Kinetic and molecular analysis of 5-epiaristolochene 1,3-dihydroxylase, a cytochrome P450 enzyme catalyzing successive hydroxylations of sesquiterpenes

The final step of capsidiol biosynthesis is catalyzed by 5-epiaristolochene dihydroxylase (EAH), a cytochrome P450 enzyme that catalyzes the regio- and stereospecific insertion of two hydroxyl moieties into the bicyclic sesquiterpene 5-epiaristolochene (EA). Detailed kinetic studies using EA and the two possible monohydroxylated intermediates demonstrated the release of 1beta-hydroxy-EA ((OH)EA) at high EA concentrations and a 10-fold catalytic preference for 1beta(OH)EA versus 3alpha(OH)EA, indicative of a preferred reaction order of hydroxylation at C-1, followed by that at C-3. Sequence alignments and homology modeling identified active-site residues tested for their contribution to substrate specificity and overall enzymatic activity. Mutants EAH-S368C and EAH-S368V exhibited wild-type catalytic efficiencies for 1beta(OH)EA biosynthesis, but were devoid of the successive hydroxylation activity for capsidiol biosynthesis. In contrast to EAH-S368C, EAH-S368V catalyzed the relative equal biosynthesis of 1beta(OH)EA, 2beta(OH)EA, and 3beta(OH)EA from EA with wild-type efficiency. Moreover, EAH-S368V converted approximately 1.5% of these monohydroxylated products to their respective ketone forms. Alanine and threonine mutations at position 368 were significantly compromised in their conversion rates of EA to capsidiol and correlated with 3.6- and 5.7-fold increases in their Km values for the 1beta(OH)EA intermediate, respectively. A role for Ile486 in the successive hydroxylations of EA was also suggested by the EAH-I468A mutant, which produced significant amounts 1beta(OH)EA, but negligible amounts of capsidiol from EA. The altered product profile of the EAH-I486A mutant correlated with a 3.6-fold higher Km for EA and a 4.4-fold slower turnover rate (kcat) for 1beta(OH)EA. These kinetic and mutational studies were correlated with substrate docking predictions to suggest how Ser368 and Ile486 might contribute to active-site topology, substrate binding, and substrate presentation to the oxo-Fe-heme reaction center.     

Cloning, heterologous expression, and functional characterization of 5-epi-aristolochene-1,3-dihydroxylase from tobacco (Nicotiana tabacum)

Capsidiol is a bicyclic, dihydroxylated sesquiterpene produced by several solanaceous species in response to a variety of environmental stimuli. It is the primary antimicrobial compound produced by Nicotiana tabacum in response to fungal elicitation, and it is formed via the isoprenoid pathway from 5-epi-aristolochene. Much of the biosynthetic pathway for the formation of this compound has been elucidated, except for the enzyme(s) responsible for the conversion of 5-epi-aristolochene to its dihydroxylated form, capsidiol. Biochemical evidence from previous studies with N. tabacum (Whitehead, I. M., Threlfall, D. R., and Ewing, D. F., 1989, Phytochemistry 28, 775-779) and Capsicum annuum Hoshino, T., Yamaura, T., Imaishi, H., Chida, M., Yoshizawa, Y., Higashi, K., Ohkawa, H., Mizutani, J., 1995, Phytochemistry 38, 609-613. suggested that the oxidation of 5-epi-aristolochene to capsidiol was mediated by at least one elicitor-inducible cytochrome P450 hydroxylase. In extending these observations, we developed an in vivo assay for 5-epi-aristolochene hydroxylase activity and used it to demonstrate a dose-dependent inhibition of activity by ancymidol and ketoconazole, two well characterized inhibitors of cytochrome P450 enzymes. Using degenerate oligonucleotide primers designed to the well conserved domains found within most P450 enzymes, including the heme binding domain, cDNA fragments representing four distinct P450 families (CYP71, CYP73, CYP82, and CYP92) were amplified from a cDNA library prepared against mRNA from elicitor-treated cells using PCR. The PCR fragments were subsequently used to isolate full-length cDNAs (CYP71D20 and D21, CYP73A27 and A28, CYP82E1 and CYP92A5), and these in turn were used to demonstrate that the corresponding mRNAs were all induced in elicitor-treated cells, albeit with different induction patterns. Representative, full-length cDNAs for each of the P450s were engineered into a yeast expression system, and the recombinant yeast assessed for functional expression of P450 protein by measuring the CO difference spectra of the yeast microsomes. Only microsomal preparations from yeast expressing the CYP71D20 and CYP92A5 cDNAs exhibited significant CO difference absorbance spectra at 450 nm and were thus tested for their ability to hydroxylate 5-epi-aristolochene and 1-deoxycapsidiol, a putative mono-hydroxylated intermediate in capsidiol biosynthesis. Interestingly, the CYP71D20-encoded enzyme activity was capable of converting both 5-epi-aristolochene and 1-deoxycapsidiol to capsidiol in vitro, consistent with the notion that this P450 enzyme catalyzes both hydroxylations of its hydrocarbon substrate.     

