Evaluation of structural features in fungal cytochromes P450 predicted to rule catalytic diversification

Fungi belong to the large kingdom of lower eukaryotic organisms encompassing yeasts along with filamentous and dimorphic members. Microbial P450 enzymes have contributed to exploration of and adaptation to diverse ecological niches such as conversion of lipophilic compounds to more hydrophilic derivatives or degradation of a vast array of environmental toxicants. To better understand diversification of the catalytic behavior of fungal P450s, detailed insight into the molecular machinery steering oxidative attack on the distinctly structured endogenous and xenobiotic substrates is of preeminent interest. Based on a general, CYP102A1-related template the bulk of predicted substrate/inhibitor-binding determinants were shown to cluster near the distal heme face within the six known substrate recognition sites (SRSs) made up by the α-helical B′/F/G/I tetrad, the B′–C interhelical loop and strands of the β6-sheet, population density being highest in the structurally flexible SRS-1 and SRS-4 domains, showing a low degree of conservation. Reactivity toward ligands favorably coincides with the lipophilicity/hydrophilicity profile and bulkiness of critical amino acids acting as selective filters. Some decisive elements may also serve in maintenance of catalytic competence via their action as gatekeepers directing substrate access/positioning or stabilizers of the heme environment enabling dioxygen activation. Non-SRS residues seem to control spin state equilibria and attract redox partners by electrostatic forces. Of note, the inhibitory potency of azole-type fungicides is likely to arise from perturbation of the complex interplay of the mechanistic principles addressed above. Knowledge-supported exploitation of the topological data will be helpful in the manufacture of commodity/specialty chemicals as well as therapeutic agents. Also, engineered fungal P450s may be used to improve pollutant-specific bioremediation of contaminated soils.     

Structural determinants of aromatase cytochrome p450 inhibition in substrate recognition site-1

The porcine gonadal form of aromatase cytochrome P450 (P450arom) exhibits higher sensitivity to inhibition by the imidazole, etomidate, than the placental isozyme. The residue(s) responsible for this functional difference was mapped using chimeragenesis and point mutation analysis of the placental isozyme, and the kinetic analysis was conducted on native and mutant enzymes after overexpression in insect cells. The etomidate sensitivity of the placental isozyme was markedly increased by substitution of the predicted substrate recognition site-1 (SRS-1) and essentially reproduced that of the gonadal isozyme by substitution of SRS-1 and the predicted B helix. A single isoleucine (I) to methionine (M) substitution at position 133 of the placental isozyme (I(133)M) was proven to be the critical residue within SRS-1. Residue 133 is located in the B'-C loop and has been shown to be equally important in other steroid-metabolizing P450s. Single point mutations (including residues 110, 114, 120, 128, 137, and combinations thereof among others) and mutation of the entire B and C helixes were without marked effect on etomidate inhibitory sensitivity. The same mutation (I(133)M) introduced into human P450arom also markedly increased etomidate sensitivity. Mutation of Ile(133) to either alanine (I(133)A) or tyrosine (I(133)Y) decreased apparent enzyme activity, but the I(133)A mutant was sensitive to etomidate inhibition, suggesting that it is Ile(133) that decreases etomidate binding rather than Met(133) increasing enzyme sensitivity. Androstenedione turnover and affinity were similar for the I(133)M mutant and the native placental isozyme. These data suggest that Ile(133) is a contact residue in SRS-1 of P450arom, emphasize the functional conservation that exists in SRS-1 of a number of steroid-hydroxylating P450 enzymes, and suggest that substrate and inhibitor binding are dependent on different contact points to varying degrees.     

