title: Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1.
abstract: Aristolochic acid I (AAI) is a plant drug found in Aristolochia species that causes aristolochic acid nephropathy, Balkan endemic nephropathy and their associated urothelial malignancies. AAI is activated via nitroreduction producing genotoxic N-hydroxyaristolactam, which forms DNA adducts. The major enzymes responsible for the reductive bioactivation of AAI are
  quinone oxidoreductase and cytochromes P450 (CYP) 1A1 and 1A2. Using site-directed mutagenesis we investigated the possible mechanisms of CYP1A1/1A2/1B1-catalyzed AAI nitroreduction. Molecular modelling predicted that the hydroxyl groups of serine122/threonine124 (Ser122/Thr124) amino acids in the CYP1A1/1A2-AAI binary complexes located near to the nitro group of AAI, are mechanistically important as they provide the proton required for the stepwise reduction reaction. In contrast, the closely related CYP1B1 with no hydroxyl group containing residues in its active site is ineffective in catalyzing AAI nitroreduction. In order to construct an experimental model, mutant forms of CYP1A1 and 1A2 were prepared, where Ser122 and Thr124 were replaced by Ala (CYP1A1-S122A) and Val (CYP1A2-T124V), respectively. Similarly, a CYP1B1 mutant was prepared in which Ala133 was replaced by Ser (CYP1B1-A133S). Site-directed mutagenesis was performed using a quickchange approach. Wild and mutated forms of these enzymes were heterologously expressed in Escherichia coli and isolated enzymes characterized using UV-vis spectroscopy to verify correct protein folding. Their catalytic activity was confirmed with CYP1A1, 1A2 and 1B1 marker substrates. Using (32)P-postlabelling we determined the efficiency of wild-type and mutant forms of CYP1A1, 1A2, and 1B1 reconstituted with NADPH:CYP oxidoreductase to bioactivate AAI to reactive intermediates forming covalent DNA adducts. The S122A and T124V mutations in CYP1A1 and 1A2, respectively, abolished the efficiency of CYP1A1 and 1A2 enzymes to generate AAI-DNA adducts. In contrast, the formation of AAI-DNA adducts was catalyzed by CYP1B1 with the A133S mutation. Our experimental model confirms the importance of the hydroxyl group possessing amino acids in the active center of CYP1A1 and 1A2 for AAI nitroreduction.
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic. jan.milichovsky@natur.cuni.cz.
authors: Jan
authors: J
authors: Milichovský
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic. frantisek.barta@natur.cuni.cz.
authors: František
authors: F
authors: Bárta
authors: Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany. h.schmeiser@dkfz-heidelberg.de.
authors: Heinz H
authors: HH
authors: Schmeiser
authors: Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King's College London, London SE1 9NH, UK. volker.arlt@kcl.ac.uk.
authors: Volker M
authors: VM
authors: Arlt
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic. evafrei@t-online.de.
authors: Eva
authors: E
authors: Frei
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic. stiborov@natur.cuni.cz.
authors: Marie
authors: M
authors: Stiborová
authors: Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic. vacmar@natur.cuni.cz.
authors: Václav
authors: V
authors: Martínek
mesh_terms: D034341:Aristolochic Acids / Q000378:metabolism*; D001189:Aryl Hydrocarbon Hydroxylases / Q000235:genetics* / Q000378:metabolism*; D002384:Catalysis; D020134:Catalytic Domain / Q000235:genetics*; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065633:Cytochrome P-450 CYP1B1; D018736:DNA Adducts / Q000378:metabolism; D006801:Humans; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D010084:Oxidation-Reduction; D011994:Recombinant Proteins; D013379:Substrate Specificity
chemical_list: D034341:Aristolochic Acids; D018736:DNA Adducts; D011994:Recombinant Proteins; C000228:aristolochic acid I; D001189:Aryl Hydrocarbon Hydroxylases; D019363:Cytochrome P-450 CYP1A1; D019388:Cytochrome P-450 CYP1A2; D065633:Cytochrome P-450 CYP1B1
keywords: 1A2 and 1B1; DNA adduct formation; aristolochic acid I; aristolochic acid nephropathy; nitroreduction; site-directed mutagenesis of cytochromes P450 1A1