Characterization of CONSTITUTIVE PHOTOMORPHOGENESIS AND DWARFISM Homologs in Rice (Oryza sativa L.)

To enhance our understanding of brassinosteroid (BR) biosynthesis in rice, we attempted to identify putative rice homologs of Arabidopsis CYP90A1/ CPD and related mutants. Two candidate genes, designated CYP90A3/OsCPD1 and CYP90A4/OsCPD2, are located on chromosomes 11 (2.0 cM) and 12 (1.9 cM), respectively. Based on sequence similarity with the Arabidopsis CYP90A1/CPD gene, we predict that the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 gene products function as C-23α hydroxylases in the BR biosynthesis pathway. Both are broadly expressed in wild-type rice, and their expression is regulated by a feedback mechanism. A retrotransposon insertion mutant of CYP90A3/OsCPD1, oscpd1-1, did not produce any BR-deficient phenotype or feedback upregulation of genes for BR biosynthesis enzymes. These results indicate that if, as predicted, the CYP90A3/OsCPD1 and CYP90A4/OsCPD2 genes do function in the BR biosynthesis pathway, they may each have enough capacity to catalyze BR biosynthesis on their own. As a consequence, the oscpd1-1 mutant may not be deficient in endogenous BRs. Interestingly, BR biosynthesis enzymes except C-6 oxidase are encoded by plural genes in rice but by single genes in Arabidopsis (again, except C-6 oxidase). On the basis of these findings, we discuss the differences in BR biosynthesis between rice and Arabidopsis.     

The development of a whole-cell based medium throughput screening system for the discovery of human aldosterone synthase (CYP11B2) inhibitors: old drugs disclose new applications for the therapy of congestive heart failure, myocardial fibrosis and hypertension

Cytochrome P450 enzymes play an important role in steroid hormone biosynthesis of the human adrenal gland, e.g., the production of cortisol and aldosterone. Aldosterone, the most important human mineralocorticoid, is involved in the regulation of the salt and water homeostasis of the body and thus in the regulation of blood pressure, whereas cortisol is the most important glucocorticoid of the human body. CYP11B-dependent steroid hydroxylases are drug development targets, and since they are very closely related enzymes, the discovery of selective inhibitors has been subject to intense investigations for several years. Here we report the development of a whole-cell medium throughput screening technology for the discovery of CYP11B2 inhibitors. The new screening system displayed high reproducibility and was applied to investigate a library of pharmacologically active compounds. 1268 compounds were investigated during this study which revealed 5 selective inhibitors of CYP11B2 (after validation against CYP11B1). The new inhibitors of CYP11B2 are already existing drugs that could be used either in the treatment of hyperaldosteronism-related diseases or as lead compounds that could further be optimised to achieve safer and selective inhibitors of aldosterone synthase. Article from the Special issue on 'Targeted Inhibitors'.     

Cytochrome p450 CYP79F1 from arabidopsis catalyzes the conversion of dihomomethionine and trihomomethionine to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates

Glucosinolates are natural plant products that have received rising attention due to their role in interactions between pests and crop plants and as chemical protectors against cancer. Glucosinolates are derived from amino acids and have aldoximes as intermediates. We report that cytochrome P450 CYP79F1 catalyzes aldoxime formation in the biosynthesis of aliphatic glucosinolates in Arabidopsis thaliana. Using recombinant CYP79F1 functionally expressed in Escherichia coli, we show that both dihomomethionine and trihomomethionine are metabolized by CYP79F1 resulting in the formation of 5-methylthiopentanaldoxime and 6-methylthiohexanaldoxime, respectively. 5-methylthiopentanaldoxime is the precursor of the major glucosinolates in leaves of A. thaliana, i.e. 4-methylthiobutylglucosinolate and 4-methylsulfinylbutylglucosinolate, and a variety of other glucosinolates in Brassica sp. Transgenic A. thaliana with cosuppression of CYP79F1 have a reduced content of aliphatic glucosinolates and a highly increased level of dihomomethionine and trihomomethionine. The transgenic plants have a morphological phenotype showing loss of apical dominance and formation of multiple axillary shoots. Our data provide the first evidence that a cytochrome P450 catalyzes the N-hydroxylation of chain-elongated methionine homologues to the corresponding aldoximes in the biosynthesis of aliphatic glucosinolates.     

