Reciprocal expression of 17alpha-hydroxylase-C17,20-lyase and aromatase cytochrome P450 during bovine trophoblast differentiation: a two-cell system drives placental oestrogen synthesis

No definitive information is yet available on the steroidogenic capacity of the two morphologically distinct cell types forming the bovine trophoblast, the uninucleated trophoblast cells (UTCs) and the trophoblast giant cells (TGCs). Hence, in order to localise 17alpha-hydroxylase-C17,20-lyase (P450c17) on a cellular level and to monitor its expression as a function of gestational age, placentomes from pregnant (days 80-284; n = 19), prepartal (days 273-282; 24-36 h prior to the onset of labour; n = 3) and parturient cows (n = 5) were immunostained for P450c17 using an antiserum against the recombinant bovine enzyme. At all stages investigated, P450c17 was exclusively found in the UTCs of chorionic villi (CV), where staining was ubiquitous between days 80 and 160, but was largely restricted to primary CV and the branching sites of secondary CV between days 160 and 240. Thereafter, a distinct ubiquitous staining reoccurred in the UTCs of all CV in late pregnant, prepartal and parturient animals. Using an antiserum against human aromatase cytochrome P450 (P450arom), specific cytoplasmic staining was observed in TGCs. In placentomes from pregnant cows, staining intensity was higher in mature compared with immature TGCs and was more pronounced in the trophoblast covering big stem villi compared with the trophoblast at other sites of the villous tree. In placentomes of a parturient cow, specific staining was only found in mature TGCs that survived the normal, but substantial, prepartal decline in TGC numbers. These results clearly showed that bovine UTCs and TGCs exhibit different steroidogenic capacities, constituting a 'two-cell' organisation for oestrogen synthesis. P450c17 expression appears to be quickly down-regulated and P450arom is up-regulated when UTCs enter the TGC differentiation pathway.     

Cloning and heterologous expression of the NADPH cytochrome P450 oxidoreductase genes from an industrial dicarboxylic acid-producing Candida tropicalis

NADPH cytochrome P450 oxidoreductase (CPR) catalyses the transfer of electrons during P450-mediated oxidation, which plays an important role in the omega-oxidation pathway of Candida tropicalis. Two putative allelic genes, CPR-a and CPR-b, were cloned from the long chain dicarboxylic acid-producing Candida tropicalis 1230, using cassette PCR methods. Both the identified open reading frames predict the gene products of 679 amino acid residues. The deduced amino acid sequences of CPR-a and CPR-b are highly homologous to CPR genes from C. tropicalis ATCC 750 and Candida maltosa. Both genes were individually expressed in a cpr mutant of Saccharomyces cerevisiae with high CPR activities, in which only a small distinction was observed between recombinant CPR-a and CPR-b. Both CPR-a and CPR-b contain one CTG codon, which codes for serine (amino acid 50) in C. tropicalis rather than universal leucine. A mutated cDNA of CPR-a with a TCG codon instead of CTG codon was constructed and expressed, resulting in little increase in CPR activity. This indicates that the alteration of Ser-50 has little effect on functional expression of CPR. Furthermore, high ketoconazole sensitivity for the cpr mutant was complemented by heterologous expression of the cloned CPR-a or CPR-b.     

Control of oestradiol secretion and of cytochrome P450 aromatase messenger ribonucleic acid accumulation by FSH involves different intracellular pathways in oestrogenic bovine granulosa cells in vitro

The objective of this study was to determine the major intracellular signalling pathways used by FSH and insulin to stimulate cytochrome P450 aromatase (Cyp19) mRNA and oestradiol accumulation in oestrogenic bovine granulosa cells in vitro. Bovine granulosa cells from small follicles (2-4 mm diameter) were cultured for 6 days under non-luteinizing conditions in the presence of insulin at 100 ng/ml, or insulin (10 ng/ml) and FSH (1 ng/ml). On day 4 of culture, specific inhibitors of phosphatidylinositol 3-kinase (PI3K; LY-294002), protein kinase C (PKC; GF-109203X), protein kinase A (PKA; H-89) or mitogen-activated protein (MAP) kinase activation (PD-98059) were added. The addition of PI3K and PKC inhibitors, but not of PKA inhibitor, significantly decreased insulin-stimulated Cyp19 mRNA levels and oestradiol accumulation (P < 0.001). The PKA inhibitor significantly decreased FSH-stimulated Cyp19 mRNA abundance and oestradiol secretion, whereas PI3K and PKC inhibitors decreased oestradiol secretion without affecting Cyp19 mRNA accumulation. Inhibition of MAP kinase pathway significantly increased Cyp19 mRNA abundance in insulin- and FSH-stimulated cells. P450scc mRNA levels and progesterone secretion were not affected by any inhibitor in either experiment. Although FSH stimulates Cyp19 expression predominantly through PKA, oestradiol secretion is altered by PI3K and PKC pathways independently of Cyp19 mRNA levels. In addition, we suggest that Cyp19 is under tonic inhibition mediated through a MAP kinase pathway.     

