Biomedical subjects

J R Halpert

Publications and source records attributed to J R Halpert.

At least 37 records · Page 2Linked to original sources

PubMed1999

Identification of selective mechanism-based inactivators of cytochromes P-450 2B4 and 2B5, and determination of the molecular basis for differential susceptibility.

Rabbit cytochromes P-450 (P-450) 2B4 and 2B5 differ by only 12 amino acid residues yet they exhibit unique steroid hydroxylation profiles. Previous studies have led to the identification of active site residues that are determinants of these specificities. In this study, mechanism-based inactivators were identified that discriminate between the closely related 2B4 and 2B5 enzymes. A previously characterized inhibitor, 2-ethynylnaphthalene (2EN), was found to be selective for 2B4 inactivation. As inhibitor metabolism and the partition ratio affect susceptibility, molecular dynamics simulations were performed to assess the stability of the productive binding orientation of 2EN within 2B4 and 2B5 three-dimensional models. Although 2EN was stable within the 2B4 model, it exhibited substantial movement away from the heme moiety in the 2B5 model. However, heterologously expressed 2B5 was found to catalyze the oxidation of 2EN to the stable product 2-naphthylacetic acid. Thus, the increased mobility of 2EN may result in reduced susceptibility of 2B5 by increasing the probability that the reactive ketene intermediate hydrolyzes with water instead of reacting with active site residues. Another compound, 1-adamantyl propargyl ether (1APE), selectively inactivated 2B5. The structural basis for 2EN and 1APE susceptibility was assessed using active site mutants. Interconversion of 2EN susceptibility was observed for 2B4 or 2B5 mutants containing a single alteration at residue 363. Single substitutions in 2B4 also conferred susceptibility to 1APE; however, multiple alterations were required to reduce the susceptibility of 2B5. These alterations may influence inhibitor susceptibility by affecting the stability of the productive binding orientation.

Adamantane↗

PubMed1999

Use of the steroid derivative RPR 106541 in combination with site-directed mutagenesis for enhanced cytochrome P-450 3A4 structure/function analysis.

RPR 106541 (20R-16alpha,17alpha-[butylidenebis(oxy)]-6al pha, 9alpha-difluoro-11beta-hydroxy-17beta-(methylthio)androst a-4-en-3-one) is an airway-selective steroid developed for the treatment of asthma. Two metabolites produced by human liver microsomes were identified as R- and S-sulfoxide diastereomers based on liquid chromatography/mass spectrometry analysis, proton nuclear magnetic resonance, and cochromatography with standards. Sulfoxide formation was determined to be cytochrome P-450 (CYP) 3A4-dependent by correlation with CYP3A4-marker nifedipine oxidase activity, inhibition by cyclosporin A and troleandomycin, and inhibition of R- (70%) and S- (64%) sulfoxide formation by anti-3A antibody. Expressed CYP2C forms catalyzed RPR 106541 sulfoxidation; however, other phenotyping approaches failed to confirm the involvement of CYP2C forms in these reactions in human liver microsomes. Expressed CYP3A4 catalyzed the formation of the sulfoxide diastereomers in a 1:1 ratio, whereas CYP3A5 displayed stereoselectivity for formation of the S-diastereomer. The high rate of sulfoxidation by CYP3A4 and the blockage of oxidative metabolism at the electronically favored 6beta-position provided advantages for RPR 106541 over other substrates as an active site probe of CYP3A4. Therefore, oxidation of RPR 106541 by various CYP3A4 substrate recognition site (SRS) mutants was assessed. In SRS-4, A305V and F304A showed dramatically reduced rates of R-diastereomer formation (83 and 64% decreases, respectively), but S-diastereomer formation was affected to a lesser extent. A370V (SRS-5) showed decreased formation of the R-sulfoxide (52%) but increased formation of the S-diastereomer. In the SRS-2 region, the most dramatic change in sulfoxide ratios was observed for L210A. In conclusion, the structure of RPR 106541 imposes specific constraints on enzyme binding and activity and thus represents an improved CYP3A4 probe substrate.

Androstenes↗

PubMed1999

Structure-function analysis of human cytochrome P-450 2B6 using a novel substrate, site-directed mutagenesis, and molecular modeling.