Drug metabolism in human brain: high levels of cytochrome P4503A43 in brain and metabolism of anti-anxiety drug alprazolam to its active metabolite

Cytochrome P450 (P450) is a super-family of drug metabolizing enzymes. P450 enzymes have dual function; they can metabolize drugs to pharmacologically inactive metabolites facilitating their excretion or biotransform them to pharmacologically active metabolites which may have longer half-life than the parent drug. The variable pharmacological response to psychoactive drugs typically seen in population groups is often not accountable by considering dissimilarities in hepatic metabolism. Metabolism in brain specific nuclei may play a role in pharmacological modulation of drugs acting on the CNS and help explain some of the diverse response to these drugs seen in patient population. P450 enzymes are also present in brain where drug metabolism can take place and modify therapeutic action of drugs at the site of action. We have earlier demonstrated an intrinsic difference in the biotransformation of alprazolam (ALP) in brain and liver, relatively more alpha-hydroxy alprazolam (alpha-OHALP) is formed in brain as compared to liver. In the present study we show that recombinant CYP3A43 metabolizes ALP to both alpha-OHALP and 4-hydroxy alprazolam (4-OHALP) while CYP3A4 metabolizes ALP predominantly to its inactive metabolite, 4-OHALP. The expression of CYP3A43 mRNA in human brain samples correlates with formation of relatively higher levels of alpha-OH ALP indicating that individuals who express higher levels of CYP3A43 in the brain would generate larger amounts of alpha-OHALP. Further, the expression of CYP3A43 was relatively higher in brain as compared to liver across different ethnic populations. Since CYP3A enzymes play a prominent role in the metabolism of drugs, the higher expression of CYP3A43 would generate metabolite profile of drugs differentially in human brain and thus impact the pharmacodynamics of psychoactive drugs at the site of action.     

Cytochrome P450 Monooxygenase CYP53 Family in Fungi: Comparative Structural and Evolutionary Analysis and Its Role as a Common Alternative Anti-Fungal Drug Target

Cytochrome P450 monooxygenases (CYPs/P450s) are heme-thiolate proteins whose role as a drug target against pathogenic microbes has been explored because of their stereo- and regio-specific oxidation activity. We aimed to assess the CYP53 family''s role as a common alternative drug target against animal (including human) and plant pathogenic fungi and its role in fungal-mediated wood degradation. Genome-wide analysis of fungal species revealed the presence of CYP53 members in ascomycetes and basidiomycetes. Basidiomycetes had a higher number of CYP53 members in their genomes than ascomycetes. Only two CYP53 subfamilies were found in ascomycetes and six subfamilies in basidiomycetes, suggesting that during the divergence of phyla ascomycetes lost CYP53 P450s. According to phylogenetic and gene-structure analysis, enrichment of CYP53 P450s in basidiomycetes occurred due to the extensive duplication of CYP53 P450s in their genomes. Numerous amino acids (103) were found to be conserved in the ascomycetes CYP53 P450s, against only seven in basidiomycetes CYP53 P450s. 3D-modelling and active-site cavity mapping data revealed that the ascomycetes CYP53 P450s have a highly conserved protein structure whereby 78% amino acids in the active-site cavity were found to be conserved. Because of this rigid nature of ascomycetes CYP53 P450s'' active site cavity, any inhibitor directed against this P450 family can serve as a common anti-fungal drug target, particularly toward pathogenic ascomycetes. The dynamic nature of basidiomycetes CYP53 P450s at a gene and protein level indicates that these P450s are destined to acquire novel functions. Functional analysis of CYP53 P450s strongly supported our hypothesis that the ascomycetes CYP53 P450s ability is limited for detoxification of toxic molecules, whereas basidiomycetes CYP53 P450s play an additional role, i.e. involvement in degradation of wood and its derived components. This study is the first report on genome-wide comparative structural (gene and protein structure-level) and evolutionary analysis of a fungal P450 family.     

