Exploration of natural and artificial sequence spaces:: Towards a functional remodeling of membrane-bound cytochrome P450

被引:7
作者
Abécassis, V
Urban, P
Aggerbeck, L
Truan, G
Pompon, D [1 ]
机构
[1] CNRS, Ctr Genet Mol, UPR 2167, F-91190 Gif Sur Yvette, France
[2] CNRS, Plate Forme Puces ADN Gif Orsay, F-91190 Gif Sur Yvette, France
关键词
combinatorial library; P450; sequence mapping; CYP1A1; CYP1A2; structure-function;
D O I
10.1080/1024242031000121502
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Two complementary methods are described that associate in vitro and in vivo steps to generate sequence diversity by segment directed saturated mutagenesis and family shuffling. A high-throughput DNA chip-based procedure for the characterization and potentially the equalization of combinatorial libraries is also presented. Using these approaches, two combinatorial libraries of cytochrome P450 variants derived from the CYP1A subfamily were constructed and their sequence diversity characterized. The results of functional screening using high-throughput tools for the characterization of membrane P450-catalyzed activities, suggest that the 204-214 sequence segment of human CYP1A1 is not critical for polycyclic aromatic hydrocarbon recognition, as was hypothesized from previous data. Moreover, mutations in this segment do not alter the discrimination between alkoxyresorufins, which, for all tested mutants, remained similar to that of wild-type CYP1A1. In contrast, the constructed CYP1A1-CYP1A2 mosaic structures, containing multiple crossovers, exhibit a wide range of substrate preference and regioselectivity. These mosaic structures also discriminate between closely related alkoxyresorufin substrates. These results open the way to global high-throughput analysis of structure-function relationships using combinatorial libraries of enzymes together with libraries of structurally related substrates.
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页码:55 / 66
页数:12
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