Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology
Molecular Lego: design of molecular assemblies of P450 enzymes for nanobiotechnology
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DOI：
10.1016/s0956-5663(01)00286-x
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发表时间：
2002-01-01
影响因子：
12.6
通讯作者：
Giannini, S
中科院分区：
文献类型：
--
作者：
Gilardi, G;Meharenna, YT;Giannini, S
本文报道了分子乐高方法在P450酶中的应用。蛋白质结构域用作催化（P450 BM 3血红素结构域和人P450 2 EI）或电子转移（黄素氧还蛋白和P450 BM 3还原酶）模块。我们的目标是建立具有改进的电化学性能的组件，构建可溶性人P450酶，并产生基于P450 BM 3的新P450催化模块库。从巨大芽孢杆菌细胞色素P450 BM 3（BMP）和脱硫弧菌黄素氧还蛋白（FLD）的可溶性血红素结构域中获得了一个合理设计的基因融合组装体（BMP FLD）。组装成功表达，并以其活性形式表征。显示出改善的电化学性能。通过将2 EI酶的关键元件与P450 BM 3的选定部分融合，实现了人膜结合P450 2 E1（2 E1）的溶解。构建了含有P450 BM 3的前54个残基、P450 2 E1从残基81开始的全序列和P450 BM 3的还原酶结构域的组装体。2 E1-BM 3组装体在大肠杆菌胞质中成功表达。可溶形式的2 E1-BM 3在一氧化碳气氛中被还原，并在450 nm处显示出典型的吸收峰，这是折叠和活性P450酶的特征。最后，本实验室先前开发的碱法用于在P450 BM 3的随机突变体库中筛选P450活性。许多变体对非生理底物具有活性。例如杀虫剂和多环芳烃。提供新的P450催化模块。这三个研究领域的结合为纳米生物技术的开发提供了有趣的工具。(C)2002 Elsevier Science B. V.保留所有权利。
This paper reports on the application of the molecular Lego approach to P450 enzymes. Protein domains are used as catalytic (P450 BM3 haem domain and human P450 2EI) or electron transfer (flavodoxin and P450 BM3 reductase) modules. The objectives are to build assemblies with improved electrochemical properties, to construct soluble human P450 enzymes, and to generate libraries of new P450 catalytic modules based on P450 BM3. A rationally designed, gene-fused assembly (BMP-FLD) was obtained from the soluble haem domain of cytochrome P450 BM3 from Bacillus megaterium (BMP) and flavodoxin from Desulfovibrio vulgaris (FLD). The assembly was expressed successfully and characterised in its active form. displaying improved electrochemical properties. Solubilisation of the human, membrane-bound P450 2E1 (2E1) was achieved by fusing key elements of the 2EI enzyme with selected parts of P450 BM3. An assembly containing the first 54 residues of P450 BM3, the whole sequence of P450 2E1 from residue 81 and the reductase domain of P450 BM3 was constructed. The 2E1 - BM3 assembly was successfully expressed in the cytosol of Escherichia coli. The soluble form of 2E1-BM3 was reduced in carbon monoxide atmosphere and displayed the typical absorption peak at 450 nm, characteristic of a folded and active P450 enzyme. Finally, the alkali method previously developed in this laboratory was used to screen for P450 activity within a library of random mutants of P450 BM3. A number of variants active towards non-physiological substrates. Such as pesticides and polyaromatic hydrocarbons were identified. providing new P450 catalytic modules. The combination of these three areas of research provide interesting tools for exploitation in nanobiotechnology. (C) 2002 Elsevier Science B.V. All rights reserved.