Cytochromes P450 in phenolic metabolism

Three independent cytochrome P450 enzyme families catalyze the three rate-limiting hydroxylation steps in the phenylpropanoid pathway leading to the biosynthesis of lignin and numerous other phenolic compounds in plants. Their characterization at the molecular and enzymatic level has revealed an unexpected complexity of phenolic metabolism as the major route involves shikimate/quinate esters and alcohol/aldehyde intermediates. Engineering expression of CYP73s (encoding cinnamate 4-hydroxylase), CYP98s (encoding 4-coumaroylshikimate 3′-hydroxylase) or CYP84s (encoding coniferaldehyde 5-hydroxylase) leads to modified lignin and seed phenolic composition. In particular CYP73s and CYP98s also play essential roles in plant growth and development, while CYP84 constitutes a check-point for the synthesis of syringyl lignin and sinapate esters. Although recent data shed new light on the main path for lignin synthesis, they also raised new questions. Mutants and engineered plants revealed the existence of (an) alternative pathway(s), which most likely involve(s) different precursors and oxygenases. On the other hand, phylogenetic analysis of plant genomes show the existence of P450 gene duplications in each family, which may have led to the acquisition of novel or additional physiological functions in planta. In addition to the main lignin pathway, P450s contribute to the biosynthesis of many bioactive phenolic derivatives, with potential applications in medicine and plant defense, including lignans, phenylethanoids, benzoic acids, xanthones or quinoid compounds. A very small proportion of these P450s have been characterized so far, and rarely at a molecular level. The possible involvement of P450s in salicylic acid is discussed.     

cDNA cloning and functional characterisation of CYP98A14 and NADPH:cytochrome P450 reductase from <Emphasis Type="Italic">Coleus blumei</Emphasis> involved in rosmarinic acid biosynthesis

The final reactions of rosmarinic acid biosynthesis, the introduction of the aromatic 3- and 3′-hydroxyl groups, are catalysed by cytochrome P450-dependent hydroxylases. The cDNAs encoding CYP98A14 as well as a NADPH:cytochrome P450 reductase (CPR) were isolated from Coleus blumei and actively expressed in Saccharomyces cerevisiae. The CYP98A14-cDNA showed an open reading frame of 1521 nucleotides with high similarities to 4-coumaroylshikimate/quinate 3-hydroxylases. Yeast microsomes harbouring the CYP98A14 protein catalysed the 3-hydroxylation of 4-coumaroyl-3′,4′-dihydroxyphenyllactate and the 3′-hydroxylation of caffeoyl-4′-hydroxyphenyllactate, in both cases forming rosmarinic acid. Apparent K _m-values for 4-coumaroyl-3′,4′-dihydroxyphenyllactate and caffeoyl-4′-hydroxyphenyllactate were determined to be at 5 μM and 40 μM, respectively. CYP98A14 differs from CYP98s from other plants, since 4-coumaroylshikimate or -quinate were not accepted as substrates. Coexpression of the Coleus blumei CPR and CYP98A14 in the same yeast cells increased the hydroxylation activity up to sevenfold. CYP98A14 from Coleus blumei is a novel bifunctional cytochrome P450 specialised for rosmarinic acid biosynthesis.     

Differential production of meta hydroxylated phenylpropanoids in sweet basil peltate glandular trichomes and leaves is controlled by the activities of specific acyltransferases and hydroxylases