The cytochrome P450ALK multigene family of an n-alkane-assimilating yeast, Yarrowia lipolytica: cloning and characterization of genes coding for new CYP52 family members

Genes encoding cytochromes P450 of the CYP52 family in the n-alkane-assimilating yeast Yarrowia lipolytica have been cloned and analyzed. Degenerate PCR primers which were designed for the conserved amino acid sequences of cytochromes P450ALK of Candida species were used for amplification and isolation of genes encoding P450ALK from a genomic DNA library of Y. lipolytica CX161-1B. Seven new genes (YlALK2-YlALK8) were isolated. Five of the seven YlALK genes were induced by n-alkane under the culture conditions used in this study, whereas their expression was strictly repressed by glycerol but not by glucose, similar to the case of YlALK1, reported previously. Disruption of YlALK2, YlALK3, YlALK4 or YlALK6 did not change the growth of Y. lipolytica on medium containing n-alkanes of various chain lengths. A mutant with disruptions in both YlALK1 and YlALK2 did not grow well on n-hexadecane, whereas one with disruptions in both YlALK1 and YlALK3, which has the same phenotype as the YlALK1 singly disrupted mutant, grew well on n-hexadecane. These results suggest that the presence of multiple P450ALK species is a rather common phenomenon among the n-alkane-assimilating yeasts and that in the n-alkane assimilation of Y. lipolytica, YlALK1 functions to assimilate n-decane and longer molecules, whereas YlALK2 is involved in the assimilation of molecules longer than n-dodecane; other YlALKs are not significantly involved in the assimilation of C10-C16 n-alkanes.     

Cloning and characterization of an n-alkane-inducible cytochrome P450 gene essential for n-decane assimilation by Yarrowia lipolytica

A gene encoding cytochrome P450 involved in n-alkane utilization was cloned from an n-alkane assimilating yeast, Yarrowia lipolytica CX161-1B. The RT-PCR was performed on the mRNA prepared from the cells grown on n-alkane as a template using degenerated PCR primers designed for the conserved amino acid sequences of the CYP52 family. The RT-PCR amplified fragment was then used as a probe to isolate genes coding for P450 of the CYP52 family from the genomic DNA library of the strain CX161-1B. The nucleotide sequence of one of the positive clones was determined. An open reading frame which had the same nucleotide sequence as the RT-PCR-amplified fragment was identified. It was of 523 amino acid residues, 60·2 kDa in molecular mass, and had 30–45% sequence identity with the other members of the CYP52 family of Candida species so far analysed. The expression of the P450 gene that was named as YlALK1 was induced by n-tetradecane and repressed by glycerol. A YlALK1 gene disruptant did not grow well on n-decane, but grew on longer-chain n-alkanes such as hexadecane as a sole carbon source. Introduction of YlALK1 on a plasmid to the disruptant restored the decane assimilation. These results suggest that the YlALK1 gene product is the major P450Alk to metabolize short-chain n-alkanes such as decane and dodecane in Y. lipolytica. © 1998 John Wiley & Sons, Ltd.     

Gene expression for LH receptor, 17 alpha-hydroxylase and StAR in the theca interna of preantral and early antral follicles in the bovine ovary