Probing sesquiterpene hydroxylase activities in a coupled assay with terpene synthases

5-epi-Aristolochene dihydroxylase (EAH) catalyzes unique stereo- and regiospecific hydroxylations of a bicyclic sesquiterpene hydrocarbon to generate capsidiol. To define functional and mechanistic features of the EAH enzyme, the utility of a coupled assay using readily available sesquiterpene synthases and microsomes from yeast overexpressing the EAH enzyme was determined. Capsidiol and deoxycapsidiol biosyntheses were readily measured in coupled assays consisting of 5-epi-aristolochene synthase and EAH as determined by the incorporation of radiolabeled farnesyl diphosphate into thin-layer chromatography-isolated products and verified by gas chromatography-mass spectrometry analysis. The assays were dependent on the amounts of synthase and hydroxylase protein added, the incubation times, and the presence of nicotinamide adenine dinucleotide phosphate. The utility of this coupled assay was extended by examining the relative efficiency of the EAH enzyme to catalyze hydroxylations of different sesquiterpene skeletons generated by other terpene synthases.     

Controlling the regiospecificity and coupling of cytochrome P450cam: T185F mutant increases coupling and abolishes 3-hydroxynorcamphor product.

Cytochrome P450cam (P450CIA1) catalyzes the hydroxylation of camphor and several substrate analogues such as norcamphor and 1-methyl-norcamphor. Hydroxylation was found experimentally at the 3, 5, and 6 positions of norcamphor, but only at the 5 and 6 positions of 1-methyl-norcamphor. In the catalytic cycle, the hydroxylation of substrate is coupled to the consumption of NADH. For camphor, the degree of coupling is 100%, but for both norcamphor and 1-methyl-norcamphor, the efficiency is dramatically lowered to 12% and 50%, respectively. Based on an examination of the active site of P450cam, it appeared that mutating position 185 might dramatically alter the product specificity and coupling of hydroxylation of norcamphor by P450cam. Analysis of molecular dynamics trajectories of norcamphor bound to the T185F mutant of cytochrome P450cam predicted that hydroxylation at the 3 position should be abolished and that the coupling should be dramatically increased. This mutant was constructed and the product profile and coupling experimentally determined. The coupling was doubled, and hydroxylation at the 3 position was essentially abolished. Both of these results are in agreement with the prediction.     

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.     

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.     