Sweet basil (Ocimum basilicum) peltate glandular trichomes produce a variety of small molecular weight phenylpropanoids, such as eugenol, caffeic acid, and rosmarinic acid, that result from meta hydroxylation reactions. Some basil lines do not synthesize eugenol but instead synthesize chavicol, a phenylpropanoid that does not contain a meta hydroxyl group. Two distinct acyltransferases, p-coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase and p-coumaroyl-coenzyme A:4-hydroxyphenyllactic acid p-coumaroyl transferase, responsible for the production of p-coumaroyl shikimate and of p-coumaroyl 4-hydroxyphenyllactate, respectively, were partially purified and shown to be specific for their substrates. p-Coumaroyl-coenzyme A:shikimic acid p-coumaroyl transferase is expressed in basil peltate glands that are actively producing eugenol and is not active in glands of noneugenol-producing basil plants, suggesting that the levels of this activity determine the levels of synthesis of some meta-hydroxylated phenylpropanoids in these glands such as eugenol. Two basil cDNAs encoding isozymes of cytochrome P450 CYP98A13, which meta hydroxylates p-coumaroyl shikimate, were isolated and found to be highly similar (90% identity) to the Arabidopsis homolog, CYP98A3. Like the Arabidopsis enzyme, the basil enzymes were found to be very specific for p-coumaroyl shikimate. Finally, additional hydroxylase activities were identified in basil peltate glands that convert p-coumaroyl 4-hydroxyphenyllactic acid to its caffeoyl derivative and p-coumaric acid to caffeic acid.     

CYP98A6 from Lithospermum erythrorhizon encodes 4-coumaroyl-4'-hydroxyphenyllactic acid 3-hydroxylase involved in rosmarinic acid biosynthesis

Rosmarinic acid is the dominant hydroxycinnamic acid ester accumulated in Boraginaceae and Lamiaceae plants. A cytochrome P450 cDNA was isolated by differential display from cultured cells of Lithospermum erythrorhizon, and the gene product was designated CYP98A6 based on the deduced amino acid sequence. After expression in yeast, the P450 was shown to catalyze the 3-hydroxylation of 4-coumaroyl-4'-hydroxyphenyllactic acid, one of the final two steps leading to rosmarinic acid. The expression level of CYP98A6 is dramatically increased by addition of yeast extract or methyl jasmonate to L. erythrorhizon cells, and its expression pattern reflected the elicitor-induced change in rosmarinic acid production, indicating that CYP98A6 plays an important role in regulation of rosmarinic acid biosynthesis.     

A coumaroyl-ester-3-hydroxylase insertion mutant reveals the existence of nonredundant meta-hydroxylation pathways and essential roles for phenolic precursors in cell expansion and plant growth

Cytochromes P450 monooxygenases from the CYP98 family catalyze the meta-hydroxylation step in the phenylpropanoid biosynthetic pathway. The ref8 Arabidopsis (Arabidopsis thaliana) mutant, with a point mutation in the CYP98A3 gene, was previously described to show developmental defects, changes in lignin composition, and lack of soluble sinapoyl esters. We isolated a T-DNA insertion mutant in CYP98A3 and show that this mutation leads to a more drastic inhibition of plant development and inhibition of cell growth. Similar to the ref8 mutant, the insertion mutant has reduced lignin content, with stem lignin essentially made of p-hydroxyphenyl units and trace amounts of guaiacyl and syringyl units. However, its roots display an ectopic lignification and a substantial proportion of guaiacyl and syringyl units, suggesting the occurrence of an alternative CYP98A3-independent meta-hydroxylation mechanism active mainly in the roots. Relative to the control, mutant plantlets produce very low amounts of sinapoyl esters, but accumulate flavonol glycosides. Reduced cell growth seems correlated with alterations in the abundance of cell wall polysaccharides, in particular decrease in crystalline cellulose, and profound modifications in gene expression and homeostasis reminiscent of a stress response. CYP98A3 thus constitutes a critical bottleneck in the phenylpropanoid pathway and in the synthesis of compounds controlling plant development. CYP98A3 cosuppressed lines show a gradation of developmental defects and changes in lignin content (40% reduction) and structure (prominent frequency of p-hydroxyphenyl units), but content in foliar sinapoyl esters is similar to the control. The purple coloration of their leaves is correlated to the accumulation of sinapoylated anthocyanins.     

