Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells
Reaction mechanisms of non-heme diiron hydroxylases characterized in whole cells
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DOI：
10.1016/j.jinorgbio.2005.06.020
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发表时间：
2005-10-01
影响因子：
3.9
通讯作者：
Austin, RN
中科院分区：
文献类型：
--
作者：
Bertrand, E;Sakai, R;Austin, RN
表达非血红素二铁羟化酶AlkB和甲苯4-单加氧酶（T4 MO）的全细胞用于探测酶反应机制。AlkB催化自由基时钟底物双环[4.1.0]庚烷（降冰片烷）、螺辛烷和1，1-二乙基环丙烷的羟基化，并且不催化自由基时钟1，1-二甲基环丙烷或1，1，2，2-四甲基环丙烷的羟基化。降冰片烷的羟基化产生的产物分布与大肠杆菌中表达的野生型恶臭假单胞菌GPo 1和来自恶臭假单胞菌GPol的AlkB中的酶的“氧反弹”机制一致。在反应机制中存在基于底物的自由基的证据是清楚的。对于降冰片烷，该自由基的寿命随实验条件而变化。实验与较高的基板浓度产生较短的自由基寿命（约1纳秒），而实验与较低的基板浓度产生较长的自由基寿命（约19纳秒）。使用野生型或配备AlkB的宿主生物体，使用“静息细胞”或“生长细胞”方法获得一致的结果。T4 MO在E.大肠杆菌还催化降冰片烷的羟基化，自由基寿命约为0.07 ns。没有自由基寿命依赖于衬底浓度。来自用二乙基环丙烷、螺辛烷、二甲基环丙烷和二乙基环丙烷的实验的结果与AIkB的受限活性位点一致。(c)2005年爱思唯尔公司All rights reserved.
Whole cells expressing the non-heme diiron hydroxylases AlkB and toluene 4-monooxygenase (T4MO) were used to probe enzyme reaction mechanisms. AlkB catalyzes the hydroxylation of the radical clock substrates bicyclo[4.1.0]heptane (norcarane), spirooctane and 1,1-diethylcyclopropane, and does not catalyze the hydroxylation of the radical clocks 1,1-dimethylcyclopropane or 1,1,2,2-tetramethylcyclopropane. The hydroxylation of norcarane yields a distribution of products consistent with an "oxygen-rebound" mechanism for the enzyme in both the wild type Pseudomonas putida GPo1 and AlkB from P. putida GPol expressed in Escherichia coli. Evidence for the presence of a substrate-based radical during the reaction mechanism is clear. With norcarane, the lifetime of that radical varies with experimental conditions. Experiments with higher substrate concentrations yield a shorter radical lifetime (approximate to 1 ns), while experiments with lower substrate concentrations yield a longer radical lifetime (approximate to 19 ns). Consistent results were obtained using either wild type or AlkB-equipped host organisms using either "resting cell" or "growing cell" approaches. T4MO expressed in E. coli also catalyzes the hydroxylation of norcarane with a radical lifetime of approximate to 0.07 ns. No radical lifetime dependence on substrate concentration was seen. Results from experiments with diethylcyclopropane, spirooctane, dimethylcyclopropane, and diethylcyclopropane are consistent with a restricted active site for AIkB. (c) 2005 Elsevier Inc. All rights reserved.