Physiological Regulation, Xenobiotic Induction, and Heterologous Expression of P450 Monooxygenase Gene pc-3 (CYP63A3), a New Member of the CYP63 Gene Cluster in the White-rot FungusPhanerochaete chrysosporium

In order to characterize the functional diversity in CYP63 cluster of tandemly linked P450 genes (pc-1, pc-2, and pc-3) in Phanerochaete chrysosporium, here we report the functional characterization of pc-3 (CYP63A3), a newly cloned member of this group. pc-3 expression was favored in nutrient-limited versus nutrient-rich media in 3–6-day-old cultures and was upregulated by starch as a carbon source or by oxygenation of cultures. pc-3 was induced by various xenobiotics in defined nutrient-limited (3–9-fold) and nutrient-rich (2–5-fold) cultures. Particularly, a range of unsubstituted and substituted aliphatic hydrocarbons (alkanes and fatty acids) induced the expression under the two nutrient conditions albeit in a differential manner. Interestingly, pc-3 was also inducible by certain oxygenated mono aromatics (nitrophenol, benzoate, and resorcinol), lower molecular weight (2 to 4 ring size) polycyclic aromatic hydrocarbons (PAHs) and alkali-treated lignin derivatives in nutrient-rich malt extract cultures. The study further establishes that the three CYP63 genes (CYP63A1, A2, and A3) are independently regulated despite being members of the tandem gene cluster with high gene structural similarity (13–14 introns) and protein sequence homology (59–85%). The pc-3 cDNA (1,812 bp) was expressed in E. coli as a His-tagged protein (∼ 74 kDa). This constitutes the first report on heterologous expression of a P450 monooxygenase enzyme from this model white-rot fungus.     

Phylogenetic analysis of Bacillus P450 monooxygenases and evaluation of their activity towards steroids

Cytochrome P450 (P450) open reading frames (ORFs) identified in genome sequences of Bacillus species are potential resources for new oxidation biocatalysts. Phylogenetic analysis of 29 Bacillus P450 ORFs revealed that the P450s consist of a limited number of P450 families, CYP102, CYP106, CYP107, CYP109, CYP134, CYP152, and CYP197. Previously, we identified the catalytic activities of three P450s of Bacillus subtilis towards steroids by rapid substrate screening using Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR/MS). Here, we further applied this method to evaluate the activity of Bacillus cereus P450s towards steroids. Five P450 genes were cloned from B. cereus ATCC 10987 based on its genomic sequence and were expressed in Escherichia coli. These P450s were reacted with a mixture of 30 compounds that mainly included steroids, and the reaction mixtures were analyzed using FT-ICR/MS. We found that BCE_2659 (CYP106) catalyzed the monooxygenation of methyltestosterone, progesterone, 11-ketoprogesterone, medroxyprogesterone acetate, and chlormadinone acetate. BCE_2654 (CYP107) monooxygenated testosterone enanthate, and BCE_3250 (CYP109) monooxygenated testosterone and compactin. Based on the phylogenetic relationship and the known substrate specificities including ones identified in this study, we discuss the catalytic potential of Bacillus P450s towards steroids.     

Expression profiling of <Emphasis Type="Italic">Streptomyces peucetius</Emphasis> metabolic genes using DNA microarray analysis

Doxorubicin (DXR) and daunorubicin (DNR) are anthracycline antibiotics produced by Streptomyces peucetius and widely used as cancer chemotherapeutic agents. To improve their productivity, regulation of DXR/DNR synthesis genes as well as central metabolic pathway genes must be understood more clearly. So far, studies have focused on DXR/DNR gene regulation. To investigate the correlation between the central metabolic pathway genes and DXR/DNR productivity, we selected 265 genes involved in glycolysis, fermentation, the citric acid cycle, butanoate metabolism, etc., and searched for their sequences in the S. peucetius genome by comparing gene sequences to those of Streptomyces coelicolor. The homologous genes were amplified by PCR and arrayed on glass microarray slides. Gene expression was monitored under two different growth media conditions, R2YE and NDYE. Genes involved in the production of malonyl-CoA and propionyl-CoA, the main precursors for doxorubicin synthesis, were mainly upregulated in NDYE media. Genes related to acetyl-CoA and the urea cycle were also upregulated. These changes in gene expression were confirmed by real-time RT-PCR.     