6α-Hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The V_max for 6α-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent K_m was 90 μM. Cytochrome b₅ was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6α-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r²=0.97) and testosterone 6β-hydroxylation (r²=0.9). There was also a strong correlation between 6α-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6β-hydroxylation (r²=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6α-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 μM concentration. Other inhibitors, such as α-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent K_m for 6α-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 μM). This might give an explanation for the limited formation of 6α-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6α-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Identification of a novel rat microsomal vitamin D3 25-hydroxylase

Vitamin D3 requires the 25-hydroxylation in the liver and the subsequent 1alpha-hydroxylation in the kidney to exert its biological activity. Vitamin D3 25-hydroxylation is hence an essential modification step for vitamin D3 activation. Until now, three cytochrome P450 molecular species (CYP27A1, CYP2C11, and CYP2D25) have been characterized well as vitamin D3 25-hydroxylases. However, their physiological role remains unclear because of their broad substrate specificities and low activities toward vitamin D3 relative to other substrates. In this study, we purified vitamin D3 25-hydroxylase from female rat liver microsomes. The activities of the purified fraction toward vitamin D3 and 1alpha-hydroxyvitamin D3 were 1.1 and 13 nmol/min/nmol of P450, respectively. The purified fraction showed a few protein bands in a 50-60-kDa range on SDS-PAGE, typical for a cytochrome P450. The tryptic peptide mass fingerprinting of a protein band (56 kDa) with matrix-assisted laser desorption ionization/time of flight mass spectrometry identified this band as CYP2J3. CYP2J3 was heterologously expressed in Escherichia coli. Purified recombinant CYP2J3 showed strong 25-hydroxylation activities toward vitamin D3 and 1alpha-hydroxyvitamin D3 with turnover numbers of 3.3 and 22, respectively, which were markedly higher than those of P450s previously characterized as 25-hydroxylases. Quantitative PCR analysis showed that CYP2J3 mRNA is expressed at a level similar to that of CYP27A1 without marked sexual dimorphism. These results strongly suggest that CYP2J3 is the principal P450 responsible for vitamin D3 25-hydroxylation in rat liver.     

Characterization in vitro and in vivo of the putative multigene 4-coumarate:CoA ligase network in Arabidopsis: syringyl lignin and sinapate/sinapyl alcohol derivative formation

A recent in silico analysis revealed that the Arabidopsis genome has 14 genes annotated as putative 4-coumarate:CoA ligase isoforms or homologues. Of these, 11 were selected for detailed functional analysis in vitro, using all known possible phenylpropanoid pathway intermediates (p-coumaric, caffeic, ferulic, 5-hydroxyferulic and sinapic acids), as well as cinnamic acid. Of the 11 recombinant proteins so obtained, four were catalytically active in vitro, with fairly broad substrate specificities, confirming that the 4CL gene family in Arabidopsis has only four members. This finding is in agreement with our previous phylogenetic analyses, and again illustrates the need for comprehensive characterization of all putative 4CLs, rather than piecemeal analysis of selected gene members. All 11 proteins were expressed with a C-terminal His6-tag and functionally characterized, with one, At4CL1, expressed in native form for kinetic property comparisons. Of the 11 putative His6-tagged 4CLs, isoform At4CL1 best utilized p-coumaric, caffeic, ferulic and 5-hydroxyferulic acids as substrates, whereas At4CL2 readily transformed p-coumaric and caffeic acids into the corresponding CoA esters, while ferulic and 5-hydroxyferulic acids were converted quite poorly. At4CL3 also displayed broad substrate specificity efficiently converting p-coumaric, caffeic and ferulic acids into their CoA esters, whereas 5-hydroxyferulic acid was not as effectively utilized. By contrast, while At4CL5 is the only isoform capable of ligating sinapic acid, the two preferred substrates were 5-hydroxyferulic and caffeic acids. Indeed, both At4CL1 and At4CL5 most effectively utilized 5-hydroxyferulic acid with kenz approximately 10-fold higher than that for At4CL2 and At4CL3. The remaining seven 4CL-like homologues had no measurable catalytic activity (at approximately 100 microg protein concentrations), again bringing into sharp focus both the advantages to, and the limitations of, current database annotations, and the need to unambiguously demonstrate true enzyme function. Lastly, although At4CL5 is able to convert both 5-hydroxyferulic and sinapic acids into the corresponding CoA esters, the physiological significance of the latter observation in vitro was in question, i.e. particularly since other 4CL isoforms can effectively convert 5-hydroxyferulic acid into 5-hydroxyferuloyl CoA. Hence, homozygous lines containing T-DNA or enhancer trap inserts (knockouts) for 4cl5 were selected by screening, with Arabidopsis stem sections from each mutant line subjected to detailed analyses for both lignin monomeric compositions and contents, and sinapate/sinapyl alcohol derivative formation, at different stages of growth and development until maturation. The data so obtained revealed that this "knockout" had no significant effect on either lignin content or monomeric composition, or on the accumulation of sinapate/sinapyl alcohol derivatives. The results from the present study indicate that formation of syringyl lignins and sinapate/sinapyl alcohol derivatives result primarily from methylation of 5-hydroxyferuloyl CoA or derivatives thereof rather than sinapic acid ligation. That is, no specific physiological role for At4CL5 in direct sinapic acid CoA ligation could be identified. How the putative overlapping 4CL metabolic networks are in fact organized in planta at various stages of growth and development will be the subject of future inquiry.     