Can peroxygenase and microperoxidase substitute cytochrome P450 in biosensors

Aromatic peroxygenase (APO) from the basidiomycetous mushroom Agrocybe aegerita (AaeAPO) and microperoxidases (MPs) obtained from cytochrome c exhibit a broad substrate spectrum including hydroxylation of selected aromatic substrates, demethylation and epoxidation by means of hydrogen peroxide. It overlaps with that of cytochrome P450 (P450), making MPs and APOs to alternate recognition elements in biosensors for the detection of typical P450 substrates. Here, we discuss recently developed approaches using microperoxidases and peroxygenases in view of their potential to supplement P450 enzymes as recognition elements in biosensors for aromatic compounds. Starting as early as the 1970s, the direct electron transfer between electrodes and the heme group of heme peptides called microperoxidases has been used as a model of oxidoreductases. These MP-modified electrodes are used as hydrogen peroxide detectors based on the catalytic current generated by electrically contacted microperoxidase molecules. A similar catalytic reaction has been obtained for the electrode-immobilised heme protein AaeAPO. However, up to now, no MP-based sensors for substrates have been described. In this review, we present biosensors which indicate 4-nitrophenol, aniline, naphthalene and p-aminophenol based on the peroxide-dependent substrate conversion by electrode-immobilised MP and AaeAPO. In these enzyme electrodes, the signal is generated by the conversion of all substrates, thus representing in complex media an overall parameter. The performance of these sensors and their further development are discussed in comparison with P450-based electrodes.     

CYP2E1 active site residues in substrate recognition sequence 5 identified by photoaffinity labeling and homology modeling

Despite its biological importance, our knowledge of active site structure and relevance of critical amino acids in CYP2E1 catalytic processes remain limited. In this study, we identified CYP2E1 active site residues using photoaffinity labeling with 7-azido-4-methylcoumarin (AzMC) coupled with a CYP2E1 homology model. In the absence of light, AzMC was an effective competitor against substrate p-nitrophenol oxidation by CYP2E1. Photoactivation of AzMC led to a concentration-dependent loss in CYP2E1 activity and structural integrity resulting from the modification of both heme and protein. The photo-labeling reaction degraded heme and produced a possible heme adduct. Probe incorporation into the protein occurred at multiple sites within substrate recognition sequence 5 (SRS-5). Based on a CYP2E1 homology model, we hypothesize AzMC labels SRS-5 residues, Leu363, Val364, and Leu368, in the active site. In addition, we propose a series of phenylalanines, especially Phe106, mediate contacts with the coumarin.     

Crystal structure of CYP199A2, a para-substituted benzoic acid oxidizing cytochrome P450 from Rhodopseudomonas palustris

CYP199A2, a cytochrome P450 enzyme from Rhodopseudomonas palustris, oxidatively demethylates 4-methoxybenzoic acid to 4-hydroxybenzoic acid. 4-Ethylbenzoic acid is converted to a mixture of predominantly 4-(1-hydroxyethyl)-benzoic acid and 4-vinylbenzoic acid, the latter being a rare example of CC bond dehydrogenation of an unbranched alkyl group. The crystal structure of CYP199A2 has been determined at 2.0-Å resolution. The enzyme has the common P450 fold, but the B′ helix is missing and the G helix is broken into two (G and G′) by a kink at Pro204. Helices G and G′ are bent back from the extended BC loop and the I helix to open up a clearly defined substrate access channel. Channel openings in this region of the P450 fold are rare in bacterial P450 enzymes but more common in eukaryotic P450 enzymes. The channel is hydrophobic except for the basic residue Arg246 at the entrance, which probably plays a role in the specificity of this enzyme for charged benzoates over neutral phenols and benzenes. The substrate binding pocket is hydrophobic, with Ser97 and Ser247 being the only polar residues. Computer docking of 4-ethylbenzoic acid into the active site suggests that the substrate carboxylate oxygens interact with Ser97 and Ser247, and the β-methyl group is located over the heme iron by Phe185, the side chain of which is only 6.35 Å above the iron in the native structure. This binding orientation is consistent with the observed product profile of exclusive attack at the para substituent. Putidaredoxin of the CYP101A1 system from Pseudomonas putida supports substrate oxidation by CYP199A2 at ∼6% of the activity of the physiological ferredoxin. Comparison of the heme proximal faces of CYP199A2 and CYP101A1 suggests that charge reversal surrounding the surface residue Leu369 in CYP199A2 may be a significant factor in this low cross-activity.     