Cloning,expression in yeast,and functional characterization of CYP71D14, a root-specific cytochrome P450 from <Emphasis Type="Italic">Petunia hybrida</Emphasis>

Alkaloids, which are naturally occurring amines, are biosynthesized and accumulated in plant tissues such as roots, leaves, and stems. Many alkaloids have pharmacological effects on humans and animals. Cytochrome P450 (P450 or CYP) monooxygenases are known to play key roles in the biosynthesis of alkaloids in higher plants. A cDNA clone encoding a P450 protein consisting of 502 amino acids was isolated from Petunia hybrida. The deduced amino acid sequence of the cDNA clone showed a high level of similarity with the other P450 species in the CYP71D family; hence, this novel P450 was named CYP71D14. Among plant P450 species, CYP71D14 had 45.7% similarity in its amino acid sequence to CYP71D12, which is involved in the biosynthesis of the indole alkaloids vinblastine and vincristine. Expression of CYP71D14 mRNA in Petunia plants was examined by Northern blot analysis by using a full-length cDNA of CYP71D14 as a probe. CYP71D14 mRNA was expressed most abundantly in the roots. The nucleotide sequence of CYP71D14 has been submitted to the DDBJ, EMBL, and GenBank nucleotide databases under the accession number AB028462. An erratum to this article can be found at     

Overexpression of barley <Emphasis Type="Italic">hva1</Emphasis> gene in creeping bentgrass for improving drought tolerance

The objectives of this study were to test the feasibility of introducing barley hva1 gene, a LEA3 member, into perennial grass species using the Agrobacterium-mediated transformation technique and to determine whether heterologous expression of hva1 would alleviate water-deficit injury in grass species. Creeping bentgrass (Agrostis stolonifera var. palustris), a drought-intolerant grass species, was transformed transiently or stably using three different promoters in conjunction with the downstream report/target genes. Two abscisic acid (ABA)-inducible promoters, ABA1 and ABA2 derived from ABA-response complex (ABRC3) were used to examine stress-responsive expression of the green fluorescent protein (GFP). Transient expression of GFP demonstrated the inducibility of ABA1 and ABA2 promoters in response to exogenous ABA application. The ABA2 promoter was further studied for stress-responsive expression of hva1 and a maize Ubi-1 promoter was tested for constitutive expression of the gene. In the T₀ generation, the Ubi-1::hva1 transformants displayed variable expression levels of HVA1 protein under normal growth conditions. The hva1 gene in the ABA2::hva1 transformants maintained low expression under well-watered conditions, but was upregulated under water-deficit conditions. The tolerance to water deficit of T₀ transgenic lines was assessed by measuring leaf relative water content and visually rating the severity of leaf wilting during to water stress. Under water-stressed conditions, some transgenic lines maintained high water content in leaves and showed significantly less extent of leaf wilting compared with non-transgenic control plants. These results indicated that the introduction of barley hva1 gene using constitutive or stress-inducible promoters lessened water-deficit injury in creeping bentgrass, suggesting that heterologous expression of LEA3 protein genes may enhance the survival ability of creeping bentgrass in water limiting environments.     

甘薯细胞色素P450单加氧酶基因IbCYP714E1和IbCYP714E2的鉴定及表达分析

CYP714基因在植物赤霉素合成与代谢过程中发挥着重要作用。该研究从甘薯基因组中鉴定出2个CYP714基因,对基因的结构和编码蛋白质的理化性质等进行了生物信息学分析,并利用荧光定量PCR(qRT-PCR)技术分析基因在不同组织和非生物胁迫条件下的表达特征,为解析甘薯CYP714基因的生物学功能提供帮助。结果表明:(1)2个基因为分别编码518个和521个氨基酸的碱性亲水蛋白,被亚细胞定位于细胞质中;(2)2个蛋白质均含有CYP714蛋白亚家族的3个特征结构域,与毛白杨的PtCYP714E2、PtCYP714E4和PtCYP714E5蛋白聚为一类,分别定名为IbCYP714E1和IbCYP714E2;(3)荧光定量PCR分析显示,IbCYP714E1和IbCYP714E2基因的表达部位存在一定差异,IbCYP714E1在柴根、初生根和叶片中表达量较高,而IbCYP714E2基因只在柴根和花上表达量较高,在盐和干旱胁迫下,IbCYP714E1基因表达量均增加,而IbCYP714E2基因只在盐胁迫条件下表达量增加。IbCYP714E1和IbCYP714E2基因可能参与赤霉素的降解和对非生物胁迫的应答。     

Isolation and characterization of a <Emphasis Type="Italic">Paramecium</Emphasis> cDNA clone encoding a putative serine/threonine protein kinase

Mitogen-activated protein (MAP) kinases, a closely related family of protein kinases, are involved in cell cycle regulation and differentiation in yeast and human cells. They have not been documented in ciliates. We used PCR to amplify DNA sequences of a ciliated protozoan—Paramecium caudatum—using primers corresponding to amino acid sequences that are common to MAP kinases. We isolated and sequenced one putative MAP kinase-like serine/threonine kinase cDNA from P. caudatum. This cDNA, called pcstk1 (Paramecium caudatum Serine/Threonine Kinase 1) shared approximately 35% amino acid identity with MAP kinases from yeast. MAP kinases are activated by phosphorylation of specific threonine and tyrosine residues. These two amino acid residues are conserved in the PCSTK1 sequence at positions Thr 159 and Tyr 161. The PSTAIRE motif, which is characteristic of the CDK2 gene family, cannot be found in ORF of PCSTK1. The highest homology score was to human STK9, which contains MAP type kinase domains. Comparisons of expression level have shown that pcstk1 is expressed equally in cells at different stages (sexual and asexual). We discussed the possibility, as in other organisms, that a family of MAP kinase genes exists in P. caudatum.     

