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Rational mutagenesis was used to improve the thermal stability of human cytochrome P450 2B6 and canine P450 2B11. Comparison of the amino acid sequences revealed seven sites that are conserved between the stable 2B1 and 2B4 but different from those found in the less stable 2B6 and 2B11. P334S was the only mutant that showed increased heterologous expression levels and thermal stability in both 2B6 and 2B11. The mechanism of this effect was explored with pressure-perturbation spectroscopy. Compressibility of the heme pocket in variants of all four CYP2B enzymes containing proline at position 334 are characterized by lower compressibility than their more stable serine 334 counterpart. Therefore, the stabilizing effect of P334S is associated with increased conformational flexibility in the region of the heme pocket. Improved stability of P334S 2B6 and 2B11 may facilitate the studies of these enzymes by X-ray crystallography and biophysical techniques., (2009 Elsevier Inc. All rights reserved.)

CYP2C9 polymorphisms result in reduced enzyme catalytic activity and greater activation by effector molecules as compared to wild-type protein, with the mechanism(s) for these changes in activity not fully elucidated. Through T(1) NMR and spectral binding analyses, mechanism(s) for these differences in behavior of the variant proteins (CYP2C9.2, CYP2C9.3, and CYP2C9.5) as compared to CYP2C9.1 were assessed. Neither altered binding affinity nor substrate (flurbiprofen) proton to heme-iron distances differed substantially among the four enzymes. Co-incubation with dapsone resulted in reduced substrate proton to heme-iron distances for all enzymes, providing at least a partial mechanism for the activation of CYP2C9 variants by dapsone. In summary, neither altered binding affinity nor substrate orientation appear to be major factors in the reduced catalytic activity noted in the CYP2C9 variants, but dapsone co-incubation caused similar changes in substrate proton to heme-iron distances suggesting at least partial common mechanisms in the activation of the CYP2C9 forms.

Modifications at the N-terminus of the rabbit CYP4B1 gene resulted in expression levels in Escherichia coli of up to 660 nmol/L. Solubilization of the enzyme from bacterial membranes led to substantial conversion to cytochrome P420 unless alpha-naphthoflavone was added as a stabilizing ligand. Mass spectrometry analysis and Edman sequencing of purified enzyme preparations revealed differential N-terminal post-translational processing of the various constructs expressed. Notably, bacterial expression of CYP4B1 produced a holoenzyme with >98.5% of its heme prosthetic group covalently linked to the protein backbone. The near fully covalently linked hemoproteins exhibited similar rates and regioselectivities of lauric acid hydroxylation to that observed previously for the partially heme processed enzyme expressed in insect cells. These studies shed new light on the consequences of covalent heme processing in CYP4B1 and provide a facile system for future mechanistic and structural studies with the enzyme.

Dog CYP2B11, rat CYP2B1, and rabbit CYP2B5 have been expressed in Escherichia coli from cDNAs modified at the N-terminus (Barnes et al., 1991, Proc. Natl. Acad. Sci. USA 88, 5597-5601). Using 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (Chaps), solubilized membranes representing > 100 nmol of P450 2B11, > 35 nmol of P450 2B1, and > 7 nmol of P450 2B5 were efficiently extracted (40-70% yield) from a 1-liter culture. Chaps-solubilized preparations produced a reduced CO/reduced difference spectrum devoid of P420 and were used directly in a reconstituted system. The E. coli-expressed 2B enzymes retained the same functional characteristics as the purified hepatic enzymes or enzymes expressed in COS cells in terms of androstenedione metabolite profiles. Hydroxylation rates were determined under a variety of conditions, including two concentrations of NADPH-cytochrome P450 reductase (2 and 16 nmol/nmol P450) and the absence or presence of cytochrome b5 (2 nmol/nmol P450). The androstenedione hydroxylase activities of expressed 2B1 and 2B5 were stimulated by cytochrome b5, whereas P450 2B11 was inhibited slightly by cytochrome b5. Purified expressed 2B11 (specific content, 8 nmol/mg protein) had similar activities as the Chaps-solubilized membrane preparation. The solubilized membranes containing 2B11 were also tested with 2,2',4,4',5,5'-hexachlorobiphenyl (245-HCB). Three major metabolites, 2-hydroxy-4,5,2',4',5'-pentachlorobiphenyl, 3-hydroxy-2,4,5,2',4',5'-hexachlorobiphenyl, and 2-hydroxy-3,4,5,2',4',5'-hexachlorobiphenyl were produced from 245-HCB. These metabolites are identical to those produced by 2B11 purified from liver microsomes. The 245-HCB hydroxylation rates were similar for E. coli-expressed 2B11, dog liver microsomes, and purified liver 2B11. When only the second codon in the 2B1 was changed to GCT, > 25 nmol of P450 was extracted from a 1-liter culture, suggesting that the full Barnes et al. modification scheme may not be necessary for high-level expression. An efficient method of expressing, extracting, and analyzing different P450 2B enzymes has thus been achieved. In addition, rabbit P450 2B5, which has never been purified from liver, as well as different P450 2B mutants can now be expressed at much higher levels than previously reported. The ability to express different 2B wild-type and mutant P450s in E. coli provides an excellent opportunity to study the molecular basis of species differences in substrate metabolism.