CYP17 inhibitors for prostate cancer therapy

Prostate cancer (PC) is now the second most prevalent cause of death in men in the USA and Europe. At present, the major treatment options include surgical or medical castration. These strategies cause ablation of the production of testosterone (T), dihydrotestosterone (DHT) and related androgens by the testes. However, because these procedures do not affect adrenal, prostate and other tissues' androgen production, they are often combined with androgen receptor antagonists to block their action. Indeed, recent studies have unequivocally established that in castration-resistant prostate cancer (CRPC) many androgen-regulated genes become re-expressed and tissue androgen levels increase despite low serum levels. Clearly, inhibition of the key enzyme which catalyzes the biosynthesis of androgens from pregnane precursors, 17α-hydroxy/17,20-lyase (hereafter referred to as CYP17) could prevent androgen production from all sources. Thus, total ablation of androgen production by potent CYP17 inhibitors may provide effective treatment of prostate cancer patients. This review highlights the role of androgen biosynthesis in the progression of prostate cancer and the impact of CYP17 inhibitors, such as ketoconazole, abiraterone acetate, VN/124-1 (TOK-001) and TAK-700 in the clinic and in clinical development. Article from the special issue on Targeted Inhibitors.     

Polymorphic Cytochrome P450 Enzymes (CYPs) and Their Role in Personalized Therapy

The cytochrome P450 (CYP) enzymes are major players in drug metabolism. More than 2,000 mutations have been described, and certain single nucleotide polymorphisms (SNPs) have been shown to have a large impact on CYP activity. Therefore, CYPs play an important role in inter-individual drug response and their genetic variability should be factored into personalized medicine. To identify the most relevant polymorphisms in human CYPs, a text mining approach was used. We investigated their frequencies in different ethnic groups, the number of drugs that are metabolized by each CYP, the impact of CYP SNPs, as well as CYP expression patterns in different tissues. The most important polymorphic CYPs were found to be 1A2, 2D6, 2C9 and 2C19. Thirty-four common allele variants in Caucasians led to altered enzyme activity. To compare the relevant Caucasian SNPs with those of other ethnicities a search in 1,000 individual genomes was undertaken. We found 199 non-synonymous SNPs with frequencies over one percent in the 1,000 genomes, many of them not described so far. With knowledge of frequent mutations and their impact on CYP activities, it may be possible to predict patient response to certain drugs, as well as adverse side effects. With improved availability of genotyping, our data may provide a resource for an understanding of the effects of specific SNPs in CYPs, enabling the selection of a more personalized treatment regimen.     

Molecular cloning and characterization of methylenedioxy bridge-forming enzymes involved in stylopine biosynthesis in Eschscholzia californica

(S)-stylopine is an important intermediate in the biosynthesis of benzophenanthridine alkaloids, such as sanguinarine. Stylopine biosynthesis involves the sequential formation of two methylenedioxy bridges. Although the methylenedioxy bridge-forming P450 (CYP719) involved in berberine biosynthesis has been cloned from Coptis japonica[Ikezawa N, Tanaka M, Nagayoshi M, Shinkyo R, Sakaki T, Inouye K & Sato F (2003) J Biol Chem278, 38557-38565], no information is available regarding the genes for methylenedioxy bridge-forming enzymes in stylopine biosynthesis. Two cytochrome P450 cDNAs involved in stylopine biosynthesis were isolated using degenerate primers designed for C. japonica CYP719 from cultured Eschscholzia californica cells. Heterologous expression in Saccharomyces cerevisiae showed that both CYP719A2 and CYP719A3 had stylopine synthase activity to catalyze methylenedioxy bridge-formation from cheilanthifoline to stylopine, but not cheilanthifoline synthase activity to convert scoulerine to cheilanthifoline. Functional differences and expression patterns of CYP719A2 and CYP719A3 were examined to investigate their physiological roles in stylopine biosynthesis. Enzymatic analysis showed that CYP719A2 had high substrate affinity only toward (R,S)-cheilanthifoline, whereas CYP719A3 had high affinity toward three similar substrates (R,S)-cheilanthifoline, (S)-scoulerine, and (S)-tetrahydrocolumbamine. An expression analysis in E. californica plant tissues showed that CYP719A2 and CYP719A3 exhibited expression patterns similar to those of three stylopine biosynthetic genes (CYP80B1, berberine bridge enzyme, and S-adenosyl-l-methionine : 3'-hydroxy-N-methylcoclaurine 4'-O-methyltransferase), whereas the specific expression of CYP719A3 in root was notable. Treatment of E. californica seedlings with methyl jasmonate resulted in the coordinated induction of CYP719A2 and CYP719A3 genes. The physiological roles of CYP719A2 and CYP719A3 in stylopine biosynthesis are discussed.     