The structural basis for functional differences between human cytochrome P-450 2B6 and rat 2B1 was investigated. An amino acid sequence alignment predicted the location of 2B6 substrate recognition site (SRS) residues. Ten residues within these SRSs unique to 2B6 compared with 2B1, 2B4, and 2B11 were chosen for mutagenesis. Two additional sites that differ between 2B6 and 2B1 and are known to have a role in 2B1 substrate specificity were also mutated. The 2B6 mutants were expressed in Spodoptera frugiperda cells and characterized using the 2B6-specific substrate RP 73401 [3-cyclopentyloxy-N-(3,5-dichloro-4-pyridyl)-4-methoxybenzamide], the 2B1-selective substrate androstenedione, and the common substrate 7-ethoxy-4-trifluoromethylcoumarin. Mutants F107I and L363V exhibited decreased RP 73401 hydroxylation but retained most of the wild-type level of 2B6 7-ethoxy-4-trifluoromethylcoumarin O-deethylase activity. In addition, SRS exchanges were studied in which the amino acid sequence of 2B6 SRSs was converted to the sequence of 2B1. Each of these constructs, having two to seven substitutions, expressed at levels similar to 2B6 but did not acquire significant androstenedione hydroxylase activity. Docking of RP 73401 into the active site of a 2B6 homology model suggested a direct interaction with residue L363 but not with F107. Findings from this study suggest that 1) residues F107 and L363 are necessary for 2B6 RP 73401 hydroxylase activity, 2) 2B6 is able to tolerate multiple SRS substitutions without compromising protein expression levels or protein stability, and 3) conferring androstenedione hydroxylase function to cytochrome P-450 2B6 is more complex than altering a single SRS.

Animals↗

PubMed1999

Induction of cytochrome P-450 enzymes after repeated exposure to 4-vinylcyclohexene in B6C3F1 mice.

4-Vinylcyclohexene (VCH), an ovarian toxicant in mice, is known to irreversibly deplete ovarian follicles as a consequence of VCH diepoxide formation. Because ovotoxicity requires repeated dosing of VCH, the effect of consecutive daily doses of VCH (7.5 mmol/kg/day) on mouse liver microsomal activities and VCH epoxidation was determined. Cytochromes P-450 2B and 2A (CYP2B and CYP2A), principle isoforms involved in the bioactivation of VCH, as well as CYP2E1 and CYP3A were evaluated. VCH exposure increased total cytochrome P-450 content (35-83% above control levels) after either 5, 10, or 15 days of treatment. Western blot analysis revealed an induction of CYP2A, CYP2B, and CYP2E1 at day 10. Elevated levels of CYP2A and CYP2B correlated with marker androstenedione and testosterone 16alpha- and 16beta-hydroxylase activities. Microsomes prepared from mice pretreated with VCH for 10 days demonstrated an increase (>/=2-fold) in the rate of VCH monoepoxide and diepoxide formation. Microsomal VCH epoxidation was increased to a similar extent by phenobarbital, acetone, and dexamethasone treatment. An increase in cytosolic glutathione S-transferase activity was observed after repeated VCH treatment, an enzyme potentially involved in detoxification of the VCH epoxides. Interestingly, preliminary studies indicated that circulating levels of the monoepoxide (vinylcyclohexene 1, 2-monoepoxide) and diepoxide of VCH were elevated after repeated dosing of VCH. Overall, the results indicate that repeated exposure of VCH in mice induces cytochrome P-450-dependent activities, and in turn induction of its metabolism. Additional studies examining the toxicokinetics of VCH after repeated exposure are required to further delineate the relevance of induction in VCH-induced ovotoxicity.

Animals↗

PubMed1998

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe.

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.

Aflatoxin B1↗

PubMed1998

Analysis of human cytochrome P450 3A4 cooperativity: construction and characterization of a site-directed mutant that displays hyperbolic steroid hydroxylation kinetics.

