Site-directed mutagenesis of cytochrome P450s CYP2A1 and CYP2A2: influence of the distal helix on the kinetics of testosterone hydroxylation.

Cytochrome P450s CYP2A1 and CYP2A2 exhibit 88% sequence similarity, yet CYP2A1 metabolizes testosterone almost exclusively (90%) at the 7 alpha-position, whereas CYP2A2 forms several metabolites, with 15 alpha-hydroxytestosterone as a major metabolite. One of the regions with relatively low sequence homology corresponds by sequence alignment to the I and J helices of P450cam. Since this region is known to be part of the active site for P450cam, 26 single point and two double point mutants were prepared where the amino acid for one form was substituted with that of the other. Mutant and wild-type enzymes were expressed in Hep G2 cells using the vaccinia virus vector. Analysis of testosterone regioselectivity revealed that 25 of the mutants show the same regioselectivity as the parent wild-type enzymes and three are inactive, suggesting that no single amino acid in this region is totally responsible for the different selectivities of CYP2A1 and CYP2A2. Kinetic analysis of the CYP2A1 mutants showed that four of the mutants with changes near the conserved oxygen-binding region had Km values with much higher and Vmax values much lower than those of the wild-type enzyme and one mutant had a Vmax value twice as high as that of the wild-type enzyme. Deuterium isotope effects on 7 alpha-hydroxxylation were used to determine changes in the rate of reduction and estimate the relative amount of excess water formation. Changes in reduction rates and the amount of water produced are not sufficient to account for the differences in Vmax values, suggesting that the amount of hydrogen peroxide released is a primary determinant for changes in Vmax.     

Chimeric cDNA expression and site directed mutagenesis studies of cytochrome P450s CYP2A1 and CYP2A2

Construction of chimeras and site directed mutagenesis were used to study the regioselectivity and kinetics of testosterone hydroxylation by the cytochrome P450s CYP2A1 and CYP2A2. Although these enzymes exhibit 88% sequence similarity, they catalyze very different regioselective hydroxylations of testosterone. Active chimeras inwhich the first 355 amino acids do not correspond to a single enzyme show broad radioselectivity, whereas the specificity of the parent enzyme is obtained if the first 355 amino acids are unchanged. Therefore, the region between amino acids 275 and 355 is important in maintaining regioselectivity. Single point mutants were constructed for the 13 amino acid differences in this region. For 26 single point and 2 double mutants all active mutants have the same regioselectivity as the parent enzymes. However, kinetic analysis of the CYP2A1 mutants showed that 4 single point mutants and 1 double mutant had kinetic parameters very different from the parent enzyme. All of these substitutions are associated with the conserved dioxygen binding region of the putative I helix predicted from the crystal structure of P450_cam. Deuterium isotope effects were used to determine any changes in the rate of reduction and to estimate the relative amount of excess water formation. Changes in reduction rates are not sufficient to account for the differences in V_max values. Therefore, it is likely that the amount of hydrogen peroxide formed is a primary determinant of V_max.     

Cloning and expression analysis of a new member of the cytochrome P450, CYP2A15 from the Chinese hamster, encoding testosterone 7alpha-hydroxylase.

We cloned a new cytochrome P450 cDNA encoding testosterone 7alpha-hydroxylase in the Chinese hamster, designated CYP2A15 which shares significant amino acid sequence homology with members of the CYP2A subfamily. The CYP2A15 cDNA was isolated by screening a liver cDNA library and the sequence contains an open reading frame of 1482 nucleotides encoding a polypeptide of 493 amino acids with a calculated molecular mass of 56,295 Da. This is flanked by a 5'-untranslated region of 2 bp and a 3' untranslated region of 191 bp including the poly(A) tail. We determined the catalytic activity of CYP2A15 using microsomes obtained by transient expression of its cDNA in transfected COS-7 cells. The heterologously expressed CYP2A15 was found to hydroxylate testosterone at position 7alpha in a reconstituted system. RT-PCR experiments revealed that the mRNA of CYP2A15 was expressed in liver, but not detected in kidney, lung, or small intestine. The expression of CYP2A15 mRNA was slightly induced by treatment with either rifampicin or 3-methylcholanthrene.     

Cellular localization of cytochrome P450IIA1 in testes of mature Sprague-Dawley rats.