Overexpression of CYP710A1 and CYP710A4 in transgenic <Emphasis Type="Italic">Arabidopsis</Emphasis> plants increases the level of stigmasterol at the expense of sitosterol

Sitosterol and stigmasterol are major sterols in vascular plants. An altered stigmasterol:sitosterol ratio has been proposed to influence the properties of cell membranes, particularly in relation to various stresses, but biosynthesis of stigmasterol is poorly understood. Recently, however, Morikawa et al. (Plant Cell 18:1008–1022, 2006) showed in Arabidopsis thaliana that synthesis of stigmasterol and brassicasterol is catalyzed by two separate sterol C-22 desaturases, encoded by the genes CYP710A1 and CYP710A2, respectively. The proteins belong to a small cytochrome P450 subfamily having four members, denoted by CYP710A1-A4, and are related to the yeast sterol C-22 desaturase Erg5p acting in ergosterol synthesis. Here, we report on our parallel investigation of the Arabidopsis CYP710A family. To elucidate the function of CYP710A proteins, transgenic Arabidopsis plants were generated overexpressing CYP710A1 and CYP710A4. Compared to wild-type plants, both types of transformant displayed a normal phenotype, but contained increased levels of free stigmasterol and a concomitant decrease in the level of free sitosterol. CYP710A1 transformants also displayed higher levels of esterified forms of stigmasterol, cholesterol, 24-methylcholesterol and isofucosterol. The results confirm the findings of Morikawa et al. (Plant Cell 18:1008–1022, 2006) regarding the function of CYP710A1 in stigmasterol synthesis, and show that CYP710A4 also has this capacity. Furthermore, our results suggest that an increased stigmasterol level alone is sufficient to stimulate esterification of other major sterols.     

Genotoxic activation of 2-phenylbenzotriazole-type compounds by human cytochrome P4501A1 and N-acetyltransferase expressed in Salmonella typhimurium umu strains

Four 2-phenylbenzotriazole (PBTA)-type compounds (PBTA-4, PBTA-6, PBTA-7, and PBTA-8) were identified as major mutagens in blue cotton/rayon-adsorbed substances collected at sites below textile dyeing factories or municipal water treatment plants treating domestic waste and effluents from textile dyeing factories in several rivers in Japan. The main purpose of this study is to understand the basis of the roles of human cytochrome P450 (CYP) and N-acetyltransferases (NATs) in genotoxic activation of PBTA derivatives. We compared the induction of umuC gene expression as a measure of genotoxicity using Salmonella typhimurium TA1535/pSK1002 (parental strain), NM2009 (bacterial O-acetyltransferase-overexpressing strain) established in our laboratories. PBTA-4, PBTA-6, PBTA-7, and PBTA-8 induced the umuC gene expression more strongly in the bacterial O-acetyltransferase-overproducing strain than in the parental strain in the presence of rat S9 mix. We determined the activation of PBTA derivatives by cDNA-based recombinant (Trichoplusia ni) systems expressing human or rat cytochrome P450 enzymes (P450 or CYP) and NADPH-P450 reductase using S. typhimurium NM2009. The results showed that human recombinant CYP1A1 enzyme was much more active than CYP1A2 and CYP3A4 in the genotoxic activation of PBTA-4, PBTA-6, PBTA-7, and PBTA-8. Similarly, rat recombinant CYP1A1 enzyme catalyzed the activation of these chemicals at high rates. α-Naphthoflavone, a known inhibitor of CYP1A1, was found to inhibit genotoxic activation caused by PBTA derivatives. We further determined the activation of PBTA derivatives using S. typhimurium NM6001 (human NAT1-expressing strain), S. typhimurium NM6002 (human NAT2-expressing strain), and S. typhimurium NM6000 (O-AT-deficient parent strain) in the presence of S9 mix. PBTA-4 showed almost similar sensitivity in the NAT1-expressing strain and the NAT2-expressing strain, although NAT2-expressing strain exhibited relatively higher sensitivity to PBTA-6, PBTA-7, and PBTA-8 than NAT1-expressing strain. The results support the view that O-acetylation by human NAT1 and NAT2 enzymes is involved in the genotoxic activation of PBTA compounds. These results demonstrate for the first time that human P4501A1 and NATs (NAT1 and NAT2) contribute significantly to the activation of PBTA-type compounds to genotoxic metabolites that induce umuC gene expression in S. typhimurium tester strains.     

