The effect of reciprocal active site mutations in human cytochromes P450 1A1 and 1A2 on alkoxyresorufin metabolism Jianguo Liu, a Spencer S. Ericksen, a Meena Sivaneri, a Dan Besspiata, a Charles W. Fisher, b,1 and Grazyna D. Szklarz a, * a Department of Basic Pharmaceutical Sciences, School of Pharmacy, West Virginia University, Morgantown, WV 26506-9530, USA b Department of Pharmaceutical Sciences, Texas Tech Health Sciences Center, School of Pharmacy, Amarillo, TX 79106-1920, USA Received 28 August 2003, and in revised form 8 December 2003 Abstract Five reciprocal active site mutants of P450 1A1 and 1A2 and an additional mutant, Val/Leu-382 ! Ala, were constructed, ex- pressed in Escherichia coli, and purified by Ni–NTA affinity chromatography. In nearly every case, the residue replacement led to loss of 7-methoxy- and 7-ethoxyresorufin O-dealkylase activity compared to the wild-type enzymes, except for the P450 1A1 S122T mutation which increased both activities. Mutations at position 382 in both P450 1A1 and 1A2 shifted substrate specificity from one enzyme to another, confirming the importance of this residue. Changes in activity of P450 1A enzymes upon amino acid replacement were, in general, consistent with molecular dynamics analyses of substrate motion in the active site of homology models. Ó 2004 Elsevier Inc. All rights reserved. Keywords: Cytochrome P450; Site-directed mutagenesis; Structure–function relationships; Substrate specificity; Alkoxyresorufin O-dealkylation; Molecular modeling; Molecular dynamics Cytochromes P450 (P450) 2 are versatile catalysts that play an important role in the oxidation of a vast array of xenobiotic and endogenous compounds. There is a considerable interest in the function of these enzymes due to their involvement in the detoxification of foreign compounds and bioactivation of drugs and carcinogens. For example, cytochromes P450 of the 1A subfamily are responsible for the metabolic activation of known pro- carcinogenic environmental chemicals, toxins, and toxic drugs [1]. In humans, the two members of the P450 1A subfamily, P450 1A1 and 1A2, share 72% amino acid sequence identity, but display different substrate speci- ficities and inhibitor susceptibilities [2,3]. P450 1A1 mainly metabolizes benzo[a]pyrene and other polycyclic aromatic hydrocarbons (PAHs) to their toxic derivatives [2–4], while 1A2 preferentially oxidizes heterocyclic and aromatic amines [5,6]. Some substrates are metabolized by both enzymes, but with different efficiencies. For example, P450 1A1 effectively oxidizes 7-ethoxyresoru- fin, while P450 1A2, although able to metabolize 7-ethoxyresorufin, exhibits preference towards the 7- methoxyresorufin [7]. It would be of great interest to determine the structural basis for these differences in specificity, and whether the substitutions of key residues of one enzyme to those of another will convert the ac- tivity. Modeling and experimental studies with highly related P450 2B4 and 2B5 showed that single reciprocal mutants displayed altered substrate specificities [8], al- though multiple active-site mutations were necessary to completely interconvert activities [9]. Recently, we have constructed a homology model of P450 1A1 based on the structure of P450 2C5 [10]. * Corresponding author. Fax: 1-304-293-2576. E-mail address: gszklarz@hsc.wvu.edu (G.D. Szklarz). 1 Present address: CEDRA Corporation, 8609 Cross Park Drive, Austin, TX 78754, USA. 2 Abbreviations used: P450, cytochrome P450; NADPH, b-nico- tinamide adenine dinucleotide phosphate reduced form; IPTG, isopro- pyl-b-D-thiogalactopyranoside; ALA, d-aminolevulinic acid; CHAPS, 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate; EDTA, ethlenediaminetetraacetic acid; DLPC, dilauroyl-L-3-phosphatidyl- choline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel electrophoresis; WT, wild type; ssDNA, single strand DNA; dsDNA, double strand DNA; EROD, 7-ethoxyresorufinO-deethylase; MROD, 7-methoxyresorufin O-demethylase. 0003-9861/$ - see front matter Ó 2004 Elsevier Inc. All rights reserved. doi:10.1016/j.abb.2003.12.040 Archives of Biochemistry and Biophysics 424 (2004) 33–43 ABB www.elsevier.com/locate/yabbi