Substrate recognition sites in 14alpha-sterol demethylase from comparative analysis of amino acid sequences and X-ray structure of Mycobacterium tuberculosis CYP51.

The crystal structure of 14alpha-sterol demethylase from Mycobacterium tuberculosis (MTCYP51) [Proc. Natl. Acad. Sci. USA 98 (2001) 3068-3073] provides a template for analysis of eukaryotic orthologs which constitute the CYP51 family of cytochrome P450 proteins. Putative substrate recognition sites (SRSs) were identified in MTCYP51 based on the X-ray structures and have been compared with SRSs predicted based on Gotoh's analysis [J. Biol. Chem. 267 (1992) 83-90]. While Gotoh's SRS-4, 5, and 6 contribute in formation of the putative MTCYP51 substrate binding site, SRS-2 and 3 likely do not exist in MTCYP51. SRS-1, as part of the open BC loop, in the conformation found in the crystal can provide only limited contacts with the sterol. However, its role in substrate binding might dramatically increase if the loop closes in response to substrate binding. Thus, while the notion of SRSs has been very useful in leading to our current understanding of P450 structure and function, their identification by sequence alignment between distant P450 families will not necessarily be a good predictor of residues associated with substrate binding. Localization of CYP51 mutation hotspots in Candida albicans azole resistant isolates was analyzed with respect to SRSs. These mutations are found to be outside of the putative substrate interacting sites indicating the preservation of the protein active site under the pressure of azole treatment. Since the mutations residing outside the putative CYP51 active side can profoundly influence ligand binding within the active site, perhaps they provide insight into the basis of evolutionary changes which have occurred leading to different P450s.     

A comparative approach to structure-function studies of mammalian aromatases

To date, structure–function studies of aromatase cytochrome P450 (P450arom) have been advanced by point mutation analyses utilizing almost exclusively the human enzyme, in conjunction with computer-generated models of the three-dimensional form of the enzyme based on prokaryotic cytochromes P450. Recent studies have identified duplicated isozymes of porcine P450arom, the gonadal and placental forms of which appear to differ substantially in substrate utilization and inhibitor sensitivity. We present a comparative approach to define regions of P450arom responsible for specific functional characteristics using complimentary DNAs encoding the porcine isozymes. Constructs encoding the native and chimeric porcine and human P450arom enzymes were transiently expressed and activity was assessed using the tritiated water assay. Sensitivity to inhibition by the imidazole etomidate was investigated, and P450arom expression was assessed by immunoblot analysis. All constructs yielded active P450arom, suggesting that exchanging entire structural elements does not preclude catalytic function. The activity of the gonadal isozyme was shown to be inhibited by etomidate at concentrations 185 and 300-fold lower than those required to induce a similar inhibition of the placental and human enzymes, respectively. In contrast, there was only a two-fold difference in the sensitivity of the gonadal and placental isozymes to inhibition by CGS16949A. Analysis of chimeric constructs indicated that the sensitivity to etomidate was associated with residues in the B, B′ and C helices of the gonadal P450arom encompassing only one of six putative substrate recognition sites. Additionally, sensitivity to etomidate was not correlated with enzyme activity among the chimeric enzymes. Therefore, it appears that residues of the porcine gonadal P450arom that are responsible for etomidate binding may be distinct from those involved in substrate recognition and metabolism. These data support the notion that a comparative approach employing the use of chimeric enzymes provides a useful tool in directing point mutational analysis to determine residues important in inhibitor and perhaps substrate recognition of P450 enzymes such as P450arom. These studies are currently in progress.     

Comparison of Drosophila cytochrome P450 metabolism of natural and model substrates

Metabolism of some insecticides and toxic natural plant compounds is known to involve cytochrome P450 enzymes. Correlations between insecticide resistance and deethylation of the model substrate, 7-ethoxycoumarin, have prompted its use in screens for potentially resistant insect populations. The applicability of this model substrate as an indicator of the enzyme activities and inductive responsiveness of cytochrome P450 isoforms involved in the metabolism of carnegine was investigated. This toxic isoquinoline alkaloid is found in the host-plants of some species of cactophilic Drosophila. The results show that the ethoxycoumarin (ECOD) assay does not accurately predict carnegine metabolism either quantitatively or with respect to the overall pattern of activity. Therefore, the ECOD assay may be as isozyme-specific in insects as has already been demonstrated in mammals and its use as an indicator of general P450 activity is questionable.     