Cloning and tissue distribution of a novel human cytochrome p450 of the CYP3A subfamily, CYP3A43

On the basis of the detection of an expressed sequence tag ('EST') similar to the human cytochrome P450 3A4 cDNA, we have identified a novel member of the human cytochrome P450 3A subfamily. The coding region is 1512-bp long and shares 84, 83, and 82% sequence identity on the cDNA level with CYP3A4, 3A5, and 3A7, respectively, with a corresponding amino acid identity of 76, 76, and 71%. Quantitative real time based mRNA analysis revealed CYP3A43 expression levels at about 0.1% of CYP3A4 and 2% of CYP3A5 in the liver, with significant expression in 70% of the livers examined. Gene specific PCR of cDNA from extrahepatic tissues showed, with the exception of the testis, only low levels of CYP3A43 expression. The CYP3A43 cDNA was heterologously expressed in yeast, COS-1 cells, mouse hepatic H2.35 cells and in human embryonic kidney (HEK) 293 cells, but in contrast to CYP3A4 which was formed in all cell types, no detectable CYP3A43 protein was produced. This indicates a nonfunctional protein or specific conditions required for proper folding. It is concluded that CYP3A43 mRNA is expressed mainly in liver and testis and that the protein would not contribute significantly to human drug metabolism.     

A gel-free MS-based quantitative proteomic approach accurately measures cytochrome P450 protein concentrations in human liver microsomes

The human cytochrome P450 (P450) superfamily consists of membrane-bound proteins that metabolize a myriad of xenobiotics and endogenous compounds. Quantification of P450 expression in various tissues under normal and induced conditions has an important role in drug safety and efficacy. Conventional immunoquantification methods have poor dynamic range, low throughput, and a limited number of specific antibodies. Recent advances in MS-based quantitative proteomics enable absolute protein quantification in a complex biological mixture. We have developed a gel-free MS-based protein quantification strategy to quantify CYP3A enzymes in human liver microsomes (HLM). Recombinant protein-derived proteotypic peptides and synthetic stable isotope-labeled proteotypic peptides were used as calibration standards and internal standards, respectively. The lower limit of quantification was approximately 20 fmol P450. In two separate panels of HLM examined (n = 11 and n = 22), CYP3A, CYP3A4 and CYP3A5 concentrations were determined reproducibly (CV or=0.87) and marker activities (r(2)>or=0.88), including testosterone 6beta-hydroxylation (CYP3A), midazolam 1'-hydroxylation (CYP3A), itraconazole 6-hydroxylation (CYP3A4) and CYP3A5-mediated vincristine M1 formation (CYP3A5). Taken together, our MS-based method provides a specific, sensitive and reliable means of P450 protein quantification and should facilitate P450 characterization during drug development, especially when specific substrates and/or antibodies are unavailable.     