Microsomal preparations of Aspergillus ochraceus TS oxidised benzo(a)pyrene very efficiently in the presence of NADPH and 02 and exhibits a pH optimum of 8.0-8.2. The hydroxylation is also effected in presence of NaI04. Hydroxylation was inhibited by metyrapone, SKF-525A, PCMB, imidazole, carbon monoxide and flavone but not by cyanide, azide and antimycin A indicating thereby the involvement of cytochrome P-450 in this reaction. Inhibition by cytochrome C is consistent with the participation of NADPH-cytochrome C reductase in this hydroxylation. Reduced microsomes and its solubilized preparation, when treated with carbon monoxide, showed absorption maxima at 453 and 449 respectively. Different classical inducers of cytochrome P-450 induce the benzo(a)-pyrene hydroxylase activity to varying degree and as such suggests the existence of multiple forms of cytochrome P-450 in this fungus.

Hepatic microsomal cytochrome P-450 from the untreated coastal marine fish scup, Stenotomus chrysops, was solubilized and resolved into five fractions by ion-exchange chromatography. The major fraction, cytochrome P-450E (Mr = 54,300), was further purified to a specific content of 11.7 nmol heme/mg protein and contained a chromophore absorbing at 447 nm in the CO-ligated, reduced difference spectrum. NH2-terminal sequence analysis of cytochrome P-450E by Edman degradation revealed no homology with any known cytochrome P-450 isozyme in the first nine residues. S. chrysops liver NADPH-cytochrome P-450 reductase, purified 225-fold (Mr = 82,600), had a specific activity of 45-60 U/mg with cytochrome c, contained both FAD and FMN, and was isolated as the one-electron reduced semiquinone. Purified cytochrome P-450E metabolized several substrates including 7-ethoxycoumarin, acetanilide, and benzo[a]pyrene when reconstituted with lipid and hepatic NADPH-cytochrome P-450 reductase from either S. chrysops or rat. The purified, reconstituted monooxygenase system was sensitive to inhibition by 100 microM 7,8-benzoflavone, and analysis of products in reconstitutions with purified rat epoxide hydrolase indicated a preference for oxidation on the benzo-ring of benzo[a]pyrene consistent with the primary features of benzo[a]pyrene metabolism in microsomes. Cytochrome P-450E is identical to the major microsomal aromatic hydrocarbon-inducible cytochrome P-450 by the criteria of molecular weight, optical properties, and catalytic profile. It is suggested that substantial quantities of this aromatic hydrocarbon-inducible isozyme exist in the hepatic microsomes of some untreated S. chrysops. The characterization of this aryl hydrocarbon hydroxylase extends our understanding of the metabolism patterns observed in hepatic microsomes isolated from untreated fish.

Mouse skin contains a NADPH-dependent, aryl hydrocarbon hydroxylase (AHH), which is inducible by polycyclic aromatic hydrocarbons. In general, unsubstituted polycyclic hydrocarbons caused a greater induction of epidermal AHH than substituted one (1,2,3,4-dibenzanthracene greater than 1,2,5,6-dibenzanthracene greater than benz (a)anthracene equal 3-methylcholanthrene greater than 7,12-dimethlbenz (a)anthracene) which did not correlate with their ability to initiate tumors in mouse skin. Two different techniques were used to isolate epidermis and similar results were obtained with both. However, the technique of isolating epidermis using a mild heat treatment required that the temperature be maintained at 52 degrees C for 30 sec. If the temperature was raised to 54 degrees C or above, there was a large reduction in the AHH activity. Isolated epidermis has 4 to 5 times the AHH activity as dermis and about twice that of whole skin. This was true for control mice or mice in which AHH was induced by pretreatment with benz(a)anthracene.