Comparative Proteomics Among Cytochrome P450 Family 1 for Differential Substrate Specificity

Apart from playing key roles in drug metabolism and adverse drug–drug interactions, CYPs are potential drug targets to treat a variety of diseases. The intervention of over expression of P450 1A1 (CYP1A1) in tumor cells is identified as a novel strategy for anticancer therapy. We investigated three isoforms of CYP1 family (CYP1A1, CYP1A2, and CYP1B1) for their substrate specificity. The understanding of macromolecular features that govern substrate specificity is required to understand the interplay between the protein function and dynamics. This can help in design of new antitumor molecule specifically metabolized by CYP1A1 to mediate their antitumor activity. In the present study, we carried out the comparative protein structure analysis of the three isoforms. Sequence alignment, root mean square deviation (RMSD) analysis, B-factor analysis was performed to give a better understanding of the macromolecular features involved in substrate specificity and to understand the interplay between protein dynamics and functions which will have important implications on rational design of anticancer drugs. We identified the differences in amino acid residues among the three isoforms of CYP1 family, which may account for differential substrate specificity. Six putative substrate recognition sequences are characterized along with the regions they form in the protein structure. Further the RMSD and B-factor analysis provides the information about the identified residues having the maximum RMSD and B-factor deviations.     

CYP2D6 polymorphisms in patients with porphyrias

The cytochrome P-450 (CYP) isoenzymes, a superfamily of heme proteins which are the terminal oxidases of the mixed function oxidases system, metabolize more than 70% of all clinically approved drugs. The highly polymorphic CYP2D6 isoform metabolizes more than 25% of most common drugs, and the phenotypes of the 70-plus allelic variants range from compromised to excessive enzymatic activity. Porphyrias are a group of inherited or acquired metabolic disorders of heme biosynthesis, due to a specific decrease in the activity of one of the enzymes of the heme pathway. Clinical signs and symptoms of porphyrias are frequently associated with exposure to precipitating agents, including clinically approved drugs. CYP enzymes, including CYP2D6, participate in the metabolism of some porphyrinogenic drugs, leading to the deregulation of heme biosynthesis. Considering that some of the drugs not recommended for use in porphyric patients are metabolized by CYP2D6, the presence of CYP2D6 polymorphisms in porphyric patients would influence the triggering of the disease when these individuals receive a precipitating agent that is metabolized by CYP2D6. To investigate CYP2D6 polymorphisms in porphyric patients, healthy Argentinean volunteers, porphyric patients, and a group of individuals with high levels of iron were studied. Results indicated that the CYP2D6*3 and CYP2D6*4 alleles, in particular, would be linked to the onset of disease. Predictive genotyping for CYP2D6 in porphyric patients holds promise as a method to improve the clinical efficacy of drug therapy and to personalize drug administration for these patients.     

Characterization of CYP76M5-8 indicates metabolic plasticity within a plant biosynthetic gene cluster

