Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam)).
Oxidation of polychlorinated benzenes by genetically engineered CYP101 (cytochrome P450(cam)).
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基因工程 CYP101（细胞色素 P450（cam））氧化多氯苯。
DOI：
10.1046/j.1432-1327.2001.02018.x
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发表时间：
2001
期刊：
影响因子：
--
通讯作者：
L. Wong
中科院分区：
文献类型：
--
作者：
J. P. Jones;Ellen J. O’Hare;L. Wong
多氯苯是难降解的环境污染物，主要是因为它们对微生物通常用于芳香族化合物生物降解的双加氧酶的攻击具有抗性。我们已经调查了血红素单加氧酶CYP 101（细胞色素P450（凸轮））从恶臭假单胞菌的突变体的多氯苯的氧化，其目的是产生新的系统，其生物降解。野生型CYP 101对二氯苯和三氯苯氧化为氯酚的活性较低，但对高氯化苯没有检测到产物。通过Y 96 F突变增加活性位点的疏水性，使活性增加了100倍，五氯苯和六氯苯都被缓慢氧化为五氯苯酚。用F87 W突变减少活性位点顶部的可用空间以迫使底物更靠近血红素结合，导致大多数底物的活性进一步增加10倍。同样在活性位点的顶部引入F98 W突变，降低了NADH-周转率，但增加了偶联效率，并且观察到F87 W-Y 96 F-F98 W突变体对1，3-二氯苯和1，3，5-三氯苯的偶联> 90%。V247 L突变体通常增加了NADH的周转率，F87 W-Y 96 F-V247 L突变体在不需要表面活性剂或有机助溶剂的情况下，对高度不溶性的五氯苯表现出相当快的NADH周转率（229分钟-1）。由于所有的氯酚都被微生物降解，因此可以构建新型的生物降解系统，其中CYP 101突变体将惰性多氯苯转化为酚，然后通过自然途径容易地降解。
Polychlorinated benzenes are recalcitrant environmental pollutants primarily because they are resistant to attack by dioxygenases commonly used by micro-organisms for the biodegradation of aromatic compounds. We have investigated the oxidation of polychlorinated benzenes by mutants of the haem mono-oxygenase CYP101 (cytochrome P450(cam)) from Pseudomonas putida with the aim of generating novel systems for their biodegradation. Wild-type CYP101 had low activity for the oxidation of dichlorobenzenes and trichlorobenzenes to the chlorophenols, but no products were detected for the heavily chlorinated benzenes. Increasing the active-site hydrophobicity with the Y96F mutation increased the activity up to 100-fold, and both pentachlorobenzene and hexachlorobenzene were oxidized slowly to pentachlorophenol. Decreasing the space available at the top of the active site with the F87W mutation to force the substrate to be bound closer to the haem resulted in a further 10-fold increase in activity with most substrates. Introducing the F98W mutation, also at the top of the active site, decreased the NADH-turnover rates but increased the coupling efficiencies, and > 90% coupling was observed for 1,3-dichlorobenzene and 1,3,5-trichlorobenzene with the F87W--Y96F--F98W mutant. The V247L mutation generally increased the NADH-turnover rates, and the F87W--Y96F--V247L mutant showed reasonably fast NADH turnover (229 min(-1)) with the highly insoluble pentachlorobenzene without the need for surfactants or organic cosolvents. As all chlorophenols are degraded by micro-organisms, novel biodegradation systems could be constructed in which CYP101 mutants convert the inert polychlorinated benzenes to the phenols, which are then readily degraded by natural pathways.
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影响因子：
5.6
作者：
POULOS, TL;FINZEL, BC;HOWARD, AJ
通讯作者：
HOWARD, AJ
影响因子：
4.1
作者：
Rietjens, IMCM;denBesten, C;vanBladeren, PJ
通讯作者：
vanBladeren, PJ
通过定点诱变揭示细胞色素 P-450cam 活性位点氢键的作用。
DOI：
--
发表时间：
1988
期刊：
The Journal of biological chemistry
影响因子：
--
作者：
Atkins,WM;Sligar,SG
通讯作者：
Sligar,SG
影响因子：
2.9
作者：
LOIDA, PJ;SLIGAR, SG
通讯作者：
SLIGAR, SG