Cytochrome P450 3A4 is generally considered to be the most important human drug-metabolizing enzyme and is known to catalyze the oxidation of a number of substrates in a cooperative manner. An allosteric mechanism is usually invoked to explain the cooperativity. Based on a structure-activity study from another laboratory using various effector-substrate combinations and on our own studies using site-directed mutagenesis and computer modeling of P450 3A4, the most likely location of effector binding is in the active site along with the substrate. Our study was designed to test this hypothesis by replacing residues Leu-211 and Asp-214 with the larger Phe and Glu, respectively. These residues were predicted to constitute a portion of the effector binding site, and the substitutions were designed to mimic the action of the effector by reducing the size of the active site. The L211F/D214E double mutant displayed an increased rate of testosterone and progesterone 6beta-hydroxylation at low substrate concentrations and a decreased level of heterotropic stimulation elicited by alpha-naphthoflavone. Kinetic analyses of the double mutant revealed the absence of homotropic cooperativity with either steroid substrate. At low substrate concentrations the steroid 6beta-hydroxylase activity of the wild-type enzyme was stimulated by a second steroid, whereas L211F/D214E displayed simple substrate inhibition. To analyze L211F/D214E at a more mechanistic level, spectral binding studies were carried out. Testosterone binding by the wild-type enzyme displayed homotropic cooperativity, whereas substrate binding by L211F/D214E displayed hyperbolic behavior.

Binding Sites↗

PubMed1998

Probing the active site of cytochrome P450 2B1: metabolism of 7-alkoxycoumarins by the wild type and five site-directed mutants.

A series of 7-alkoxycoumarins (chain length of 1-7 carbon atoms) was utilized as active site probes of purified Escherichia coli-expressed cytochrome P450 2B1 wild type and five site-directed mutants (I114V, F206L, V363A, V363L, and G478S). The production of 7-hydroxycoumarin, the O-dealkylation product, by the wild-type enzyme exhibited a rank order of C2 > C4 > C3 > C1 > C5 > C6 = C7. The pattern observed for the P450 I114V mutant was similar to that of the wild-type enzyme, whereas with F206L and G478S mutants, the rate of O-dealkylation was low with all the compounds. In contrast, with V363A, the highest rate of product formation was observed with 7-butoxycoumarin. The V363L mutant preferentially catalyzed the O-dealkylation of 7-methoxy- and 7-ethoxycoumarin, and a further increase in the length of the alkyl chain led to a marked decrease in product formation. The stoichiometry of 7-butoxycoumarin oxidation by V363L suggested that products other than 7-hydroxycoumarin were also formed. HPLC and GC-EIMS analyses revealed that P450 2B1 V363L produced 7-(3-hydroxybutoxy)coumarin and 7-(4-hydroxybutoxy)coumarin as major oxidation products, while the V363A mutant mainly catalyzed the O-dealkylation of 7-butoxycoumarin. Docking of alkoxycoumarins into the active site of a P450 2B1 homology model confirmed the importance of the studied residues in substrate dealkylation and explained the formation of novel 7-butoxycoumarin products by the V363L mutant.

7-Alkoxycoumarin O-Dealkylase↗

PubMed1998

Structural determinants of progesterone hydroxylation by cytochrome P450 2B5: the role of nonsubstrate recognition site residues.

The highly related rabbit cytochromes P450 2B4 and 2B5 differ in only 12 amino acid positions, but only 2B5 has activity toward progesterone. Previously, simultaneous site-directed mutagenesis of four key substrate recognition site (SRS) residues (114, 294, 363, and 367) was shown to result in interconversion of the androstenedione hydroxylase specificities of cytochrome P450 2B4 and 2B5. However, the progesterone metabolite profiles of the 2B4 quadruple mutant or of a quintuple mutant in which residue 370 was also mutated to the 2B5 residue were not identical to that of P450 2B5. Therefore, single mutants of P450 2B5 at the remaining seven positions were constructed, expressed in Escherichia coli, and studied with progesterone as the substrate. The single mutants at positions 120 and 221, which are outside any known SRS, exhibited a significant alteration in progesterone hydroxylation. Based on these results, Ile-114, Arg-120, Ser-221, Ser-294, Ile-363, and Val-367 in cytochrome P450 2B4 were replaced simultaneously with Phe, His, Pro, Thr, Val, and Ala, respectively, from 2B5. This yielded a mutant with a very similar progesterone metabolite profile to that of 2B5, although the total activity was lower. An additional substitution at residue 370 produced a multiple mutant P450 2B4 I114F-R120H-S221P-S294T-I363V-V367A- T370M with very similar or identical substrate specificity, regio- and stereospecificity and kinetic properties to that of P450 2B5 wild type.

Animals↗

PubMed1998

Analysis of four residues within substrate recognition site 4 of human cytochrome P450 3A4: role in steroid hydroxylase activity and alpha-naphthoflavone stimulation.