Arabidopsis CYP90B1 catalyses the early C-22 hydroxylation of C27, C28 and C29 sterols

Arabidopsis dwf4 is a brassinosteroid (BR)-deficient mutant, and the DWF4 gene encodes a cytochrome P450, CYP90B1. We report the catalytic activity and substrate specificity of CYP90B1. Recombinant CYP90B1 was produced in Escherichia coli, and CYP90B1 activity was measured in an in vitro assay reconstituted with NADPH-cytochrome P450 reductase. CYP90B1 converted campestanol (CN) to 6-deoxocathasterone, confirming that CYP90B1 is a steroid C-22 hydroxylase. The substrate specificity of CYP90B1 indicated that sterols with a double bond at positions C-5 and C-6 are preferred substrates compared with stanols, which have no double bond at the position. In particular, the catalytic efficiency (k(cat)/K(m)) of CYP90B1 for campesterol (CR) was 325 times greater than that for CN. As CR is more abundant than CN in planta, the results suggest that C-22 hydroxylation of CR before C-5alpha reduction is the main route of BR biosynthetic pathway, which contrasts with the generally accepted route via CN. In addition, CYP90B1 showed C-22 hydroxylation activity toward various C(27-29) sterols. Cholesterol (C27 sterol) is the best substrate, followed by CR (C28 sterol), whereas sitosterol (C29 sterol) is a poor substrate, suggesting that the substrate preference of CYP90B1 may explain the discrepancy between the in planta abundance of C27/C28/C29 sterols and C27/C28/C29 BRs.     

A single active-site mutation of P450BM-3 dramatically enhances substrate binding and rate of product formation

Identifying key structural features of cytochromes P450 is critical in understanding the catalytic mechanism of these important drug-metabolizing enzymes. Cytochrome P450BM-3 (BM-3), a structural and mechanistic P450 model, catalyzes the regio- and stereoselective hydroxylation of fatty acids. Recent work has demonstrated the importance of water in the mechanism of BM-3, and site-specific mutagenesis has helped to elucidate mechanisms of substrate recognition, binding, and product formation. One of the amino acids identified as playing a key role in the active site of BM-3 is alanine 328, which is located in the loop between the K helix and β 1-4. In the A328V BM-3 mutant, substrate affinity increases 5-10-fold and the turnover number increases 2-8-fold compared to wild-type enzyme. Unlike wild-type enzyme, this mutant is purified from E. coli with endogenous substrate bound due to the higher binding affinity. Close examination of the crystal structures of the substrate-bound native and A328V mutant BMPs indicates that the positioning of the substrate is essentially identical in the two forms of the enzyme, with the two valine methyl groups occupying voids present in the active site of the wild-type substrate-bound structure.     

Regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by cytochrome P-450 isozymes purified from phenobarbital-induced rat liver

The regioselectivity and stereoselectivity of androgen hydroxylations catalyzed by five isozymes of cytochrome P-450 purified from phenobarbital-induced rat liver were studied in a reconstituted monooxygenase system using testosterone (T) and androst-4-ene-3,17-dione (delta 4-A) as substrates. P-450 PB-3, an isozyme exhibiting low catalytic activity with many xenobiotic substrates, catalyzed efficient (turnover = 15.7 to 18.5 min-1 P-450-1 at 25 microM substrate) and highly stereoselective B-ring hydroxylations of both steroid substrates, with the corresponding 7 alpha- and 6 alpha-hydroxy alcohols formed in ratios of approximately 20 to 30:1, respectively. P-450 PB-2c metabolized testosterone to a mixture of 16 alpha OH-T, 2 alpha OH-T, and delta 4-A (product ratio = 1.0/0.78/0.33; turnover = 10.2 min-1 P-450-1). PB-2c is present in significantly larger amounts in mature male rats as compared to immature males, and probably catalyzes the male-specific testosterone 16 alpha-hydroxylase activity known to be induced at puberty and subject to endocrine control. P-450 PB-4, the major phenobarbital-induced isozyme in rat liver, catalyzed efficient D-ring hydroxylations, yielding 16 beta OH- delta 4-A as the predominant product with delta 4-A as substrate (turnover = 12.0 min-1 P-450-1) and a mixture of 16 beta OH-T, 16 alpha OH-T, and delta 4-A (the latter compound presumably formed via 17 alpha hydroxylation) with testosterone as substrate (turnover = 5.2 min-1 P-450-1). P-450 isozymes PB-1 and PB-5 hydroxylated both steroids with essentially the same regioselectivity as PB-4 but at only 5 to 10% the catalytic rate. Cytochrome b5 stimulated most of these steroid hydroxylations up to 2-fold with no change in regio- or stereoselectivity. The identification of specific steroid metabolites as diagnostic of particular P-450 isozymes should be useful for the assessment of isozymic contributions to microsomal activities and, in addition, facilitate comparisons of P-450 isozymes isolated in different laboratories.     