Substrate Recognition by the Multifunctional Cytochrome P450 MycG in Mycinamicin Hydroxylation and Epoxidation Reactions

The majority of characterized cytochrome P450 enzymes in actinomycete secondary metabolic pathways are strictly substrate-, regio-, and stereo-specific. Examples of multifunctional biosynthetic cytochromes P450 with broader substrate and regio-specificity are growing in number and are of particular interest for biosynthetic and chemoenzymatic applications. MycG is among the first P450 monooxygenases characterized that catalyzes both hydroxylation and epoxidation reactions in the final biosynthetic steps, leading to oxidative tailoring of the 16-membered ring macrolide antibiotic mycinamicin II in the actinomycete Micromonospora griseorubida. The ordering of steps to complete the biosynthetic process involves a complex substrate recognition pattern by the enzyme and interplay between three tailoring modifications as follows: glycosylation, methylation, and oxidation. To understand the catalytic properties of MycG, we structurally characterized the ligand-free enzyme and its complexes with three native metabolites. These include substrates mycinamicin IV and V and their biosynthetic precursor mycinamicin III, which carries the monomethoxy sugar javose instead of the dimethoxylated sugar mycinose. The two methoxy groups of mycinose serve as sensors that mediate initial recognition to discriminate between closely related substrates in the post-polyketide oxidative tailoring of mycinamicin metabolites. Because x-ray structures alone did not explain the mechanisms of macrolide hydroxylation and epoxidation, paramagnetic NMR relaxation measurements were conducted. Molecular modeling based on these data indicates that in solution substrate may penetrate the active site sufficiently to place the abstracted hydrogen atom of mycinamicin IV within 6 Å of the heme iron and ∼4 Å of the oxygen of iron-ligated water.     

Effect of mutations at Lys250, Arg251, and Lys253 of cytochrome P450 1A2 on the catalytic activities and the bindings of bifunctional axial ligands.

Some eukaryotic cytochromes P450 (P450s) have a series of ionic amino acids, corresponding to Lys250, Arg251, and Lys253 residues in the P450 1A2 sequence. To understand the roles of those ionic amino acids in the catalytic function of P450, three single mutants, Lys250Leu, Arg251Leu, and Lys253Leu of P450 1A2 were obtained from yeast (Saccharomyces cerevisiae) expression system. Turnover numbers of the Arg251Leu mutant in dealkylation reactions of methoxy- and ethoxyresorufin catalyzed by the P450 reconstituted system were remarkably increased by sixfold compared to those of the wild type. The Lys250Leu and Lys253Leu mutants also showed turnover numbers higher than those of the wild type by three- to fourfold. Those catalytic activities were inhibited competitively by pyridine derivatives, nitrogenous axial ligands to the P450 heme. From those findings together with other spectral data, it was suggested that the ionic site of Lys250, Arg251, and Lys253 may be somehow located near the substrate recognition site and/or near the axial-ligand access channel of this enzyme.     

Structural basis for juvenile hormone biosynthesis by the juvenile hormone acid methyltransferase

Juvenile hormone (JH) acid methyltransferase (JHAMT) is a rate-limiting enzyme that converts JH acids or inactive precursors of JHs to active JHs at the final step of JH biosynthesis in insects and thus presents an excellent target for the development of insect growth regulators or insecticides. However, the three-dimensional properties and catalytic mechanism of this enzyme are not known. Herein, we report the crystal structure of the JHAMT apoenzyme, the three-dimensional holoprotein in binary complex with its cofactor S-adenosyl-l-homocysteine, and the ternary complex with S-adenosyl-l-homocysteine and its substrate methyl farnesoate. These structures reveal the ultrafine definition of the binding patterns for JHAMT with its substrate/cofactor. Comparative structural analyses led to novel findings concerning the structural specificity of the progressive conformational changes required for binding interactions that are induced in the presence of cofactor and substrate. Importantly, structural and biochemical analyses enabled identification of one strictly conserved catalytic Gln/His pair within JHAMTs required for catalysis and further provide a molecular basis for substrate recognition and the catalytic mechanism of JHAMTs. These findings lay the foundation for the mechanistic understanding of JH biosynthesis by JHAMTs and provide a rational framework for the discovery and development of specific JHAMT inhibitors as insect growth regulators or insecticides.     

