Cytochrome P450 (CYP) mutants and substrate-specificity alterations: segment-directed mutagenesis applied to human CYP1A1.
Cytochrome P450 (CYP) enzymes represent a large superfamily that displays extraordinarily diverse substrate specificities. After a concise review about CYPs of the CYP1A subfamily, which plays a crucial role in procarcinogen activation, this paper presents segment-directed mutagenesis. This approach generates a library of random combinatorial mutants limited to a precise region of human CYP1A1, namely amino acids 204-214 in which nine positions differ between CYP1A1 and CYP1A2. The resulting mutants present all combinations possible among these nine positions shifting mutated residues to their CYP1A2 counterpart. The mutants were cloned and expressed in an engineered Saccharomyces cerevisiae strain that has a microsomal oxido-reduction environment optimized for CYPs. This procedure resulted in yeast transformants that express a library of mutant CYP1A1. A subset of transformants were chosen at random, assayed for a typical CYP1A1 activity and the plasmidic DNA of functional clones was rescued and sequenced. In this approach, no preconceived idea is made as to which combination of amino acid residues controls substrate selectivity. The functional mutants were analysed further for alteration of substrate specificity with a series of heterocyclic and polycyclic aromatic hydrocarbons. Some of the implications of these analyses are discussed for the role of this region in substrate specificity, since it corresponds to a putative loop and is not part of one of the CYP substrate-recognition sites.
Urban
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Centre de Génétique Moléculaire, Centre National de la Recherche Scientifique, UPR 2137, Avenue de la terrasse, 91190 Gif-sur-Yvette, France. urban@cgm.cnrs-gif.fr
Jobert
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AS
Lainé
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Pompon
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D
D000595:Amino Acid Sequence; D019363:Cytochrome P-450 CYP1A1 / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed / Q000235:genetics*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D013329:Structure-Activity Relationship; D013379:Substrate Specificity / Q000235:genetics
D019363:Cytochrome P-450 CYP1A1