Previous studies have shown that a prominent site of extrahepatic cytochrome P450IIA1 in male rats is the testis. We investigated the cellular location of cytochrome P450IIA1 in the testes of adult rats. Using specific isolation of testicular compartments and individual cell types, as well as in vivo removal of Leydig cells by ethane dimethyl sulfonate, we determined the cellular location of cytochrome P450IIA1 using testosterone hydroxylation assay, Western immunoblotting, and immunohistochemical analysis. Enriched Leydig cell fractions had the greatest testosterone 7 alpha-hydroxylase activity as well as immunoreactivity. Immunohistochemical analysis confirmed that the cellular location of cytochrome P450IIA1 was specific to Leydig cells. The specific localization of enzyme systems that are involved in xenobiotic activation may have important implications for inducing specific cell toxicity by compounds that exert their effects in the testes.     

The importance of SRS-1 residues in catalytic specificity of human cytochrome P450 3A4

The structural basis for the regioselective hydroxylation of Delta-4-3-ketosteroids by human CYP3A4 was investigated. Prior studies had suggested that the chemical reactivity of the allylic 6beta-position might have a greater influence than steric constraints by the enzyme. Six highly conserved CYP3A residues from substrate recognition site 1 were examined by site-directed mutagenesis. F102A and A117L showed no spectrally detectable P450. V101G and T103A exhibited a wild-type progesterone metabolite profile. Of five mutants at residue N104, only N104D yielded holoenzyme and exhibited the same steroid metabolite profile as wild-type. Of four mutants at position S119 (A, L, T, V), the three hydrophobic ones produced 2beta-OH rather than 6beta-OH progesterone or testosterone as the major metabolite. Kinetic analysis showed S(50) values similar to wild-type for S119A (progesterone) and S119V (testosterone), whereas the V(max) values for 2beta-hydroxysteroid formation were increased in both cases. All four mutants exhibited an altered product profile for 7-hexoxycoumarin side-chain hydroxylation, whereas the stimulation of steroid hydroxylation by alpha-naphthoflavone was similar to the wild-type. The results indicate that the highly conserved residue S119 is a key determinant of CYP3A4 specificity and reveal an important role of the active site topology in steroid 6beta-hydroxylation.     

Construction and engineering of a thermostable self-sufficient cytochrome P450

CYP175A1 is a thermophilic cytochrome P450 and hydroxylates β-carotene. We previously identified a native electron transport system for CYP175A1. In this report, we constructed two fusion proteins consisting of CYP175A1, ferredoxin (Fdx), and ferredoxin-NADP⁺ reductase (FNR): H₂N-CYP175A1-Fdx-FNR-COOH (175FR) and H₂N-CYP175A1-FNR-Fdx-COOH (175RF). Both 175FR and 175RF were expressed in Escherichia coli and purified. The V_max value for β-carotene hydroxylation was 25 times higher with 175RF than 175FR and 9 times higher with 175RF than CYP175A1 (non-fused protein), although the k_m values of these enzymes were similar. 175RF retained 50% residual activity even at 80 °C. Furthermore, several mutants of the CYP175A1 domain of 175RF were prepared and one mutant (Q67G/Y68I) catalyzed the hydroxylation of an unnatural substrate, testosterone. Thus, this is the first report of a thermostable self-sufficient cytochrome P450 and the engineering of a thermophilic cytochrome P450 for the oxidation of an unnatural substrate.     

Determination of 3-keto-4-ene steroids and their hydroxylated metabolites catalyzed by recombinant human cytochrome P450 1B1 enzyme using gas chromatography-mass spectrometry with trimethylsilyl derivatization

A protocol utilizing gas chromatography with selected ion monitoring mass spectrometric detection (GC-SIM-MS) using a simplified trimethylsilyl (TMS) derivatization protocol was developed and validated for the determination of hydroxylated metabolites of 3-keto-4-ene steroids such as testosterone, progesterone and androstenedione. Hydroxylated metabolites catalyzed by human CYP1B1 were extracted with methylene chloride and derivatized with BSTFA-10% TMCS. To get an optimal derivatizing condition, the effect of various incubation times and temperatures was evaluated. When the incubation temperature and time in the presence of the TMS derivatizing agent were increased, the 3-keto group became derivatized with TMS to form a 3-TMS derivative. To minimize the formation of the TMS ether on the 3-keto group, a reaction condition of 56 degrees C for 10 min was used for the routine measurement of the steroids and their hydroxylated metabolite. Performance studies including linearity of calibration curves, extraction efficiency and precision were performed. Linearity of the calibration curves was satisfactory from 0.125 to 5 microM for most compounds except 21-hydroxyprogesterone and 16alpha-hydroxyandrostenedione which deviated from linearity at the lower concentrations. Mean percentage extraction recoveries were greater than 80% for all compounds. Most compounds showed good precisions with C.V.s of within-day precision of less than 5% and C.V.s of between-day precision of less than 10%. The selected ion chromatograms from the recombinant human CYP1B1 incubations with testosterone, progesterone and androstenedione showed evidence of 6beta-, 16alpha-, 2alpha-, and 15alpha-hydroxytestosterone, 6alpha- and 16alpha-hydroxyprogesterone and 6alpha- and 16alpha-hydroxyandrostenedione, respectively. There was no significant interference associated with Escherichia coli membrane extracts in detecting hydroxylated metabolites. This procedure provides a rapid and sensitive method for the evaluation of steroid hydroxylation by CYP isoenzymes.     

