Directed Evolution of Mammalian Cytochrome P450 2B1
نویسندگان
چکیده
منابع مشابه
Engineering mammalian cytochrome P450 2B1 by directed evolution for enhanced catalytic tolerance to temperature and dimethyl sulfoxide.
The previously laboratory-evolved cytochrome P450 2B1 quadruple mutant V183L/F202L/L209A/S334P (QM), which showed enhanced H(2)O(2)-mediated substrate oxidation, has now been shown to exhibit a >3.0-fold decrease in K(m,HOOH) for 7-ethoxy-4-trifluoromethylcoumarin (7-EFC) O-deethylation compared with the parental enzyme L209A. Subsequently, a streamlined random mutagenesis and a high-throughput...
متن کاملDirected evolution of mammalian cytochrome P450 2B1: mutations outside of the active site enhance the metabolism of several substrates, including the anticancer prodrugs cyclophosphamide and ifosfamide.
Cytochrome P450 2B1 has been subjected to directed evolution to investigate the role of amino acid residues outside of the active site and to engineer novel, more active P450 catalysts. A high throughput screening system was developed to measure H(2)O(2)-supported oxidation of the marker fluorogenic substrate 7-ethoxy-4-trifluoromethylcoumarin (7-EFC). Random mutagenesis by error-prone polymera...
متن کاملCytochrome P450 2B1 mediates complement-dependent sublytic injury in a model of membranous nephropathy.
Membranous nephropathy is a disease that affects the filtering units of the kidney, the glomeruli, and results in proteinuria accompanied by loss of kidney function. Passive Heymann nephritis is an experimental model that mimics membranous nephropathy in humans, wherein the glomerular epithelial cell (GEC) injury induced by complement C5b-9 leads to proteinuria. We examined the role of cytochro...
متن کاملCovalent modification of Thr302 in cytochrome P450 2B1 by the mechanism-based inactivator 4-tert-butylphenylacetylene.
The mechanism of inactivation of cytochrome P450 2B1 (CYP2B1) by 4-tert-butylphenylacetylene (BPA) has been characterized previously to be caused by the covalent binding of a reactive intermediate to the apoprotein rather than heme destruction (J Pharmacol Exp Ther 331:392-403, 2009). The identification of a BPA-glutathione conjugate and the increase in the mass of the BPA-adducted apoprotein h...
متن کاملCatalytic properties of an expressed cytochrome P450 2B1 from a Wistar-Kyoto rat liver cDNA library.
Cytochrome P450 2B1 clones were isolated from a phenobarbital-induced Wistar-Kyoto (WKY) hepatic cDNA library and were found to contain a Glu-322 --> Val substitution, compared with wild-type 2B1 from Sprague-Dawley rats. After heterologous expression in Escherichia coli and purification, activities of this 2B1 E322V variant were determined for ethoxycoumarin and androstenedione. The total acti...
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ژورنال
عنوان ژورنال: Journal of Biological Chemistry
سال: 2005
ISSN: 0021-9258
DOI: 10.1074/jbc.m500158200