Insights into electron leakage in the reaction cycle of cytochrome P450 BM3 revealed by kinetic modeling and mutagenesis

As a single polypeptide, cytochrome P450 BM3 fuses oxidase and reductase domains and couples each domain's function to perform catalysis with exceptional activity upon binding of substrate for hydroxylation. Mutations introduced into the enzyme to change its substrate specificity often decrease coupling efficiency between the two domains, resulting in unproductive consumption of cofactors and formation of water and/or reactive species. This phenomenon can correlate with leakage, in which P450 BM3 uses electrons from NADPH to reduce oxygen to water and/or reactive species even without bound substrate. The physical basis for leakage is not yet well understood in this particular member of the cytochrome P450 family. To clarify the relationship between leakage and coupling, we used simulations to illustrate how different combinations of kinetic parameters related to substrate‐free consumption of NADPH and substrate hydroxylation can lead to either minimal effects on coupling or a dramatic decrease in coupling as a result of leakage. We explored leakage in P450 BM3 by introducing leakage‐enhancing mutations and combining these mutations to assess whether doing so increases leakage further. The variants in this study provide evidence that while a transition to high spin may be vital for coupled hydroxylation, it is not required for enhanced leakage; substrate binding and the consequent shift in spin state are not necessary as a redox switch for catalytic oxidation of NADPH. Additionally, the variants in this study suggest a tradeoff between leakage and stability and thus evolvability, as the mutations we investigated were far more deleterious than other mutations that have been used to change substrate specificity.     

Radical rebound mechanism in cytochrome P-450-catalyzed hydroxylation of the multifaceted radical clocks alpha- and beta-thujone

Alpha-thujone (1alpha) and beta-thujone (1beta) were used to investigate the mechanism of hydrocarbon hydroxylation by cytochromes P-450(cam) (CYP101) and P-450(BM3) (CYP102). The thujones are hydroxylated by these enzymes at various positions, but oxidation at C-4 gives rise to both rearranged and unrearranged hydroxylation products. Rearranged products result from the formation of a radical intermediate that can undergo either inversion of stereochemistry or ring opening of the adjacent cyclopropane ring. Both of these rearrangements, as well as a C-4 desaturation reaction, are observed. The ring opening clock gives oxygen rebound rates that range from 0.2 x 10(10) to 2.8 x 10(10) s(-1) for the different substrate and enzyme combinations. The C-4 inversion reaction provides independent confirmation of a radical intermediate. The phenol product expected if a C-4 cationic rather than radical intermediate is formed is not detected. The results are consistent with a two-state process and provide support for a radical rebound but not a hydroperoxide insertion mechanism for cytochrome P-450 hydroxylation.     

Putative helix F contributes to regioselectivity of hydroxylation in mitochondrial cytochrome P450 27A1.