Alteration of cytochrome P-450 2C11-dependent testosterone metabolism in Gunn rat liver.

Cytochrome P-450 2C11 (CYP2C11) is the main isozyme present in adult male rat liver and specifically hydroxylates testosterone in positions 2 alpha and 16 alpha. In Gunn rats, this isozyme is recognized by the anti-CYP2C11 antibody but its activity towards testosterone is dramatically decreased. Moreover, peptide mapping, after digestion of microsomal fractions by V8 protease and probing with anti-CYP2C11 antibody, exhibit a pattern which differs from that obtained with Wistar rats. Taken together, data suggest that the protein sequence of CYP2C11 from the Gunn rat differs from that of the Wistar rat.     

A novel polymorphic cytochrome P450 formed by splicing of CYP3A7 and the pseudogene CYP3AP1

The cytochrome P450 3A7 (CYP3A7) is the most abundant CYP in human liver during fetal development and first months of postnatal age, playing an important role in the metabolism of endogenous hormones, drugs, differentiation factors, and potentially toxic and teratogenic substrates. Here we describe and characterize a novel enzyme, CYP3A7.1L, encompassing the CYP3A7.1 protein with the last four carboxyl-terminal amino acids replaced by a unique sequence of 36 amino acids, generated by splicing of CYP3A7 with CYP3AP1 RNA. The corresponding CYP3A7-3AP1 mRNA had a significant expression in liver, kidney, and gastrointestinal tract, and its presence was found to be tissue-specific and dependent on the developmental stage. Heterologous expression in yeast revealed that CYP3A7.1L was a functional enzyme with a specific activity similar to that of CYP3A7.1 and, in some conditions, a different hydroxylation specificity than CYP3A7.1 using dehydroepiandrosterone as a substrate. CYP3A7.1L was found to be polymorphic due to a mutation at position -6 of the first splicing site of CYP3AP1 (CYP3A7_39256T-->A), which abrogates the pseudogene splicing. This polymorphism had pronounced interethnic differences and was in linkage disequilibrium with other functional polymorphisms described in the CYP3A locus: CYP3A7*2 and CYP3A5*1. Therefore, the resulting CYP3A haplotypes express different sets of enzymes within the population. In conclusion, a novel mechanism, consisting of the splicing of the pseudogene CYP3AP1 to CYP3A7, causes the formation of the novel CYP3A7.1L having a different tissue distribution and functional properties than the parent CYP3A7 enzyme, with possible developmental, physiological, and toxicological consequences.     

Ordered chimerogenesis applied to CYP2B P450 enzymes

BackgroundStructural studies on CYP2B enzymes identified some of the features that are related to their high plasticity. The aim of this work was to understand further the possible relationships between combinations of structural elements and functions by linking shift in substrate specificity with sequence element swaps between CYP2B6 and CYP2B11.MethodsA series of 15 chimeras in which a small CYP2B6 sequence segment was swapped with its equivalent in CYP2B11 were constructed. All chimeras produced were thus mostly of CYP2B11 sequence. Time course studies were carried out with two typical CYP2B substrates, cyclophosphamide and 7-ethoxy-4-trifluoromethylcoumarin. Steady-state kinetic parameters were determined for all chimeras expressed in yeast.ResultsMost of the chimeras exhibit a high affinity for cyclophosphamide, as CYP2B11 does. A few exhibit an affinity similar to that of CYP2B6 without altered behavior toward the other substrate assayed. The swapped elements that control this specificity shift are discussed in terms of F′/G′ cassette role and substrate access channels.ConclusionsSome sequence segments control precisely the shift in affinity for cyclophosphamide between CYP2B6, which has a typical low affinity, and CYP2B11 which has a typical high affinity.General significanceThe result provides a new basis for determining the structural elements that control functions in complex enzymes.     

