Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: Potential mononuclear non-heme iron coordination sites
Site-directed mutagenesis of conserved amino acids in the alpha subunit of toluene dioxygenase: Potential mononuclear non-heme iron coordination sites
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DOI：
10.1128/jb.178.11.3133-3139.1996
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发表时间：
1996-06-01
影响因子：
3.2
通讯作者：
Gibson, DT
中科院分区：
文献类型：
--
作者：
Jiang, HY;Parales, RE;Gibson, DT
恶臭假单胞菌F1甲苯双加氧酶的末端加氧酶组分是一种需要单核铁来维持酶活性的铁硫蛋白（ISPTOL）。对末端加氧酶的大（α）亚基的所有可用预测氨基酸序列的比对显示了一个保守的单核铁结合残基簇。它们位于ISPTOL α亚基（TodC1）的氨基酸210和230之间。保守的氨基酸Glu-214、Asp-219、tyrr -221、His-222和His-228分别通过定点诱变被丙氨酸残基独立取代。TodC1中的tyr266被认为是铁配体，以相同的方式处理。为了测定TodC1及其突变体存在下的甲苯双加氧酶活性，我们开发并优化了从TodC1和纯化的TodC2 （β亚基）重建野生型ISPTOL活性的条件。Glu-214、Asp-219、His-222或His-228的突变完全破坏了甲苯双加氧酶的活性。在tyr1 -221或tyr1 -266上有丙氨酸取代的TodC1保留了部分酶活性（分别为42%和12%）。在[C-14]甲苯的实验中，两个Tyr- >Ala突变导致顺式-[C-14]-甲苯二氢醇的形成量减少，而在Glu-214， Asp-219， His-222或His-228突变消除了顺式-甲苯二氢醇的形成。经十二烷基硫酸钠-聚丙烯酰胺凝胶电泳和免疫印迹（Western blot）分析，所有突变TodC1蛋白的表达水平与野生型TodC1相当。这些结果，结合预测的22个加氧酶组分的氨基酸序列，表明保守基序Glu-X(3-4)-Asp-X(2)-His- x (4-5)-His对催化功能至关重要，谷氨酸、天冬氨酸和组氨酸残基可能在氧激活位点充当单核铁配体。
The terminal oxygenase component of toluene dioxygenase from Pseudomonas putida F1 is an iron-sulfur protein (ISPTOL) that requires mononuclear iron for enzyme activity. Alignment of all available predicted amino acid sequences for the large (alpha) subunits of terminal oxygenases showed a conserved cluster of potential mononuclear iron-binding residues. These were between amino acids 210 and 230 in the alpha subunit (TodC1) of ISPTOL. The conserved amino acids, Glu-214, Asp-219, Tyr-221, His-222, and His-228, were each independently replaced with an alanine residue by site-directed mutagenesis. Tyr-266 in TodC1, which has been suggested as an iron ligand, was treated in an identical manner. To assay toluene dioxygenase activity in the presence of TodC1 and its mutant forms, conditions for the reconstitution of wild-type ISPTOL activity from TodC1 and purified TodC2 (beta subunit) were developed and optimized. A mutation at Glu-214, Asp-219, His-222, or His-228 completely abolished toluene dioxygenase activity. TodC1 with an alanine substitution at either Tyr-221 or Tyr-266 retained partial enzyme activity (42 and 12%, respectively). In experiments with [C-14]toluene, the two Tyr-->Ala mutations caused a reduction in the amount of cis-[C-14]-toluene dihydrodiol formed, whereas a mutation at Glu-214, Asp-219, His-222, or His-228 eliminated cis-toluene dihydrodiol formation. The expression level of all of the mutated TodC1 proteins was equivalent to that of wild-type TodC1 as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western blot (immunoblot) analyses. These results, in conjunction with the predicted amino acid sequences of 22 oxygenase components, suggest that the conserved motif Glu-X(3-4)-Asp-X(2)-His-X(4-5)-His is critical for catalytic function and the glutamate, aspartate, and histidine residues may act as mononuclear iron ligands at the site of oxygen activation.