Heterologous expression of cytochrome P450 2D6 mutants, electron transfer, and catalysis of bufuralol hydroxylation: the role of aspartate 301 in structural integrity

Cytochrome P450 (P450) 2D6 is a polymorphic human enzyme involved in the oxidation of >50 drugs, most of which contain a basic nitrogen. In confirmation of previous work by others, substitutions at Asp301 decreased rates of substrate oxidation by P450 2D6. An anionic residue (Asp, Glu) at this position was found to be important in proper protein folding and heme incorporation, and positively charged residues were particularly disruptive in bacterial and also in baculovirus expression systems. Truncation of 20 N-terminal amino acids had no significant effect on catalytic activity except to attenuate P450 2D6 interaction with membranes and NADPH-P450 reductase. The truncation of the N-terminus increased the level of bacterial expression of wild-type P450 2D6 (Asp301) but markedly reduced expression of all codon 301 mutants, including Glu301. Reduction of ferric P450 2D6 by NADPH-P450 reductase was enhanced in the presence of the prototypic substrate bufuralol. Bacterial flavodoxin, an NADPH-P450 reductase homolog, binds tightly to P450 2D6 but is inefficient in electron transfer to the heme. These results collectively indicate that the acidic residue at position 301 in P450 2D6 has a structural role in addition to any in substrate binding and that the N-terminus of P450 2D6 is relatively unimportant to catalytic activity beyond a role in facilitating binding to NADPH-P450 reductase.     

Toward understanding the inactivation mechanism of monooxygenase P450 BM-3 by organic cosolvents: a molecular dynamics simulation study

Cytochrome P450 BM-3 from Bacillus megaterium is an extensively studied enzyme for industrial applications. A major focus of current protein engineering research is directed to improving the catalytic performance of P450 BM-3 toward nonnatural substrates of industrial importance in the presence of organic solvents or cosolvents. For the latter reason, it is important to study the effect of organic cosolvent molecules on the structure and dynamics of the enzyme, in particular, the effect of cosolvent molecules on the active site's structure and dynamics. In this paper, we have studied, using molecular dynamics (MD) simulations, the F87A mutant of P450 BM-3 in the presence of DMSO as cosolvent, to understand the role of the F87A substitution for its catalytic activity. This mutant exhibits an altered regioselectivity and substrate specificity compared with wild-type; however, it has lower tolerance toward DMSO. The simulation results offer an explanation for the DMSO sensitivity of the F87A mutant. Our simulation results show that the F87 side chain prevents the disturbance of the water molecule bound to the heme iron by DMSO molecules. The absence of the phenyl ring in F87A mutant promotes interactions of the DMSO molecule with the heme iron resulting in water displacement by DMSO at the catalytic heme center.     

Functional interpretation and structural insights of Arabidopsis lyrata cytochrome P450 CYP71A13 involved in auxin synthesis

Cytochrome P450 CYP71A13 of Arabidopsis lyrata is a heme protein involved in biosynthesis of indole-3-acetonitrile which leads to the formation of indolyl-3-acetic acid. It catalyzes a unique reaction: formation of a carbon-nitrogen triple bond and dehydration of indolyl-3-acetaldoxime. Homology model of this 57 kDa polypeptide revealed that the heme existed between H-helix and J- helix in the hydrophobic pocket, although both helixes are involved in catalytic activity, where Gly305 and Thr308, 311 of H- helix were involved in its stabilization. The substrate indole-3-acetaldoxime was tightly fitted into the substrate pocket with the aromatic ring being surrounded by amino acid residues creating a hydrophobic environment. The smaller size of the substrate binding pocket in cytochrome P450 CYP71A13 was due to the bulkiness of the two amino acid residues Phe182 and Trp315 pointing into the substrate binding cavity. The apparent role of the heme in cytochrome P450 CYP71A13 was to tether the substrate in the catalysis by indole-3-acetaldoxime dehydratase. Since the crystal structure of cytochrome P450 CYP71A13 has not yet been solved, the modeled structure revealed mechanism of substrate recognition and catalysis.     