17 beta-estradiol hydroxylation catalyzed by human cytochrome P450 1A1: a comparison of the activities induced by 2,3,7,8-tetrachlorodibenzo-p-dioxin in MCF-7 cells with those from heterologous expression of the cDNA.

Exposure of MCF-7 breast cancer cells to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) causes an elevated cytochrome P450 content and a marked increase in the microsomal hydroxylation of 17 beta-estradiol (E2) at the C-2, C-4, C-15 alpha, and C-6 alpha positions. In this study we investigated the involvement of cytochromes P450 of the 1A gene subfamily in this metabolism of E2. Hydroxylation at each of these four positions of E2 was inhibited by P450 1A-subfamily inhibitors, alpha-naphthoflavone, benzo[a]pyrene, and 7-ethoxyresorufin. Northern blots showed that treatment of MCF-7 cells with TCDD resulted in production of the 2.6-kb CYP1A1 mRNA, but not the 3.0-kb CYP1A2 mRNA. Immunoblot analyses with anti-P450 1A antibodies confirmed the production of P450 1A1 protein in TCDD-treated MCF-7 cells. Anti-rat P450 1A IgG inhibited the hydroxylation of E2 at C-2, C-15 alpha, and C-6 alpha, but not hydroxylation at C-4. E2 hydroxylation by human cytochromes P450 1A1 and P450 1A2 was assessed in experiments with microsomes from Saccharomyces cerevisiae after transformation with cDNAs encoding the two cytochromes. The major hydroxylase activities of expressed human P450 1A1 were at the C-2, C-15 alpha, and C-6 alpha positions of E2; expressed human P450 1A2 catalyzed hydroxylation predominately at C-2. While both expressed P450s 1A1 and 1A2 had minor hydroxylase activities at the C-4 position, neither catalyzed a low-Km hydroxylation at C-4 similar to that observed with microsomes from TCDD-treated MCF-7 cells. These results provide strong evidence that P450 1A1 catalyzes the hydroxylations of E2 at the C-2, C-15 alpha, and C-6 alpha in incubations with microsomes from TCDD-treated MCF-7 cells, but suggest TCDD may also induce a cytochrome P450 E2 4-hydroxylase that is distinct from P450 1A1 or P450 1A2.     

Gene conversion and differential regulation in the rat P-450 IIA gene subfamily. Purification, catalytic activity, cDNA and deduced amino acid sequence, and regulation of an adult male-specific hepatic testosterone 15 alpha-hydroxylase

Previous studies on regulation of the rat hepatic P-450 IIA1 cDNA have provided evidence for a second gene closely related to but regulated in a manner quite distinct from P-450 IIA1. Experiments were carried out to isolate the cDNA for this second P-450 gene, designated IIA2, in order to study more directly its regulation and relationship to IIA1. A full length cDNA to IIA2 was isolated from an adult male rat liver lambda gt11 library and sequenced completely. The IIA2 cDNA shared 93% nucleotide and 88% deduced amino acid similarities with the previously characterized IIA1 cDNA (Nagata, K., Matsunaga, T., Gillette, J., Gelboin, H. V., and Gonzalez, F. J. (1987) J. Biol. Chem. 262, 2787-2793). The protein, deduced from the cDNA, contained 492 amino acids and a calculated Mr of 56,352. Comparison of the IIA1 and IIA2 cDNAs revealed areas of low nucleotide similarity interspersed with areas of absolute identity, suggesting that gene conversions have played a role in the evolution of the IIA subfamily. Expression of IIA1 and IIA2 mRNAs in rat liver during development was studied with use of specific oligonucleotide probes. IIA1 mRNA was increased within 1 week after birth in both male and female rats; however, its postpubertal expression was decreased in males yet remained elevated in females. In contrast, IIA2 mRNA was markedly induced in male rat liver at puberty but was not detectable in females at any age examined. Furthermore, only IIA1 mRNA was induced by treatment of rats with 3-methylcholanthrene. Although IIA1 and IIA2 mRNAs were actively expressed in hepatic tissue, no evidence for their expression was found in lung, kidney, or intestine, suggesting that the IIA genes have tissue-specific promoters. Reconstituted enzyme assays on the purified protein products P-450 IIA1 and P-450 IIA2 showed that, although both enzymes share considerable sequence similarity, their positional specificities toward the prototype substrate testosterone are strikingly different.     