On the basis of alignment with structurally characterized cytochromes P450 (P450s), we have identified the putative F and G helices of mitochondrial P450s 27A1 and 11A. We introduced substitutions at Phe-207, Ile-211, and Phe-215 within putative helix F and at Trp-235 and Tyr-238 within putative helix G in P450 27A1 and compared wild type and mutants with respect to catalytic activity, product pattern, substrate binding, formation of hydrogen peroxide, and interaction with redox partner. Results indicate that the mutated residues are important for delivery of the correctly oriented substrate to the P450 active site. The I211K and F215K mutations, for example, affected the regioselectivity of P450 27A1-dependent hydroxylation reactions and conferred the P450 capacity to cleave the C-C bond of the substrate during the catalytic cycle. Studies of P450 11A1 indicate that Phe-202 has functions similar to those of its counterpart in P450 27A1 (Phe-215). We propose that putative helices F and G form the sides of the substrate-access channel, thus providing the additional mechanism to control regioselectivity of hydroxylation in mitochondrial P450s.     

Reduction of 7-alkoxyresorufins by NADPH-cytochrome P450 reductase and its differential effects on their O-dealkylation by rat liver microsomal cytochrome P450

Antibody-inhibition experiments established that the induction of cytochrome P450c is largely responsible for the marked increase in liver microsomal 7-ethoxyresorufin O-dealkylation in rats treated with 3-methylcholanthrene, whereas the induction of cytochrome P450b and/or P450e is largely responsible for the marked increase in 7-pentoxy- and 7-benzyloxyresorufin O-dealkylation in rats treated with phenobarbital. When reconstituted with NADPH-cytochrome P450 reductase and lipid, purified cytochrome P450c catalyzed the O-dealkylation of 7-ethoxyresorufin at a rate of approximately 30 nmol/nmol P450/min, which far exceeded the rate catalyzed by either purified cytochromes P450b and P450e or microsomal cytochrome P450c. In contrast, purified cytochrome P450b and P450e were poor catalysts of the O-dealkylation of 7-pentoxy- and 7-benzyloxyresorufin. However, purified cytochrome P450b is an excellent catalyst of several other reactions, such as the N-demethylation of benzphetamine, the hydroxylation of testosterone, and the O-dealkylation of 7-ethoxycoumarin. The low rate of 7-pentoxyresorufin O-dealkylation catalyzed by purified cytochrome P450b did not reflect a requirement for cytochrome b5, and could not be ascribed to an artifact of the method used to measure the formation of resourufin. The catalytic activity of purified cytochrome P450b toward 7-pentoxyresorufin was consistently low over a range of substrate and lipid concentrations, and was not stimulated by sodium deoxycholate (which stimulates the N-demethylation of benzphatamine by purified cytochrome P450b). Evidence is presented which indicates that cytochrome P450c catalyzes the O-dealkylation of both the oxidized and reduced forms of 7-ethoxyresorufin, with perhaps a slight preference for the reduced form. In contrast, cytochrome P450b preferentially catalyzes the O-dealkylation of the oxidized form of 7-pentoxyresorufin. Conditions that favored formation of the reduced form of 7-ethoxyresorufin tended to stimulate its O-dealkylation by purified cytochrome P450c, whereas conditions that favored formation of the reduced form of 7-pentoxyresorufin decreased its rate of O-dealkylation by purified cytochrome P450b. Such conditions included a molar excess of NADPH-cytochrome P450 reductase over cytochrome P450, the presence of superoxide dismutase, and the presence of DT-diaphorase (liver cytosol).(ABSTRACT TRUNCATED AT 400 WORDS)     

Resolution of two forms of cytochrome P-450 from liver microsomes of rabbit treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin.

Two forms of cytochrome P-450 with different substrate specificities were isolated from liver microsomes of rabbits treated with 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD). A specific antibody was produced toward the major form of the cytochrome. The antibody inhibits microsomal acetanilide hydroxylation (80%). It does not cross-react with the minor fraction of the cytochrome or inhibit the hydroxylation of 3,4-benzpyrene or coumarin, the N-demethylation of aminopyrine or the O-deethylation of 7-ethoxycoumarin catalyzed by rabbit liver microsomes. The major form has an estimated Mr = 54,000 and displays an n-octylamine difference spectrum with an absorption maximum at 426 nm and a minimum at 391 nm. When reconstituted, this cytochrome catalyzes acetanilide hydroxylation at a higher rate than microsomes or the minor fraction. The n-octylamine difference spectrum of the minor fraction displays an absorption maximum at 431 nm and a minimum at 410 nm. When reconstituted, this fraction catalyzes the hydroxylation of 3,4-benzpyrene and the O-deethylation of 7-ethoxycoumarin. The two cytochromes appear to be distinct entities and function in different catalytic pathways.     