Conservation in the CYP51 family. Role of the B' helix/BC loop and helices F and G in enzymatic function

CYP51 (sterol 14 alpha-demethylase) is an essential enzyme in sterol biosynthetic pathways and the only P450 gene family having catalytically identical orthologues in different biological kingdoms. The proteins have low sequence similarity across phyla, and the whole family contains about 40 completely conserved amino acid residues. Fifteen of these residues lie in the secondary structural elements predicted to form potential substrate recognition sites within the P450 structural fold. The role of 10 of these residues, in the B' helix/BC loop, helices F and G, has been studied by site-directed mutagenesis using as a template the soluble sterol 14 alpha-demethylase of known structure, CYP51 from Mycobacterium tuberculosis (MT) and the human orthologue. Single amino acid substitutions of seven residues (Y76, F83, G84, D90, L172, G175, and R194) result in loss of the ability of the mutant MTCYP51 to metabolize lanosterol. Residual activity of D195A is very low, V87A is not expressed as a P450, and A197G has almost 1 order of magnitude increased activity. After purification, all of the mutants show normal spectral properties, heme incorporation, and the ability to be reduced enzymatically and to interact with azole inhibitors. Profound influence on the catalytic activity correlates well with the spectral response to substrate binding, effect of substrate stabilization on the reduced state of the P450, and substrate-enhanced efficiency of enzymatic reduction. Mutagenesis of corresponding residues in human CYP51 implies that the conserved amino acids might be essential for the evolutionary conservation of sterol 14 alpha-demethylation from bacteria to mammals.     

A substrate-specific cytochrome P450 monooxygenase, CYP6AB11, from the polyphagous navel orangeworm (Amyelois transitella)

The navel orangeworm Amyelois transitella (Walker) (Lepidoptera: Pyralidae) is a serious pest of many tree crops in California orchards, including almonds, pistachios, walnuts and figs. To understand the molecular mechanisms underlying detoxification of phytochemicals, insecticides and mycotoxins by this species, full-length CYP6AB11 cDNA was isolated from larval midguts using RACE PCR. Phylogenetic analysis of this insect cytochrome P450 monooxygenase established its evolutionary relationship to a P450 that selectively metabolizes imperatorin (a linear furanocoumarin) and myristicin (a natural methylenedioxyphenyl compound) in another lepidopteran species. Metabolic assays conducted with baculovirus-expressed P450 protein, P450 reductase and cytochrome b₅ on 16 compounds, including phytochemicals, mycotoxins, and synthetic pesticides, indicated that CYP6AB11 efficiently metabolizes imperatorin (0.88 pmol/min/pmol P450) and slowly metabolizes piperonyl butoxide (0.11 pmol/min/pmol P450). LC-MS analysis indicated that the imperatorin metabolite is an epoxide generated by oxidation of the double bond in its extended isoprenyl side chain. Predictive structures for CYP6AB11 suggested that its catalytic site contains a doughnut-like constriction over the heme that excludes aromatic rings on substrates and allows only their extended side chains to access the catalytic site. CYP6AB11 can also metabolize the principal insecticide synergist piperonyl butoxide (PBO), a synthetic methylenedioxyphenyl compound, albeit slowly, which raises the possibility that resistance may evolve in this species after exposure to synergists under field conditions.     

