Interaction of aflatoxin B1 with cytochrome P450 2A5 and its mutants: correlation with metabolic activation and toxicity.
Among members of the mouse cytochrome P450 2A family, P450 2A5 is the best catalyst of aflatoxin B1 (AFB1) oxidation to its 8,9-epoxide (Pelkonen, P., Lang, M., Wild, C. P., Negishi, M., and Juvonen, R. O. (1994) Eur. J. Pharmacol., Environ. Toxicol. Pharmacol. Sect. 292, 67-73). Here we studied the role of amino acid residues 209 and 365 of the P450 2A5 in the metabolism and toxicity of AFB1 using recombinant yeasts. The two sites have previously been shown to be essential in the interaction of coumarin and steroids with the P450 2A5. Reducing the size of the amino acid at position 209 or introducing a negatively charged residue at this site increased the 8,9-epoxidation of AFB1 compared to the wild type. In addition, replacing the hydrophobic amino acid at the 365 position with a positively charged lysine residue strongly decreased the metabolism of AFB1. These mutations changed the KM values generally less than the Vmax values. The changes in AFB1 metabolism contrast with the changes in coumarin 7-hydroxylation caused by these amino acid substitutions, since reducing the size of the 209 residue strongly reduced coumarin metabolism and increased the K(M) values. On the other hand, the results with AFB1 are similar to those obtained with steroid hydroxylation. This suggests that the size of the substrate is important when interacting with the residue 209 of the protein. The catalytic parameters of AFB1 correlated generally with its toxicity to the recombinant yeasts expressing the activating enzyme and with the binding of AFB1 to yeast DNA. Furthermore high affinity substrates and inhibitors (e.g., methoxsalen, metyrapone, coumarin 311, 7-methylcoumarin, coumarin, and pilocarpine) of P450 2A5 could efficiently block the toxicity of AFB1. It is suggested that the recombinant yeasts expressing engineered P450 enzymes are a useful model to understand the substrate protein interactions, to study the relationship of metabolic parameters to toxicity, and to test potential inhibitors of metabolism based toxicity.
Pelkonen
P
P
Department of Pharmacology and Toxicology, University of Kuopio, Finland.
Lang
M A
MA
Negishi
M
M
Wild
C P
CP
Juvonen
R O
RO
D016604:Aflatoxin B1 / Q000378:metabolism* / Q000633:toxicity; D000596:Amino Acids / Q000378:metabolism; D001189:Aryl Hydrocarbon Hydroxylases; D065637:Cytochrome P-450 CYP2A6; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D000072467:Cytochrome P450 Family 2; D018736:DNA Adducts / Q000235:genetics; D004271:DNA, Fungal / Q000187:drug effects / Q000378:metabolism; D006899:Mixed Function Oxygenases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009153:Mutagens / Q000378:metabolism* / Q000633:toxicity; D009154:Mutation; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000187:drug effects / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
D000596:Amino Acids; D065607:Cytochrome P-450 Enzyme Inhibitors; D018736:DNA Adducts; D004271:DNA, Fungal; D009153:Mutagens; D011994:Recombinant Proteins; D003577:Cytochrome P-450 Enzyme System; D016604:Aflatoxin B1; D006899:Mixed Function Oxygenases; D001189:Aryl Hydrocarbon Hydroxylases; C585520:Cyp2a5 protein, mouse; D065637:Cytochrome P-450 CYP2A6; D000072467:Cytochrome P450 Family 2