An A245T mutation conveys on cytochrome P450eryF the ability to oxidize alternative substrates

Cytochrome P450(eryF) (CYP107A1), which hydroxylates deoxyerythronolide B in erythromycin biosynthesis, lacks the otherwise highly conserved threonine that is thought to promote O-O bond scission. The role of this threonine is satisfied in P450(eryF) by a substrate hydroxyl group, making deoxyerythronolide B the only acceptable substrate. As shown here, replacement of Ala(245) by a threonine enables the oxidation of alternative substrates using either H(2)O(2) or O(2)/spinach ferredoxin/ferredoxin reductase as the source of oxidizing equivalents. Testosterone is oxidized to 1-, 11alpha-, 12-, and 16alpha-hydroxytestosterone. A kinetic solvent isotope effect of 2.2 indicates that the A245T mutation facilitates dioxygen bond cleavage. This gain-of-function evidence confirms the role of the conserved threonine in P450 catalysis. Furthermore, a Hill coefficient of 1.3 and dependence of the product distribution on the testosterone concentration suggest that two testosterone molecules bind in the active site, in accord with a published structure of the P450(eryF)-androstenedione complex. P450(eryF) is thus a structurally defined model for the catalytic turnover of multiply bound substrates proposed to occur with CYP3A4. In view of its large active site and defined structure, catalytically active P450(eryF) mutants are also attractive templates for the engineering of novel P450 activities.     

Steroid hormone hydroxylase specificities of eleven cDNA-expressed human cytochrome P450s

Steroid hydroxylation specificities were determined for 11 forms of human cytochrome P450, representing four gene families and eight subfamilies, that were synthesized in human hepatoma Hep G2 cells by means of cDNA-directed expression using vaccinia virus. Microsomes isolated from the P450-expressing Hep G2 cells were isolated and then assayed for their regioselectivity of hydroxylation toward testosterone, androstenedione, and progesterone. Four of the eleven P450s exhibited high steroid hydroxylase activity (150-900 pmol hydroxysteroid/min/mg Hep G2 microsomal protein), one was moderately active (30-50 pmol/min/mg) and six were inactive. In contrast, 10 of the P450s effectively catalyzed O-deethylation of 7-ethoxycoumarin, a model drug substrate, while only one (P450 2A6) catalyzed significant coumarin 7-hydroxylation. Human P450 4B1, which is expressed in lung but not liver, catalyzed the 6 beta-hydroxylation of all three steroids at similar rates and with only minor formation of other hydroxylated products. Three members of human P450 family 3A, which are expressed in liver and other tissues, also catalyzed steroid 6 beta-hydroxylation as their major activity but, additionally, formed several minor products that include 2 beta-hydroxy and 15 beta-hydroxy derivatives in the case of testosterone. These patterns are similar to those exhibited by rat family 3A P450s. Although several rodent P450s belonging to subfamilies 2A, 2B, 2C, 2D are active steroid hydroxylases, four of five human P450s belonging to these subfamilies exhibited very low activity or were inactive, as were the human 1A and 2E P450s examined in the present study. These studies demonstrate that individual human cytochrome P450 enzymes can hydroxylate endogenous steroid hormones with a high degree of stereospecificity and regioselectivity, and that some, but not all of the human cytochromes exhibit metabolite profiles similar to their rodent counterparts.     

Functional characterization of medaka CYP3A38 and CYP3A40: kinetics and catalysis by expression in a recombinant baculovirus system