Sequence alignment of human cytochrome P450 3A4 with bacterial enzymes of known structure has provided a basis from which to predict residues involved in substrate oxidation. Substitutions were made at four residues (I301, F304, A305, and T309) predicted to be located within the highly conserved substrate recognition site 4. Site-directed mutants engineered to contain carboxy-terminal histidine tags were expressed in Escherichia coli and purified on a metal affinity column. The integrity of each protein was assessed by SDS-polyacrylamide gel electrophoresis and immunoblotting. Functional analysis was performed using progesterone and testosterone as substrates and alpha-naphthoflavone as an activator. In testosterone hydroxylase assays, all of the mutants displayed rates of total product formation similar to wild-type 3A4, with several mutants showing small differences in specific products formed. However, with progesterone as the substrate, mutants F304A, A305V, and T309A exhibited altered product ratios and/or changes in the rates of product formation. F304A and A305V also displayed altered flavonoid stimulation that resulted in product ratios dramatically different from wild-type 3A4. Therefore, the kinetics of progesterone hydroxylation of these mutants and the wild-type enzyme were further assessed, and the data were analyzed with the Hill equation. Results with wild-type 3A4 and F304A indicated that at high progesterone concentrations, hydroxylation rates and product ratios are independent of the presence of alpha-NF. This suggests that progesterone may be equivalent to alpha-NF as an activator. In contrast, A305V exhibited autoactivation by progesterone but inhibition by alpha-NF.

Amino Acid Sequence↗

PubMed1998

Catalytic properties of an expressed cytochrome P450 2B1 from a Wistar-Kyoto rat liver cDNA library.

Cytochrome P450 2B1 clones were isolated from a phenobarbital-induced Wistar-Kyoto (WKY) hepatic cDNA library and were found to contain a Glu-322 --> Val substitution, compared with wild-type 2B1 from Sprague-Dawley rats. After heterologous expression in Escherichia coli and purification, activities of this 2B1 E322V variant were determined for ethoxycoumarin and androstenedione. The total activities and metabolite profiles did not differ between 2B1 E322V and wild-type 2B1 for these substrates. In addition, similar rate constants of inactivation were observed with the mechanism-based inactivators chloramphenicol, N-(2-p-nitrophenethyl)chlorofluoroacetamide, and 9-ethynylphenanthrene. These results suggest that the Glu-322 --> Val alteration in the 2B1 WKY variant does not significantly affect 2B1 activity. However, another clone obtained from the cDNA library contained two additional substitutions: Val-103 --> Ala and Glu-424 --> Lys. As residue 103 is within a predicted substrate recognition site (SRS-1), it was of interest to determine whether the Val --> Ala substitution conferred any unique catalytic activities on 2B1. No differences in the metabolism of ethoxycoumarin or androstenedione were observed. However, the Val-103 --> Ala alteration caused an approximately threefold decrease in the rate constant of inactivation for 9-ethynylphenanthrene in comparison with either 2B1 E322V or wild-type 2B1. Based on computer modeling, residue 103 is predicted to be near the active site but at a distance greater than 5A from 9-ethynylphenanthrene. Our results suggest that the Val-103 --> Ala alteration may have an indirect influence on the susceptibility of P450 2B1 to mechanism-based inactivators.

Amino Acid Substitution↗

PubMed1998

Preface

Explore the source record for details and available documents.

Journal Article↗

PubMed1998

Molecular basis of P450 inhibition and activation: implications for drug development and drug therapy.

Three-dimensional homology models of cytochromes P450 (P450) 2B1 and P450 3A4 have been utilized along with site-directed mutagenesis to elucidate the molecular determinants of substrate specificity. Most of the key residues identified in 2B enzymes fall within five substrate recognition sites (SRSs) and have counterparts in bacterial P450 residues that regulate substrate binding or access. Docking of inhibitors into 2B models has provided a plausible explanation for changes in susceptibility to mechanism-based inactivation that accompany particular amino acid side-chain replacements. These studies provide a basis for predicting drug interactions due to P450 inhibition and for rational inhibitor design. In addition, the location of P450 3A4 residues capable of influencing homotropic stimulation by substrates and heterotropic stimulation by flavonoids has been identified. Steroid hydroxylation by the wild-type enzyme exhibits sigmoidal kinetics, indicative of positive cooperativity. Based on the 3A4 model and single-site mutants, a double mutant in SRS-2 has been constructed that exhibits normal Michaelis-Menten kinetics. Results of modeling and mutagenesis studies suggest that the substrate and effector bind at adjacent sites within a single large cavity in P450 3A4. A thorough understanding of the location and structural requirements of the substrate-binding and effector sites in cytochrome P450 3A4 should prove valuable in rationalizing and predicting interactions among the multitude of drugs and other compounds that bind to the enzyme.