Quantification of Three Key Enzymes Involved in Artemisinin Biogenesis in <Emphasis Type="Italic">Artemisia annua</Emphasis> by Polyclonal Antisera-Based ELISA

To elucidate the fine-tuned temporal and spatial modulation of artemisinin production in annual wormwood (Artemisia annua), we conducted enzyme-linked immunosorbent assay-based immunoquantification of three key enzymes involved in artemisinin biosynthesis, amorpha-4,11-diene synthase (ADS), cytochrome P450 monooxygenase (CYP71AV1), and cytochrome P450 reductase (CPR), in various tissues and under different growth conditions. The field-grown plants accumulate abundant ADS and CYP71AV1 but a trace amount of CPR in all tested tissues. Furthermore, ADS and CYP71AV1 accumulations in leaves are 16- and eightfold higher than in roots, and ten- and fourfold higher than in stems, respectively, demonstrating a tissue-specific expression pattern. Interestingly, the flowering field plants and cold-acclimated cultural plants produce higher levels of ADS and CYP71AV1 than non-flowering field plants or untreated cultural plants, indicating the environmental and developmental induction on ADS and CYP71AV1 genes and providing possible explanation for the observation that elevation of artemisinin level occurs after flowering.     

Targeting of the highly conserved threonine 302 residue of cytochromes P450 2B family during mechanism-based inactivation by aryl acetylenes

Cytochromes P450 (CYPs or P450s) contain a highly conserved threonine residue in the active site, which is referred to as Thr302 in the amino acid sequence of CYP2B4. Extensive biochemical and crystallographic studies have established that this Thr302 plays a critical role in activating molecular oxygen to generate Compound I, a putative iron(IV)-oxo porphyrin cation radical, that carries out the preliminary oxygenation of CYP substrates. Because of its proximity to the center of the P450 active site, this Thr302 is susceptible to mechanism-based inactivation under certain conditions. In this article, we review recent studies on the mechanism-based inactivation of three mammalian P450s in the 2B family, CYP2B1 (rat), 2B4 (rabbit) and 2B6 (human) by tert-butylphenylacetylene (tBPA). These studies showed that tBPA is a potent mechanism-based inactivator of CYP2B1, 2B4 and 2B6 with high k_inact/K_I ratios (0.23–2.3 min⁻¹ μM⁻¹) and low partition ratios (0–5). Furthermore, mechanistic studies revealed that tBPA inactivates these three CYP2B enzymes through the formation of a single ester adduct with the Thr302 in the active site. These inhibitory properties of tBPA allowed the preparation of a modified CYP2B4 where the Thr302 was covalently and stoichiometrically labeled by a reactive intermediate of tBPA in quantities large enough to permit spectroscopic and crystallographic studies of the consequences of covalent modification of Thr302. Molecular modeling studies revealed a unique binding mode of tBPA in the active site that may shed light on the potency of this inhibition. The results from these studies may serve as a basis for designing more specific and potent inhibitors for P450s by targeting this highly conserved threonine residue which is present in the active sites of most mammalian P450s.     

Identification of the proteins related to cytochrome P450 induced by fenvalerate in a <Emphasis Type="Italic">Trichoplusia ni</Emphasis> cell line

In order to reveal the metabolic reaction to the presence of fenvalerate mediated by P450 in insects, we used the trypan blue exclusion technique and 3-(4,5-dimethylthiazol)-2,5-diphenyltrazolium bromide (MTT) reduction assay to assess the vitality of Trichoplusia ni (Tn) cells treated with fenvalerate, and observed dose- and time-dependent changes in total cellular P450s. In addition, two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) were used to identify the proteins involved in the fenvalerate reaction process. Finally, the cDNA of P450 fragments was cloned and real-time RT-PCR was performed. Our data showed that at the 0–15 μmol/L challenge concentration of fenvalerate, at which the vitality of Tn cells was not affected (p > 0.05), there was a tendency toward a dose- and time-response of total cellular P450s, which peaked at the 9 h (p < 0.05) and 12 h (p < 0.01) time points following 12.5 μmol/L stimulation with fenvalerate. The 2-DE assay detected more than 1300 protein spots in each two-dimensional gel, of which 33 spots displayed significant differences. Among the changed spots, three isoforms of P450 were identified. One of the three P450 cDNA fragments (CYP4L4) was cloned and sequenced, and its expression in treated Tn cells increased significantly (p < 0.01). It was found that fenvalerate induced the expression of P450s in insect cells. This suggests that fenvalerate could be metabolized by CYP4L4 through a hydroxylation reaction in insect cells.