Phylogenic analysis of the teleost genomic lineages has demonstrated the precedent for multiple genome duplications. Among many of the genes duplicated, cytochrome P450 genes have undergone independent diversification, which can be traced to a single ancestral gene. In teleosts, cytochrome P450s, from all major families, have been identified. Among these, the CYP3A family has been cloned in several teleost species and demonstrated to contain multiple paralogs differing in gene expression patterns and tissue distribution. Herein we characterized the catalytic and kinetic activities of two medaka CYP3A paralogs (CYP3A38 and CYP3A40) with benzyloxyresorufin (BFC), a fluorescent 3A-selective substrate, and testosterone, a known metabolic substrate for CYP3A enzymes. Recombinant CYP3A was produced using the baculovirus expression vector system in Spodoptera frugiperda (Sf9) and Trichoplusia ni (Tn5) insect cells and accounted for up to 24% of total cellular protein. Following addition of a heme-albumin conjugate to log phase cells, spectral P450 content reached a maximum of 560 and 2350 pmol/mg microsomal protein for CYP3A38 and CYP3A40, respectively. Incubations containing recombinant CYP3A, human NADPH-cytochrome P-450 oxidoreductase reductase, human cytochrome b5, and a NADPH generation system catalyzed the dealkylation of BFC and hydroxylation of testosterone with a high degree of stereoselectivity. However, efficiencies and specificities were significantly different between the two isoforms. Km and Vmax activities based on BFC-catalysis were 0.116 and 0.363 muM, and 7.95 and 7.77 nmol/min/nmol P450 for CYP3A38 and CYP3A40, respectively. CYP3A38 preferentially catalyzed testosterone hydroxylation at the 6beta-, 2beta- and 16beta-positions with minor hydroxylation at other positions within the steroid nucleus. Testosterone catalysis with CYP3A40 was limited predominantly to the 6beta- and 2beta-positions. Putative identification of CYP3A substrate recognition sites (SRS) 1-6 indicates that 12 of the 49 amino acid differences between CYP3A38 and CYP3A40 OFRs occur in SRS regions previously known to be associated with steroid hydroxylation. We suggest that differences in kinetics and catalytic activities are a result of amino acid substitutions in SRS regions 1, 3 and 5 within the CYP3A38 and CYP3A40 protein sequence.     

Identification of a novel P450 expressed in rat lung: cDNA cloning and sequence, chromosome mapping, and induction by 3-methylcholanthrene

A novel P450 cDNA was isolated from a rat lung lambda gt11 library by hybridization with the rat P450 IIB1 cDNA probe. The cDNA-deduced amino acid sequence of this clone was 71% and 73% similar to rat IIA1 and IIA2 P450s; it was, therefore, designated IIA3 as the third member of the rat IIA subfamily. IIA3 demonstrates only 55% amino acid similarity with IIB1. Interestingly, this P450 also shared 85% and 94% amino acid similarities with human IIA3 and a mouse testosterone 15 alpha-hydroxylase P450, respectively, indicating that these P450s are orthologous counterparts to rat IIA3. Chromosome mapping, using mouse-hamster somatic cell hybrids, revealed that the IIA3 gene is localized on mouse chromosome 7. The IIA3 mRNA was detected in rat lung, and its level was induced 3-fold by treatment of rats with 3-methylcholanthrene. No IIA3 mRNA was seen in the liver, kidney, or intestine, even after long exposure of Northern blot filters to X-ray film. In contrast, the orthologous mouse and human IIA3 genes are expressed in liver.     

Point mutations associated with insecticide resistance in the Drosophila cytochrome P450 Cyp6a2 enable DDT metabolism.

Three point mutations R335S, L336V and V476L, distinguish the sequence of a cytochrome P450 CYP6A2 variant assumed to be responsible for 1,1,1-trichloro-2,2-bis-(4'-chlorophenyl)ethane (DDT) resistance in the RDDT(R) strain of Drosophila melanogaster. To determine the impact of each mutation on the function of CYP6A2, the wild-type enzyme (CYP6A2wt) of Cyp6a2 was expressed in Escherichia coli as well as three variants carrying a single mutation, the double mutant CYP6A2vSV and the triple mutant CYP6A2vSVL. All CYP6A2 variants were less stable than the CYP6A2wt protein. Two activities enhanced in the RDDT(R) strain were measured with all recombinant proteins, namely testosterone hydroxylation and DDT metabolism. Testosterone was hydroxylated at the 2beta position with little quantitative variation among the variants. In contrast, metabolism of DDT was strongly affected by the mutations. The CYP6A2vSVL enzyme had an enhanced metabolism of DDT, producing dicofol, dichlorodiphenyldichloroethane and dichlorodiphenyl acetic acid. The apparent affinity of the enzymes CYP6A2wt and CYP6A2vSVL for DDT and testosterone was not significantly different as revealed by the type I difference spectra. Sequence alignments with CYP102A1 provided clues to the positions of the amino acids mutated in CYP6A2. These mutations were found spatially clustered in the vicinity of the distal end of helix I relative to the substrate recognition valley. Thus this area, including helix J, is important for the structure and activity of CYP6A2. Furthermore, we show here that point mutations in a cytochrome P450 can have a prominent role in insecticide resistance.