Animals↗

PubMed1998

Structure-function of cytochromes P450 and flavin-containing monooxygenases: implications for drug metabolism.

This article is a report on a symposium held at Experimental Biology '98 in San Francisco, California. Recent developments in site-directed mutagenesis, computer-modeling, and mechanistic analysis of cytochromes P450 and flavin-containing monooxygenases are described. A unifying theme is the elaboration of general approaches for understanding and predicting the function of individual forms of these enzymes. A related goal is the production of soluble forms of mammalian cytochromes P450 for X-ray crystallography.

Animals↗

PubMed1997

Stoichiometry of 7-ethoxycoumarin metabolism by cytochrome P450 2B1 wild-type and five active-site mutants.

Recombinant P450 2B1 wild-type and the active-site mutants I114V, F206L, V363A, V363L, and G478S were purified and studied. The efficiency of coupling of reducing equivalents to 7-hydroxycoumarin formation was decreased for all the mutants except I114V. Uncoupling to H2O was increased for F206L, V363A, and G478S, decreased for V363L, and unchanged for I114V. Uncoupling to H2O2 was increased for V363L and decreased for I114V, F206L, and V363A. The findings from this study provide firm biochemical evidence that residues 206, 363, and 478 comprise part of the substrate binding site of P450 2B1.

Binding Sites↗

PubMed1997

Reassessment of cytochrome P450 2B2: catalytic specificity and identification of four active site residues.

Cytochromes P450 2B metabolize a variety of compounds and have provided an excellent framework for identifying determinants of substrate specificity. Among the rat 2B enzymes, a puzzling difference has emerged between the reported substrate specificity of purified hepatic 2B2 and that of certain 2B1 mutants containing 2B1 --> 2B2 substitutions. To address these discrepancies, we have characterized two 2B2 variants. A cDNA clone designated 2B2FF was obtained from phenobarbital-induced Lewis rats and, like some previously isolated variants, was found to contain phenylalanine at positions 58 and 114. A second 2B2 clone was generated by restoring Leu and Ile, respectively, at these positions. These enzymes were expressed in Escherichia coli and analyzed with androstenedione, testosterone, progesterone, ethoxycoumarin, benzyloxyresorufin, and pentoxyresorufin. The expressed 2B2 variants metabolized most substrates at rates that were 1-9% of those of 2B1. When steroid regio- and stereospecificity was examined, the metabolite profiles of expressed 2B2 and 2B2FF conflicted with the 16beta- and 16alpha-hydroxylation observed for purified hepatic 2B2 from Sprague-Dawley rats. These and other results suggested that the purified hepatic 2B2 contained a small percent of the 2B1 enzyme. Masses of tryptic peptides were consistent with identity between purified hepatic 2B2 and 2B2FF. On the basis of a three-dimensional homology model and the construction and analysis of 2B2 mutants, residues 114, 363, 367, and 478 were identified as determinants of substrate specificity. In addition, 2B1 and the expressed 2B2 variants showed differential susceptibility to the mechanism-based inactivators chloramphenicol and N-(2-p-nitrophenethyl)chlorofluoroacetamide.

Animals↗

PubMed1997

Significance of glycine 478 in the metabolism of N-benzyl-1-aminobenzotriazole to reactive intermediates by cytochrome P450 2B1.