Conversion of vitamin D3 to 1alpha,25-dihydroxyvitamin D3 by Streptomyces griseolus cytochrome P450SU-1

Streptomyces griseolus cytochrome P450SU-1 (CYP105A1) was expressed in Escherichia coli at a level of 1.0 micromol/L culture and purified with a specific content of 18.0 nmol/mg protein. Enzymatic studies revealed that CYP105A1 had 25-hydroxylation activity towards vitamin D2 and vitamin D3. Surprisingly, CYP105A1 also showed 1alpha-hydroxylation activity towards 25(OH)D3. As mammalian mitochondrial CYP27A1 catalyzes a similar two-step hydroxylation towards vitamin D3, the enzymatic properties of CYP105A1 were compared with those of human CYP27A1. The major metabolite of vitamin D2 by CYP105A1 was 25(OH)D2, while the major metabolites by CYP27A1 were both 24(OH)D2 and 27(OH)D2. These results suggest that CYP105A1 recognizes both vitamin D2 and vitamin D3 in a similar manner, while CYP27A1 does not. The Km values of CYP105A1 for vitamin D2 25-hydroxylation, vitamin D3 25-hydroxylation, and 25-hydroxyvitamin D3 1alpha-hydroxylation were 0.59, 0.54, and 0.91 microM, respectively, suggesting a high affinity of CYP105A1 for these substrates.     

Cloning, expression in yeast, and functional characterization of CYP76A4, a novel cytochrome P450 of petunia that catalyzes (omega-1)-hydroxylation of lauric acid

A cDNA clone of a novel cytochrome P450, CYP76A4, was isolated from Petunia hybrida. The cDNA clone contained an open reading frame (ORF) encoding a predicted 510 amino acid polypeptide. The CYP76A4 cDNA was expressed in yeast Saccharomyces cerevisiae AH22. Recombinant yeast microsomes containing the CYP76A4 hemoprotein were found to catalyze (omega-1)-hydroxylation of lauric acid.     

A cDNA encoding a rat mitochondrial cytochrome P450 catalyzing both the 26-hydroxylation of cholesterol and 25-hydroxylation of vitamin D3: gonadotropic regulation of the cognate mRNA in ovaries

A cDNA expression library prepared from rat liver RNA was screened with a polyclonal antibody specific for mitochondrial vitamin D3 25-hydroxylase and a cDNA for rabbit liver mitochondrial cytochrome P450c26 (CYP 26), yielding cDNA clones with identical sequences. The deduced amino acid sequence derived from a 1.9-kb full-length cDNA was 73% identical to that of rabbit cytochrome P450c26. A monoclonal antibody was used to demonstrate that the product of the 1.9-kb cDNA clone was targeted to the mitochondrial compartment when expressed in COS cells. Mitochondrial membranes containing the expressed protein showed both vitamin D3 25-hydroxylase and cholesterol 26-hydroxylase activities when reconstituted with ferredoxin reductase and ferredoxin, demonstrating that the same P450, designated as P450c26/25, can catalyze both reactions. Northern blot analysis revealed that the P450c26/25 cDNA hybridizes with a 2.4-kb RNA from rat liver and unstimulated ovaries. Treatment of rats with pregnant mare's serum gonadotropin resulted in a fivefold increase in the 2.4-kb mRNA as well as the appearance of a 2.1-kb mRNA species in the ovaries. Our findings document the presence of a regulated bifunctional mitochondrial cytochrome P450 capable of catalyzing the 25-hydroxylation of vitamin D3 and the 26-hydroxylation of cholesterol.     

Evolution of coumaroyl conjugate 3‐hydroxylases in land plants: lignin biosynthesis and defense

Multiple adaptations were necessary when plants conquered the land. Among them were soluble phenylpropanoids related to plant protection and lignin necessary for upright growth and long‐distance water transport. Cytochrome P450 monooxygenase 98 (CYP98) catalyzes a rate‐limiting step in phenylpropanoid biosynthesis. Phylogenetic reconstructions suggest that a single copy of CYP98 founded each major land plant lineage (bryophytes, lycophytes, monilophytes, gymnosperms and angiosperms), and was maintained as a single copy in all lineages but the angiosperms. In angiosperms, a series of independent gene duplications and losses occurred. Biochemical assays in four angiosperm species tested showed that 4‐coumaroyl‐shikimate, a known intermediate in lignin biosynthesis, was the preferred substrate of one member in each species, while independent duplicates in Populus trichocarpa and Amborella trichopoda each showed broad substrate ranges, accepting numerous 4‐coumaroyl‐esters and ‐amines, and were thus capable of producing a wide range of hydroxycinnamoyl conjugates. The gymnosperm CYP98 from Pinus taeda showed a broad substrate range, but preferred 4‐coumaroyl‐shikimate as its best substrate. In contrast, CYP98s from the lycophyte Selaginella moellendorffii and the fern Pteris vittata converted 4‐coumaroyl‐shikimate poorly in vitro, but were able to use alternative substrates, in particular 4‐coumaroyl‐anthranilate. Thus, caffeoyl‐shikimate appears unlikely to be an intermediate in monolignol biosynthesis in non‐seed vascular plants, including ferns. The best substrate for CYP98A34 from the moss Physcomitrella patens was also 4‐coumaroyl‐anthranilate, while 4‐coumaroyl‐shikimate was converted to lower extents. Despite having in vitro activity with 4‐coumaroyl‐shikimate, CYP98A34 was unable to complement the Arabidopsis thaliana cyp98a3 loss‐of‐function phenotype, suggesting distinct properties also in vivo.     