The onset of gene expression for three proteins that play pivotal roles in theca interna function, namely the LH receptor (LH-R), cytochrome P450 17 alpha-hydroxylase (17 alpha OH) and the steroidogenic acute regulatory protein (StAR), was determined. Ovaries were obtained on day 9 of the oestrus cycle from mature synchronized dairy cows (n = 5) and gene expression in preantral and antral follicles up to 4 mm in diameter was evaluated by in situ hybridization. LH-R and 17 alpha OH mRNAs were observed first, in the theca interna of large preantral follicles (type 4), concurrent with its morphological differentiation. StAR mRNA appeared later during follicular growth, in follicles >1 mm in diameter (type 6). LH-R and 17 alpha OH mRNAs were found exclusively in the thecal cells, whereas StAR mRNA appeared in thecal cells, granulosa cells of late atretic follicles and oocytes. In early atresia, thecal cells expressed all three mRNAs, and their expression decreased gradually as atresia progressed. Atresia in granulosa cells was characterized by massive apoptosis of periantral, but not peribasal cells, that differentiated into luteal-like cells expressing StAR. In summary, our study suggests that in spite of the presence of 17 alpha OH, a key enzyme in steroidogenesis, the ability to produce steroids by bovine follicles smaller than 1 mm in diameter must be very limited due to the absence of StAR protein. During the early stages of atresia, thecal cells remain morphologically and functionally healthy, and continue to express all three studied mRNAs.     

CaALK8, an alkane assimilating cytochrome P450, confers multidrug resistance when expressed in a hypersensitive strain of Candida albicans

We report the isolation of a novel C. albicans gene designated CaALK8, by its ability to complement drug hypersensitivity of a pdr5 (ABC: ATP-binding cassette drug extrusion pump) null mutant of S. cerevisiae (JG436). CaALK8 in JG436 conferred resistance to drugs such as cycloheximide (CYH), fluconazole (FCZ), O-phenanthroline (PHE) and 4-nitroquinoline oxide (NQO). The gene was so designated because its sequence was identical to a partial sequence entry named as ALK8 in the Candida database (http://alces.med.umn.edu/candida.html). CaALK8 encodes for a putative 515 amino acid protein highly homologous to alkane-inducible cytochromes P450 (CYP52 gene family) of C. maltosa and C. tropicalis. The ability of CaALK8 to confer drug resistance was also established by its expression in another drug-hypersensitive strain of S. cerevisiae (AD 1234568), which was deleted in seven ABC efflux pumps. The homozygous disruption of CaALK8 in a wild-type C. albicans strain (CAI4) did not result in altered drug susceptibilities. The overexpression of CaALK8 in CAI4 resulted in only FCZ resistance. However, a distinct MDR phenotype was evident when CaALK8 was overexpressed in a drug-hypersensitive C. albicans strain disrupted in both CDR1 and CDR2 (ABC drug extrusion pumps of C. albicans). Alk8p, similar to other Alk proteins from C. maltosa and C. tropicalis, could hydroxylate alkanes and fatty acids. In this study we demonstrate that several drugs could compete with the hydroxylation activity by directly interacting with CaAlk8p. Taken together, our results suggest that a member of the CYP52 gene family could mediate MDR in C. albicans, although it does not seem to be involved in the development of azole resistance in clinical isolates. The nucleotide sequence reported in this paper has been submitted to GenBank under Accession No. Y14766.     

Sequential exposure of porcine cumulus cells to FSH and/or LH is critical for appropriate expression of steroidogenic and ovulation-related genes that impact oocyte maturation in vivo and in vitro

In this study, we collected follicular fluid, granulosa cells, and cumulus cells from antral follicles at specific time intervals following equine chorionic gonadotropin (eCG) and human chorionic gonadotropin (hCG) treatment of gilts. The treatment with eCG increased the production of estrogen coordinately with up-regulated proliferation of granulosa and cumulus cells. eCG also induced the expression of LHCGR and PGR in cumulus cells and progesterone accumulation was detected in follicular fluid prior to the LH/hCG surge. Moreover, progesterone and progesterone receptor (PGR) were critical for FSH-induced LHCGR expression in cumulus cells in culture. The expression of LHCGR mRNA in cumulus cells was associated with the ability of LH to induce prostaglandin production, release of epidermal growth factor (EGF)-like factors, and a disintegrin and metalloprotease with thrombospondin-like repeats 1 expression, promoting cumulus cell oocyte complexes (COCs) expansion and oocyte maturation. Based on the unique expression and regulation of PGR and LHCGR in cumulus cells, we designed a novel porcine COCs culture system in which hormones were added sequentially to mimic changes observed in vivo. Specifically, COCs from small antral follicles were pre-cultured with FSH and estradiol for 10 h at which time progesterone was added for another 10 h. After 20 h, COCs were moved to fresh medium containing LH, EGF, and progesterone. The oocytes matured in this revised COC culture system exhibited greater developmental competence to blastocyst stage. From these results, we conclude that to achieve optimal COC expansion and oocyte maturation in culture the unique gene expression patterns in cumulus cells of each species need to be characterized and used to increase the effectiveness of hormone stimulation.     