Structural analysis of CYP2R1 in complex with vitamin D3

The activation of vitamin D to its hormonal form is mediated by cytochrome P450 enzymes. CYP2R1 catalyzes the initial step converting vitamin D into 25-hydroxyvitamin D. A CYP2R1 gene mutation causes an inherited form of rickets due to 25-hydroxylase deficiency. To understand the narrow substrate specificity of CYP2R1 we obtained the hemeprotein in a highly purified state, confirmed the enzyme as a vitamin D 25-hydroxylase, and solved the crystal structure of CYP2R1 in complex with vitamin D₃. The CYP2R1 structure adopts a closed conformation with the substrate access channel being covered by the ordered B′-helix and slightly opened to the surface, which defines the substrate entrance point. The active site is lined by conserved, mostly hydrophobic residues. Vitamin D₃ is bound in an elongated conformation with the aliphatic side-chain pointing toward the heme. The structure reveals the secosteroid binding mode in an extended active site and allows rationalization of the molecular basis of the inherited rickets associated with CYP2R1.     

Conformational change of cytochrome P-450 indicated by the measurement of fluorescence-energy transfer

The conformation of cytochrome P-450 was studied in relation to the interaction with its substrate by the measurement of fluorescence energy transfer. Cytochrome P-450 I-c and cytochrome P-450 II-d, both obtained from the hepatic microsome of polychlorinated biphenyl-treated rats, were used as P-450 type and P-448 type, respectively, and benzo[a]pyrene and 7-ethoxycoumarin were used as substrate. The distance between the donor and acceptor, which was described by the F?rster equation, was calculated on the basis of the fluorescence-energy transfer between the substrate as a donor and the heme of the enzyme as an acceptor. The distance was apparently changed by incorporation of the enzyme into phospholipid or by reduction of the heme, indicating that the treatments changed the conformation of the enzyme. Differences in the conformational change were observed between the two enzymes, and the conformational change of cytochrome P-450 II-d using benzo[a]pyrene as a substrate was different from that using 7-ethoxycoumarin. The results suggest that the substrate-binding sites of the two enzymes differ in position toward the heme, and that there are at least two different substrate sites in cytochrome P-450 II-d, one for benzo[a]pyrene and another for 7-ethoxycoumarin.     

Impact of incorporating the 2C5 crystal structure into comparative models of cytochrome P450 2D6

Cytochrome P450 2D6 (CYP2D6) metabolizes approximately one third of the drugs in current clinical use. To gain insight into its structure and function, we have produced four different sets of comparative models of 2D6: one based on the structures of P450s from four different microorganisms (P450 terp, P450 eryF, P450 cam, and P450 BM3), another on the only mammalian P450 (2C5) structure available, and the other two based on alternative amino acid sequence alignments of 2D6 with all five of these structures. Principal component analysis suggests that inclusion of the 2C5 crystal structure has a profound effect on the modeling process, altering the general topology of the active site, and that the models produced differ significantly from all of the templates. The four models of 2D6 were also used in conjunction with molecular docking to produce complexes with the substrates codeine and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP); this identified Glu 216 [in the F-helix; substrate recognition site (SRS) 2] as a key determinant in the binding of the basic moiety of the substrate. Our studies suggest that both Asp 301 and Glu 216 are required for metabolism of basic substrates. Furthermore, they suggest that Asp 301 (I-helix, SRS-4), a residue thought from mutagenesis studies to bind directly to the basic moiety of substrates, may play a key role in positioning the B'-C loop (SRS-1) and that the loss of activity on mutating Asp 301 may therefore be the result of an indirect effect (movement of the B'-C loop) on replacing this residue.     

Post-translational reduction of cytochrome P450IIE by CCl4, its substrate

The molecular mechanism of cytochrome P450IIE reduction by CCl4 was reexamined by measuring its enzyme activity, immunoreactive protein contents, and mRNA levels. Aniline hydroxylase and the amounts of immunoreactive P450IIE were rapidly decreased in a time-dependent manner after a single dose of CCl4. No changes were observed in the amounts of immunoreactive P450IIC and P450IA despite significant decreases decrease in their catalytic activities. However, the decreases in P450IIE enzyme activity and immunoreactive protein by CCl4 were not accompanied by a decline in its mRNA level. The data thus suggested a post-translational reduction of P450IIE by CCl4, probably due to specific destruction of the P450IIE protein by its own substrate rather than heme moiety.