6alpha-hydroxylation of taurochenodeoxycholic acid and lithocholic acid by CYP3A4 in human liver microsomes

The aim of the present study was to identify the enzymes in human liver catalyzing hydroxylations of bile acids. Fourteen recombinant expressed cytochrome P450 (CYP) enzymes, human liver microsomes from different donors, and selective cytochrome P450 inhibitors were used to study the hydroxylation of taurochenodeoxycholic acid and lithocholic acid. Recombinant expressed CYP3A4 was the only enzyme that was active towards these bile acids and the enzyme catalyzed an efficient 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid. The Vmax for 6alpha-hydroxylation of taurochenodeoxycholic acid by CYP3A4 was 18.2 nmol/nmol P450/min and the apparent Km was 90 microM. Cytochrome b5 was required for maximal activity. Human liver microsomes from 10 different donors, in which different P450 marker activities had been determined, were separately incubated with taurochenodeoxycholic acid and lithocholic acid. A strong correlation was found between 6alpha-hydroxylation of taurochenodeoxycholic acid, CYP3A levels (r2=0.97) and testosterone 6beta-hydroxylation (r2=0.9). There was also a strong correlation between 6alpha-hydroxylation of lithocholic acid, CYP3A levels and testosterone 6beta-hydroxylation (r2=0.7). Troleandomycin, a selective inhibitor of CYP3A enzymes, inhibited 6alpha-hydroxylation of taurochenodeoxycholic acid almost completely at a 10 microM concentration. Other inhibitors, such as alpha-naphthoflavone, sulfaphenazole and tranylcypromine had very little or no effect on the activity. The apparent Km for 6alpha-hydroxylation of taurochenodeoxycholic by human liver microsomes was high (716 microM). This might give an explanation for the limited formation of 6alpha-hydroxylated bile acids in healthy humans. From the present results, it can be concluded that CYP3A4 is active in the 6alpha-hydroxylation of both taurochenodeoxycholic acid and lithocholic acid in human liver.     

Lignification in wheat roots parasitised by Gaeuntannomyces graminis and Phialophora radicicola

Preliminary results suggest a possible relationship between lignin synthesis in wheat roots and the observed interaction between Gaeumannomyces graminis (Sacc.) Arx & Olivier and Phialophora radicicola Cain var. graminicola Deacon when they parasitise wheat roots. It was found that colonisation of wheat roots by P. radicicola resulted in a qualitative change in the lignin of the root, such that the content of the p-hydroxy type of aromatic nucleus was reduced almost to zero. It was also found that some of the metabolic precursors of lignin were inhibitory to the growth of G. graminis in Petri-dish culture. Most inhibitory of these precursors was caffeic acid, which reduced the growth rate of G. graminis by half at a concentration of 37 ppm. It is tentatively suggested that colonisation by P. radicicola results in an increased activity of polyphenol oxidase in the root tissues. This would lead to a more rapid synthesis of caffeic acid, with a depletion of the level of p-coumaric acid, and probably an increase in the levels of ferulic acid and sinapic acid. As well as bringing about a change in the composition of the lignin of the root, as the results show, the possible accumulation of caffeic acid in the root tissues might explain the greater resistance to infection by G. graminis observed in roots colonised first by P. radicicola.     