Light and brassinosteroid signals are integrated via a dark-induced small G protein in etiolated seedling growth

Plant growth and development are regulated through coordinated interactions between light and phytohormones. Here, we demonstrate that a dark-induced small G protein, pea Pra2, regulates a variant cytochrome P450 that catalyzes C-2 hydroxylation in brassinosteroid biosynthesis. The cytochrome P450 is dark-induced and predominantly expressed in the rapidly elongating zone of etiolated pea epicotyls, where Pra2 is also most abundant. Transgenic plants with reduced Pra2 exhibit a dark-specific dwarfism, which is completely rescued by exogenous brassinolide. Overexpression of the cytochrome P450 results in enhanced hypocotyl growth even in the light, which phenocopies the etiolated hypocotyls. We therefore propose that Pra2 and its orthologs are molecular mediators for the cross-talk between light and brassinosteroids in the etiolation process in plants.     

Purification and properties of cytochrome P-450 generally acting as a catalyst on benzo[a]pyrene hydroxylation from liver microsomes of untreated rats

A form of cytochrome P-450 generally catalyzing benzo[a]pyrene (B[a]P) hydroxylation was purified from liver microsomes of untreated rats on the basis of the catalytic activity. The purification procedures consisted of cholate solubilization and chromatography in 3 steps, on DEAE-Toyopearl (at room temperature), hydroxylapatite, and CM-Toyopearl columns. Cytochrome P-450 purified in this way (named P-450/B[a]P) was homogeneous on SDS-polyacrylamide gel electrophoresis, and the molecular weight was estimated to be 51,000. The absorption spectra of the oxidized form of P-450/B[a]P showed a Soret peak at 417 nm, characteristic of low-spin hemoprotein, and the Soret peak of the reduced cytochrome P-450-CO complex was at 451 nm. Immunochemical analysis of P-450/B[a]P indicated that P-450/B[a]P is immunologically distinct from P-450b (a major phenobarbital-inducible form of P-450) and P-450c (a major 3-methylcholanthrene-inducible form of P-450, which highly catalyzes the hydroxylation of B[a]P). B[a]P hydroxylase activity in liver microsomes of untreated rats was inhibited to about 20% by the P-450/B[a]P antibody. These results demonstrate that P-450/B[a]P is a different form of P-450 from P-450b and P-450c, and generally catalyzes B[a]P hydroxylation in liver microsomes of untreated rats.     

Interaction between substrate and oxygen ligand responsible for effective O-O bond cleavage in bovine cytochrome P450 steroid 21-hydroxylase proved by Raman spectroscopy

We investigated structural and functional properties of bovine cytochrome P450 steroid 21-hydroxylase (P450c21), which catalyzes hydroxylation at C-21 of progesterone and 17alpha-hydroxyprogesterone. The uncoupled H(2)O(2) formation was higher in the hydroxylation of progesterone (26% of NADPH consumed) than that of 17alpha-hydroxyprogesterone (15% of NADPH consumed), indicating that 17alpha-hydroxyprogesterone can better facilitate the O-O bond scission. In relation to this, it is noted that the O-O stretching mode (nu(O-O)) of the oxygen complex of P450c21 was sensitive to the substrate; the progesterone- or 17alpha-hydroxyprogesterone-bound enzyme gave single (at 1137 cm(-1)) or split nu(O-O) bands (at 1124 and 1138 cm(-1)), respectively, demonstrating the presence of two forms for the latter. In contrast to nu(O-O), no corresponding difference was observed for the Fe-O(2) stretching mode between two different substrate-bound forms. The Fe-S(Cys) stretching mode in the ferric state was also identical (349 cm(-1)) for each substrate-bound form, suggesting that modulation through the axial thiolate by the substrate is unlikely. Therefore, it is deduced that the hydroxyl group at C-17 of 17alpha-hydroxyprogesterone forms a hydrogen bond with the terminal oxygen atom of the FeOO complex in one form, yielding a lower nu(O-O) frequency with higher reactivity for O-O cleavage, whereas the other form in which the substrate does not provide a hydrogen bond to the oxygen ligand is essentially the same between the two kinds of substrates. In the hydrogen-bonded species, the substrate changes the geometry of the FeOO moiety, thereby performing the hydroxylation reaction more effectively in 17alpha-hydroxyprogesterone than in progesterone.     

