST search on AurF using Biology Workbench (http://workbench. sdsc. edu) and identified four putative proteins that share low sequence homology with AurF (15–17% identity). Further sequence alignment by Clustal W revealed several conserved aspartic acid, glutamic acid, and histidine residues, and the conservation of two copies of an EX28–37DEXXH motif. That this motif is well conserved in several di-iron oxygenases,[6–13] suggests that AurF might be a dinuclear non-heme iron oxygenase. To test this hypothesis, we cloned the aurF gene from Streptomyces thioluteus (ATCC 12310) into pTrc99A and heterologously expressed it in Escherichia coli BL21 (DE3). In vivo AurF activity in E. coli could not be observed when using the phosphate–saline solution reaction medium, as reported previously in Streptomyces experiments.[5, 14] However, addition of a carbon source such as glucose, glycerol, acetate, or succinate to the reaction media restored in vivo AurF activity in E. coli; this suggests that a certain cellular process is needed for enzyme function. This result is different from what was observed in experiments with Streptomyces. In our laboratory as well as in other