Recent reports have revealed genomic clustering of enzymatic genes for particular biosynthetic pathways in plant specialized/secondary metabolism. Rice (Oryza sativa) carries two such clusters for production of antimicrobial diterpenoid phytoalexins, with the cluster on chromosome 2 containing four closely related/homologous members of the cytochrome P450 CYP76M subfamily (CYP76M5-8). Notably, the underlying evolutionary expansion of these CYP appears to have occurred after assembly of the ancestral biosynthetic gene cluster, suggesting separate roles. It has been demonstrated that CYP76M7 catalyzes C11α-hydroxylation of ent-cassadiene, and presumably mediates an early step in biosynthesis of the derived phytocassane class of phytoalexins. Here we report biochemical characterization of CYP76M5, -6, and -8. Our results indicate that CYP76M8 is a multifunctional/promiscuous hydroxylase, with CYP76M5 and -7 seeming to provide only redundant activity, while CYP76M6 seems to provide both redundant and novel activity, relative to CYP76M8. RNAi-mediated double knockdown of CYP76M7 and -8 suppresses elicitor inducible phytocassane production, indicating a role for these monooxygenases in phytocassane biosynthesis. In addition, our data suggests that CYP76M5, -6, and -8 may play redundant roles in production of the oryzalexin class of phytoalexins as well. Intriguingly, the preceding diterpene synthase for oryzalexin biosynthesis, unlike that for the phytocassanes, is not found in the chromosome 2 diterpenoid biosynthetic gene cluster. Accordingly, our results not only uncover a complex evolutionary history, but also further suggest some intriguing differences between plant biosynthetic gene clusters and the seemingly similar microbial operons. The implications for the underlying metabolic evolution of plants are then discussed.     

CYP90C1 and CYP90D1 are involved in different steps in the brassinosteroid biosynthesis pathway in Arabidopsis thaliana

Brassinosteroids (BRs) are plant hormones that are essential for a wide range of developmental processes in plants. Many of the genes responsible for the early reactions in the biosynthesis of BRs have recently been identified. However, several genes for enzymes that catalyze late steps in the biosynthesis pathways of BRs remain to be identified, and only a few genes responsible for the reactions that produce bioactive BRs have been identified. We found that the ROTUNDIFOLIA3 (ROT3) gene, encoding the enzyme CYP90C1, which was specifically involved in the regulation of leaf length in Arabidopsis thaliana, was required for the late steps in the BR biosynthesis pathway. ROT3 appears to be required for the conversion of typhasterol to castasterone, an activation step in the BR pathway. We also analyzed the gene most closely related to ROT3, CYP90D1, and found that double mutants for ROT3 and CYP90D1 had a severe dwarf phenotype, whereas cyp90d1 single knockout mutants did not. BR profiling in these mutants revealed that CYP90D1 was also involved in BR biosynthesis pathways. ROT3 and CYP90D1 were expressed differentially in leaves of A. thaliana, and the mutants for these two genes differed in their defects in elongation of hypocotyls under light conditions. The expression of CYP90D1 was strongly induced in leaf petioles in the dark. The results of the present study provide evidence that the two cytochrome P450s, CYP90C1 and CYP90D1, play distinct roles in organ-specific environmental regulation of the biosynthesis of BRs.     

Molecular epidemiology of sporadic breast cancer. The role of polymorphic genes involved in oestrogen biosynthesis and metabolism

The major known risk factors for female breast cancer are associated with prolonged exposure to increased levels of oestrogen. The predominant theory relates to effects of oestrogen on cell growth. Enhanced cell proliferation, induced either by endogenous or exogenous oestrogens, increases the number of cell divisions and thereby the possibility for mutation. However, current evidence also supports a role for oxidative metabolites, in particular catechol oestrogens, in the initiation of breast cancer. As observed in drug and chemical metabolism, there is considerable interindividual variability (polymorphism) in the conjugation pathways of both oestrogen and catechol oestrogens. These person-to-person differences, which are attributed to polymorphisms in the genes encoding for the respective enzymes, might define subpopulations of women with higher lifetime exposure to hormone-dependent growth promotion, or to cellular damage from particular oestrogens and/or oestrogen metabolites. Such variation could explain a portion of the cancer susceptibility associated with reproductive effects and hormone exposure. In this paper the potential role of polymorphic genes encoding for enzymes involved in oestrogen biosynthesis (CYP17, CYP19, and 17beta-HSD) and conversion of the oestrogen metabolites and their by-products (COMT, CYP1A1, CYP1B1, GSTM1, GSTM3, GSTP1, GSTT1 and MnSOD) in modulating individual susceptibility to breast cancer are reviewed. Although some of these low-penetrance genes appeared as good candidates for risk factors in the etiology of sporadic breast cancer, better designed and considerably larger studies than the majority of the studies conducted so far are evidently needed before any firm conclusions can be drawn.     