The effect of mutating Gly 478 to Ala in rat cytochrome P450 2B1 on the metabolism of N-benzyl-1-aminobenzotriazole was investigated. The 7-ethoxy-4-(trifluoromethyl)coumarin O-deethylation activity of the wild-type enzyme was completely inactivated by incubating with 1 microM BBT. The G478A mutant, however, was not inactivated by incubating with up to 10 microM BBT. Whereas metabolism of BBT by the wild-type 2B1 resulted in the formation of benzaldehyde, benzotriazole, aminobenzotriazole, and a new metabolite, the G478A mutant generated only the later. This metabolite was found by NMR, IR, and mass spectrometry to be a dimeric product formed from the reaction of two BBT molecules. Two spectral binding constants, a high-affinity constant that was the same for both enzymes (30-39 microM) and a low-affinity constant that was 5-fold lower for the mutant enzyme (0.3 mM vs 1.4 mM), were observed with BBT. The apparent Km and kcat values for the G478A mutant with BBT were 0.3 mM and 12 nmol (nmol of P450)-1 min-1, respectively. Molecular modeling studies of BBT bound in the active site of P450 2B1 suggested that a mutation of Gly 478 to Ala would result in steric hindrance and suppress oxidation of BBT at the 1-amino nitrogen. When BBT was oriented in the 2B1 active site such that oxidation at the 7-benzyl carbon could occur, no steric overlap between Ala 478 and the substrate was observed. Thus, this orientation of BBT would be preferred by the mutant leading to oxidation at the 7-benzyl carbon and subsequent dimer formation. These findings indicate that a glycine 478 to alanine substitution in P450 2B1 altered the binding of BBT such that inactivating BBT metabolites were no longer generated.

Animals↗

PubMed1997

Identification of three key residues in substrate recognition site 5 of human cytochrome P450 3A4 by cassette and site-directed mutagenesis.

Cassette mutagenesis and site-directed mutagenesis were used to investigate the importance of individual amino acid residues at positions 364-377 of cytochrome P450 3A4 in determining steroid hydroxylation or stimulation by alpha-naphthoflavone. The mutants were expressed in an Escherichia coli system, and solubilized membranes were prepared. All mutants except R365G and R365K exhibited anti-3A immunoreactivity on Western blotting, although R372S and R375K were not detected as the Fe2+-CO complex. Replacement of Arg-372 by Lys yielded a typical P450 spectrum. The results indicate that the highly conserved Arg residues at positions 365 and 375 may play a role in stabilizing the tertiary structure or in heme binding. Catalytic activities of 12 mutants were examined using progesterone and testosterone as substrates, and residues 369, 370, and 373 were found to play an important role in determining substrate specificity. Although the three mutants hydroxylated progesterone and testosterone primarily at the 6beta-position like the wild-type, replacement of Ile-369 by Val suppressed progesterone 16alpha-hydroxylase activity, whereas substitution of Ala-370 with Val enhanced progesterone 16alpha-hydroxylation. Interestingly, substitution of Leu-373 with His resulted in production of a new metabolite from both steroids. Moreover, the mutants at positions 369 and 373 were more and less responsive, respectively, than the wild-type to alpha-naphthoflavone stimulation. Alterations in activities or expression of several mutants were interpreted using a three-dimensional model of P450 3A4. The results suggest that analogy with mammalian family 2 and bacterial cytochromes P450 can be used to predict P450 3A residues that contribute to regiospecific steroid hydroxylation.

Amino Acid Sequence↗

PubMed1997

Functional interaction between amino-acid residues 242 and 290 in cytochromes P-450 2B1 and 2B11.

Previous studies have revealed the functional importance of the negatively charged amino-acid residue Asp-290 of the phenobarbital-inducible dog liver cytochrome P-450 (P-450) 2B11 (Harlow, G.R. and Halpert J.R. (1996) Arch. Biochem. Biophys. 326, 85-92). A search for P-450 2B11 residues capable of forming a charge pair with Asp-290 suggested the positively charged residue Lys-242 as a likely candidate. Replacement of Lys-242 with Asp in a P-450 2B11 fusion protein with rat NADPH-cytochrome P-450 reductase (reductase) resulted in very low holoenzyme expression levels in Escherichia coli, as did replacement of Asp-290 with Lys. Remarkably, however, expression levels of the double mutant Lys-242 --> Asp/Asp-290 --> Lys were dramatically increased above either single replacement alone. Similarly, the pair-wise substitutions Lys-242 --> Leu/Asp-290 --> Ile in P-450 2B11 and Leu-242 --> Lys/Ile-290 --> Asp in P-450 2B1 showed greater holoenzyme expression levels than the constituent single mutants, providing further evidence for the close proximity of these residues within the three-dimensional structure of these two enzymes. These results support the hypothesis that a functional interaction exists between residues 242 and 290, which may help to coordinate the relative positions of proposed helices G and I. All of the mutant combinations, including the additional P-450 2B11 double mutants Tyr-242/Asn-290 and Tyr-242/Ser-290, displayed altered stereoselectivity of androstenedione hydroxylation.

Androstenedione↗