Monoterpene biosynthesis: specificity of the hydroxylations of (-)-limonene by enzyme preparations from peppermint (Mentha piperita), spearmint (Mentha spicata), and perilla (Perilla frutescens) leaves

Microsomal preparations from the epidermal oil glands of Mentha piperita, Mentha spicata, and Perilla frutescens leaves catalyze the NADPH- and O2-dependent allylic hydroxylation of the monoterpene olefin (-)-limonene at C-3, C-6, and C-7, respectively, to produce the corresponding alcohols, (-)-trans-isopiperitenol, (-)-trans-carveol, and (-)-perillyl alcohol. These transformations are the key steps in the biosynthesis of oxygenated monoterpenes in the respective species, and the responsible enzyme systems meet most of the established criteria for cytochrome P450-dependent mixed function oxygenases. The reactions catalyzed are completely regiospecific and, while exhibiting only a modest degree of enantioselectivity, are highly specific for limonene as substrate. Of numerous monoterpene olefins tested, including several positional isomers of limonene, only the 8,9-dihydro analog served as an alternate substrate for ring (C-3 and C-6) hydroxylation, but not side chain (C-7) hydroxylation. In addition to the regiospecificity of the allylic hydroxylation, these enzymes are also readily distinguishable based on differential inhibition by substituted imidazoles.     

Kinetic analysis of hydroxylation of saturated fatty acids by recombinant P450foxy produced by an Escherichia coli expression system.

Cytochrome P450foxy (P450foxy, CYP505) is a fused protein of cytochrome P450 (P450) and its reductase isolated from the fungus Fusarium oxysporum, which catalyzes the subterminal (omega-1 approximately omega-3) hydroxylation of fatty acids. Here, we produced, purified and characterized a fused recombinant protein (rP450foxy) using the Escherichia coli expression system. Purified rP450foxy was catalytically and spectrally indistinguishable from the native protein, but most of the rP450foxy was recovered in the soluble fraction of E. coli cells unlike the membrane-bound native protein. The results are consistent with our notion that the native protein is targeted to the membrane by a post-translational modification mechanism. We also discovered that P450foxy could use shorter saturated fatty acid chains (C9 and C10) as a substrate. The regiospecificity (omega-1 approximately omega-3) of hydroxylation due to the enzymatic reaction for the short substrates (decanoate, C10; undecanoate, C11) was the same as that for longer substrates. Steady state kinetic studies showed that the kcat values for all substrates tested (C9-C16) were of the same magnitude (1200-1800 min-1), whereas the catalytic efficiency (kcat/Km) was higher for longer fatty acids. Substrate inhibition was observed with fatty acid substrates longer than C13, and the degree of inhibition increased with increasing chain length. This substrate inhibition was not apparent with P450BM3, a bacterial counterpart of P450foxy, which was the first obvious difference in their catalytic properties to be identified. Kinetic data were consistent with the inhibition due to binding of the second substrate. We discuss the inhibition mechanism based on differences between P450foxy and P450BM3 in key amino acid residues for substrate binding.