Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase.
Changes in the regiospecificity of aromatic hydroxylation produced by active site engineering in the diiron enzyme toluene 4-monooxygenase.
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DOI：
10.1021/bi971049t
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发表时间：
1997-08
期刊：
影响因子：
2.9
通讯作者：
J. Pikus;J. Studts;K. McClay;R. Steffan;B. Fox
中科院分区：
文献类型：
--
作者：
J. Pikus;J. Studts;K. McClay;R. Steffan;B. Fox
mendocina假单胞菌KR1甲苯4-单加氧酶是一种多组分二铁酶。二铁中心包含在羟化酶组分[alphabetagamma] 2的tmoA多肽中，Mr约为212 kDa。产品分布研究表明，天然异构体对甲苯（相对于字母表启动子kcat约为2 s-1）、邻二甲苯（kcat约为0.8 s-1）、间二甲苯（kcat约为0.6 s-1）和其他芳香烃的对羟基化具有高度特异性。这种程度的甲基苯的区域选择性是许多其他加氧酶无法比拟的。然而，在t4mo催化氧化对二甲苯的过程中（kcat约为0.4 s-1）， 4-甲基苄基醇是主要产物，这表明该酶可以与适当的底物催化芳香或苯基羟基化。位点定向诱变已被用于研究tmoA活性位点残基Q141、I180和F205对区域特异性的贡献。同种异构体Q141C和F205I产生了区域特异性的变化，远离了对甲酚的形成，与天然同种异构体相比，F205I使间甲酚形成的百分比增加了大约5倍。纯化后的Q141C对甲苯氧化的kcat约为0.2 s-1。在对二甲苯氧化过程中，Q141C异构体也主要作为芳香环羟化酶，这与天然异构体中观察到的主要苯基羟基化形成了直接对比，而F205I异构体在对二甲苯氧化过程中产生了几乎等量的苯基和酚类产物。对于所有被测试的底物，I180F异构体相对于天然异构体在产品分布上没有实质性的变化。根据所提出的活性位点模型，Q141和F205都位于靠近FeA铁位点的疏水区域，而I180则位于靠近FeA铁位点的疏水区域。这些研究表明，预测的最靠近FeA的疏水区域的变化会影响甲苯4-单加氧酶的区域特异性。
Pseudomonas mendocina KR1 toluene 4-monooxygenase is a multicomponent diiron enzyme. the diiron center is contained in the tmoA polypeptide of teh hydroxylase component [alphabetagamma)2,Mr approximately 212 kDa]. Product distribution studies reveal that the natural isoform is highly specific for para hydroxylation of toluene (kcat approximately 2 s-1 with respect to an alphabetagamma promoter), o-xylene (kcat approximately 0.8 s-1), m-xylene (kcat approximately 0.6 s-1), and other aromatic hydrocarbons. This degree of regioselectivity for methylbenzenes is unmatched by numerous other oxygenase enzymes. However, during the T4MO-catalyzed oxidation of p-xylene (kcat approximately 0.4 s-1), 4-methyl benzyl alcohol is the major product, showing that the enzyme could catalyze either aromatic or benzylic hydroxylation with the appropriate substrate. Site-directed mutagenesis has been used to study the contributions of tmoA active site residues Q141, I180, and F205 to the regiospecificity. Isoforms Q141C and F205I yielded shifts of regiospecificity away from p-cresol formation, with F205I giving an approximately 5-fold increase in the percentage of m-cresol formation relative to that of the natural isoform. The kcat of purified Q141C for toluene oxidation was approximately 0.2 s-1. Isoform Q141C also functioned predominantly as an aromatic ring hydroxylase during the oxidation of p-xylene, in direct contrast to the predominant benzylic hydroxylation observed for the natural isoform, while isoform F205I gave nearly equivalent amounts of benzylic and phenolic products from p-xylene oxidation. Isoform I180F gave no substantial shift in product distributions relativeto the natural isoform for all substrates tested. Upon the basis of a proposed active site model, both Q141 anf F205 are suggested to lie in a hydrophobic region closer to the FeA iron site, while I180 will be closer to FeB. These studies reveal that changes in the hydrophobic region predicted to be nearest to FeA can influence the regiospecificity observed for toluene 4-monooxygenase.