Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene
Protein engineering of toluene 4-monooxygenase of Pseudomonas mendocina KR1 for synthesizing 4-nitrocatechol from nitrobenzene
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DOI：
10.1002/bit.20185
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发表时间：
2004-09-20
影响因子：
3.8
通讯作者：
Wood, TK
中科院分区：
文献类型：
--
作者：
Fishman, A;Tao, Y;Wood, TK
在发现mendocina Pseudomonas KR1的甲苯4-单加氧酶（T4MO）能以极低的速率将硝基苯氧化为4-硝基邻苯二酚后，利用定向进化和饱和诱变技术对该反应进行了改进。利用大肠杆菌TG1/pBS(Kan)T4MO在含有硝基苯的琼脂板上对tmoAB进行易出错PCR随机突变文库中550个菌落的筛选，发现了产生硝基儿茶酚的突变体。其中一个突变体NB1含有6个氨基酸取代（TmoA Y22N、184Y、S95T、I100S、S400C; TmoB D79N）。根据我们实验室之前对洋葱伯克霍尔德菌G4的甲苯邻位单加氧酶的类似酶的研究结果，我们认为羟基化酶（TmoA）的a亚基I100位是底物反应性变化最显著的位置。该位置的饱和诱变又产生了两个硝基儿茶酚突变体I100A和I100S；在200 muM硝基苯条件下，I100A生成4-硝基儿茶酚的速率是野生型T4MO的16倍以上（0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min-mg）。HPLC和质谱分析显示，NB 1、I100A和I100S变异株主要通过间硝基苯酚产生4-硝基儿茶酚，而野生型主要产生对硝基苯酚和少量的硝基儿茶酚。与野生型T4MO相比，表达变异I100A的全细胞将硝基苯转化为间硝基酚的V-max分别为0.61 +/- 0.037和0.16 +/- 0.071 nmol/min-mg蛋白，将间硝基酚转化为硝基酚的V-max分别为3.93 +/- 0.26和0.58 +/- 0.033 nmol/min-mg蛋白。因此，随机诱变改变了硝基苯氧化的区域特异性，导致4-硝基儿茶酚的产量显著增加。甲苯氧化的区域特异性也发生了变化，所有突变体产生20%间甲酚和80%对甲酚，而野生型产生96%对甲酚。有趣的是，I100A对甲苯（酶的天然底物）的氧化速率也比I100A高65% （7.2 +/- 1.2 vs 4.4 +/- 0.3 nmol/min-mg）。基于同源性的TmoA模型表明，减小I100侧链的尺寸会导致活性位点通道的宽度增加，从而促进底物的接近并促进更灵活的取向。(C) 2004 Wiley期刊有限公司
After discovering that toluene 4-monooxygenase (T4MO) of Pseudomonas mendocina KR1 oxidizes nitrobenzene to 4-nitrocatechol, albeit at a very low rate, this reaction was improved using directed evolution and saturation mutagenesis. Screening 550 colonies from a random mutagenesis library generated by error-prone PCR of tmoAB using Escherichia coli TG1/pBS(Kan)T4MO on agar plates containing nitrobenzene led to the discovery of nitrocatechol-producing mutants. One mutant, NB1, contained six amino acid substitutions (TmoA Y22N, 184Y, S95T, I100S, S400C; TmoB D79N). It was believed that position I100 of the a subunit of the hydroxylase (TmoA) is the most significant for the change in substrate reactivity due to previous results in our lab with a similar enzyme, toluene ortho-monooxygenase of Burkholderia cepacia G4. Saturation mutagenesis at this position resulted in the generation of two more nitrocatechol mutants, I100A and I100S; the rate of 4-nitrocatechol formation by I100A was more than 16 times higher than that of wild-type T4MO at 200 muM nitrobenzene (0.13 +/- 0.01 vs. 0.008 +/- 0.001 nmol/min-mg protein). HPLC and mass spectrometry analysis revealed that variants NB 1, I100A, and I100S produce 4-nitrocatechol via m-nitrophenol, while the wild-type produces primarily p-nitrophenol and negligible amounts of nitrocatechol. Relative to wild-type T4MO, whole cells expressing variant I100A convert nitrobenzene into m-nitrophenol with a V-max of 0.61 +/- 0.037 vs. 0.16 +/- 0.071 nmol/min-mg protein and convert m-nitrophenol into nitrocatechol with a V-max of 3.93 +/- 0.26 vs. 0.58 +/- 0.033 nmol/min-mg protein. Hence, the regiospecificity of nitrobenzene oxidation was changed by the random mutagenesis, and this led to a significant increase in 4-nitrocatechol production. The regiospecificity of toluene oxidation was also altered, and all of the mutants produced 20% m-cresol and 80% p-cresol, while the wild-type produces 96% p-cresol. Interestingly, the rate of toluene oxidation (the natural substrate of the enzyme) by I100A was also higher by 65% (7.2 +/- 1.2 vs. 4.4 +/- 0.3 nmol/min-mg protein). Homology-based modeling of TmoA suggests reducing the size of the side chain of I100 leads to an increase in the width of the active site channel, which facilitates access of substrates and promotes more flexible orientations. (C) 2004 Wiley Periodicals, Inc.