Biological activity of phenolic compounds. Hepatic cytochrome P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats fed phenolic monomers, polymers, and glycosides

Eight experiments were conducted to determine effects of a phenolic polymer (Kraft wood lignin, Indulin), phenolic glycosides (cane molasses and wood molasses), and phenolic monomers (vanillin, vanillic acid, ferulic acid, and p-coumaric acid) on liver cytochromes P-450, cytochrome b5, and NADPH cytochrome c reductase in chicks and rats. Chicks fed 6.0% lignin had a higher (P less than 0.01) cytochromes P-450 content than did chicks fed 0% fiber, 6.0% wood cellulose (Solka Floc), or 6.0% arenaceous flour. NADPH cytochrome c reductase activity was not affected by treatment. Chicks fed 12.0% wood molasses had a higher (P less than 0.06) cytochromes P-450 level than did chicks fed 0% fiber or 6.0% wood molasses. Cane molasses incorporated at both 6.0 and 12.0% of the diet induced (P less than 0.05) cytochromes P-450 content over those of control-fed birds. Chicks fed 6.0% lignin, with or without antibiotic (bacitracin:neomycin sulfate, 2:1), had a higher (P less than 0.01) cytochromes P-450 level than did chicks fed control diets, with or without antibiotic. Additionally, chicks fed 6.0% lignin had lower (P less than 0.01) intestinal diaminopimelic acid (DAP) levels than did chicks fed 0% fiber. Rats fed 0% fiber, 6.0% wood cellulose, 6.0% arenaceous flour, or 6.0% lignin exhibited no difference in cytochrome level or activity among treatments. Chicks fed 0.5% vanillin, 0.5% vanillic acid, 0.5% ferulic acid, or 0.5% p-coumaric acid had comparable cytochromes level and activity compared with chicks fed no phenolics. Chicks fed 0.5% p-coumaric acid had lower (P less than 0.05) rates of gain than did chicks fed control or other phenolic-containing diets. Rats fed these phenolics had similar cytochromes P-450 content among treatments.     

Comparison of the substrate specificities of human liver cytochrome P450s 2C9 and 2C18: application to the design of a specific substrate of CYP 2C18.

A series of 2-aroylthiophenes derived from tienilic acid by replacement of its OCH2COOH substituent with groups bearing various functions have been synthesized and studied as possible substrates of recombinant human liver cytochrome P450s 2C9 and 2C18 expressed in yeast. Whereas only compounds bearing a negative charge acted as substrates of CYP 2C9 and were hydroxylated at position 5 of their thiophene ring at a significant rate, many neutral 2-aroylthiophenes were 5-hydroxylated by CYP 2C18 with kcat values of >2 min-1. Among the various compounds that were studied, those bearing an alcohol function were the best CYP 2C18 substrates. One of them, compound 3, which bears a terminal O(CH2)3OH function, appeared to be a particularly good substrate of CYP 2C18. It was regioselectively hydroxylated by CYP 2C18 at position 5 of its thiophene ring with a KM value of 9 +/- 1 microM and a kcat value of 125 +/- 25 min-1, which are the highest described so far for a CYP 2C. A comparison of the oxidations of 3, by yeast-expressed CYP 1A1, 1A2, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, 3A4, and 3A5, showed that only CYP 2C8, 2C18, and 2C19 were able to catalyze the 5-hydroxylation of 3. However, the catalytic efficiency of CYP 2C18 for that reaction was considerably higher (kcat/KM value being 3-4 orders of magnitude larger than those found for CYP 2C8 and 2C19). Several human P450s exhibited small activities for the oxidative O-dealkylation of 3. The four recombinant CYP 2Cs were the best catalysts for that reaction (kcat between 1 and 5 min-1) when compared to all the P450s that were tested, even though it is a minor reaction in the case of CYP 2C18. All these results show that compound 3 is a new, selective, and highly efficient substrate for CYP 2C18 that should be useful for the study of this P450 in various organs and tissues. They also suggest some key differences between the active sites of CYP 2C9 and CYP 2C18 for substrate recognition.