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Int. J. Mol. Sci. 2016, 17(2), 213; doi:10.3390/ijms17020213

Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1

1
Department of Biochemistry, Faculty of Science, Charles University, Albertov 2030, CZ-12843 Prague 2, Czech Republic
2
Division of Radiopharmaceutical Chemistry, German Cancer Research Center (DKFZ), Im Neuenheimer Feld 280, 69120 Heidelberg, Germany
3
Analytical and Environmental Sciences Division, MRC-PHE Centre for Environment and Health, King’s College London, London SE1 9NH, UK
*
Authors to whom correspondence should be addressed.
Academic Editor: Ute Roessner
Received: 10 December 2015 / Revised: 13 January 2016 / Accepted: 21 January 2016 / Published: 5 February 2016
(This article belongs to the Section Biochemistry, Molecular and Cellular Biology)
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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 NAD(P)H: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 32P-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. View Full-Text
Keywords: aristolochic acid nephropathy; aristolochic acid I; site-directed mutagenesis of cytochromes P450 1A1; 1A2 and 1B1; nitroreduction; DNA adduct formation aristolochic acid nephropathy; aristolochic acid I; site-directed mutagenesis of cytochromes P450 1A1; 1A2 and 1B1; nitroreduction; DNA adduct formation
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MDPI and ACS Style

Milichovský, J.; Bárta, F.; Schmeiser, H.H.; Arlt, V.M.; Frei, E.; Stiborová, M.; Martínek, V. Active Site Mutations as a Suitable Tool Contributing to Explain a Mechanism of Aristolochic Acid I Nitroreduction by Cytochromes P450 1A1, 1A2 and 1B1. Int. J. Mol. Sci. 2016, 17, 213.

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