Genomic clustering of cyanogenic glucoside biosynthetic genes aids their identification in Lotus japonicus and suggests the repeated evolution of this chemical defence pathway

Cyanogenic glucosides are amino acid-derived defence compounds found in a large number of vascular plants. Their hydrolysis by specific β-glucosidases following tissue damage results in the release of hydrogen cyanide. The cyanogenesis deficient1 (cyd1) mutant of Lotus japonicus carries a partial deletion of the CYP79D3 gene, which encodes a cytochrome P450 enzyme that is responsible for the first step in cyanogenic glucoside biosynthesis. The genomic region surrounding CYP79D3 contains genes encoding the CYP736A2 protein and the UDP-glycosyltransferase UGT85K3. In combination with CYP79D3, these genes encode the enzymes that constitute the entire pathway for cyanogenic glucoside biosynthesis. The biosynthetic genes for cyanogenic glucoside biosynthesis are also co-localized in cassava (Manihot esculenta) and sorghum (Sorghum bicolor), but the three gene clusters show no other similarities. Although the individual enzymes encoded by the biosynthetic genes in these three plant species are related, they are not necessarily orthologous. The independent evolution of cyanogenic glucoside biosynthesis in several higher plant lineages by the repeated recruitment of members from similar gene families, such as the CYP79s, is a likely scenario.     

The Arabidopsis ref2 mutant is defective in the gene encoding CYP83A1 and shows both phenylpropanoid and glucosinolate phenotypes

The Arabidopsis ref2 mutant was identified in a screen for plants having altered fluorescence under UV light. Characterization of the ref2 mutants showed that they contained reduced levels of a number of phenylpropanoid pathway-derived products: sinapoylmalate in leaves, sinapoylcholine in seeds, and syringyl lignin in stems. Surprisingly, positional cloning of the REF2 locus revealed that it encodes CYP83A1, a cytochrome P450 sharing a high degree of similarity to CYP83B1, an enzyme involved in glucosinolate biosynthesis. Upon further investigation, ref2 mutants were found to have reduced levels of all aliphatic glucosinolates and increased levels of indole-derived glucosinolates in their leaves. These results show that CYP83A1 is involved in the biosynthesis of both short-chain and long-chain aliphatic glucosinolates and suggest a novel metabolic link between glucosinolate biosynthesis, a secondary biosynthetic pathway found only in plants in the order Capparales, and phenylpropanoid metabolism, a pathway found in all plants and considered essential to the survival of terrestrial plant species.     

Herbivore‐induced poplar cytochrome P450 enzymes of the CYP71 family convert aldoximes to nitriles which repel a generalist caterpillar

Numerous plant species emit volatile nitriles upon herbivory, but the biosynthesis as well as the relevance of these nitrogenous compounds in plant–insect interactions remains unknown. Populus trichocarpa has been shown to produce a complex blend of nitrogenous volatiles, including aldoximes and nitriles, after herbivore attack. The aldoximes were previously reported to be derived from amino acids by the action of cytochrome P450 enzymes of the CYP79 family. Here we show that nitriles are derived from aldoximes by another type of P450 enzyme in P. trichocarpa. First, feeding of deuterium‐labeled phenylacetaldoxime to poplar leaves resulted in incorporation of the label into benzyl cyanide, demonstrating that poplar volatile nitriles are derived from aldoximes. Then two P450 enzymes, CYP71B40v3 and CYP71B41v2, were characterized that produce aliphatic and aromatic nitriles from their respective aldoxime precursors. Both possess typical P450 sequence motifs but do not require added NADPH or cytochrome P450 reductase for catalysis. Since both enzymes are expressed after feeding by gypsy moth caterpillars, they are likely to be involved in herbivore‐induced volatile nitrile emission in P. trichocarpa. Olfactometer experiments showed that these volatile nitriles have a strong repellent activity against gypsy moth caterpillars, suggesting they play a role in induced direct defense against poplar herbivores.