Transformation of the recalcitrant pharmaceutical compound carbamazepine by Pleurotus ostreatus: role of cytochrome P450 monooxygenase and manganese peroxidase

Carbamazepine (CBZ) is an environmentally recalcitrant compound highly stable in soil and during wastewater treatment. In this study, we examined the mechanisms by which the white-rot fungus Pleurotus ostreatus metabolizes CBZ in liquid culture using a physiological approach. P. ostreatus PC9 was grown in media known to support different levels of a multiplicity of enzyme systems such as cytochrome P450 (CYP450) and manganese peroxidase (MnP). When both CYP450 and MnP systems were active, 99% of the added CBZ was eliminated from the solution and transformed to 10,11-epoxycarbamazepine. High removal of CBZ was also obtained when either MnP or CYP450 was active. When both CYP450 and MnP were inactivated, only 10 to 30% of the added CBZ was removed. In this latter system, removal of CBZ might be partially attributed to the activity of versatile peroxidase. P. ostreatus was able to eliminate CBZ in liquid culture even when CBZ was added at an environmentally relevant concentration (1 μg L(-1)). On the basis of our study, we suggest that two families of enzymes are involved in the oxidation of CBZ in liquid culture: MnP in a Mn(2+)-dependent or independent manner and CYP450. Our study also highlights the potential of using P. ostreatus for bioremediation systems.     

Computational toxicological investigation on the mechanism and pathways of xenobiotics metabolized by cytochrome P450: a case of BDE-47

Understanding the transformation mechanism and products of xenobiotics catalyzed by cytochrome P450 enzymes (CYPs) is vital to risk assessment. By density functional theory computation with the B3LYP functional, we simulated the reaction of 2,2',4,4'-tetrabromodiphenyl ether (BDE-47) catalyzed by the active species of CYPs (Compound I). The enzymatic and aqueous environments were simulated by the polarizable continuum model. The results reveal that the addition of Compound I to BDE-47 is the rate-determining step. The addition of Compound I to the ipso and nonsubstituted C atoms forms tetrahedral σ-adducts that further transform into epoxides. Hydroxylation of the epoxides leads to hydroxylated polybrominated diphenyl ethers and 2,4-dibromophenol. The addition to the Br-substituted C2 and C4 atoms has a higher barrier than addition to the nonsubstituted C atoms, forming phenoxide and cyclohexadienone which subsequently undergo debromination/hydroxylation. A novel mechanism was identified in which the approach of Compound I to C2 led to formation of a phenoxide and an expelled Br(-) ion. The predicted products were consistent with the metabolites identified by others. As a first attempt to simulate the enzymatic transformation of a polycyclic compound, this study may enlighten a computational method to predict the biotransformation of xenobiotics catalyzed by CYPs.     

Cloning and expression of soluble cytochrome c and its role in polyvinyl alcohol degradation by polyvinyl alcohol-utilizing Sphingopyxis sp. strain 113P3

The gene encoding cytocrome c in the pva operon of Sphingopyxis sp. strain 113P3 was cloned, on the basis of the sequence of the gene for cytochrome c (GenBank accession no. AB190288). The deduced amino acid sequence of the gene showed homologies (37% and 47% identities) with two cytochromes c of different origins. The recombinant cytochrome c tagged with hexahistidines was expressed in the periplasm of Escherichia coli BL21(DE3) harboring pT-GroE, which was in accordance with the localization of cytochrome c in strain 113P3; the protein was purified to homogeneity. The purified recombinant cytochrome c was a monomeric protein with a molecular weight of 16.5 kDa. The oxidized and reduced forms of the protein showed absorption maxima at 409 nm and at 414, 520 and 550 nm, respectively. The recombinant cytochrome c was fully reduced by polyvinyl alcohol (PVA), coupled with a catalytic amount (1/10 molar concentration) of the recombinant PVA dehydrogenase (PVADH) of the same origin, suggesting that the cytochrome c involved in the pva operon is a physiological primary electron acceptor for PVADH and that PVA dehydrogenation is linked with the respiratory chain in Sphingopyxis sp. strain 113P3.     

Cytochromes P450 of the sophorose lipid-producing yeast Candida apicola: Heterogeneity and polymerase chain reaction-mediated cloning of two genes

Candida apicola belongs to a group of yeasts producing high amounts of surface-active extracellular glycolipids consisting of sophorose and long-chain-ω- and (ω-1)-hydroxy fatty acids. The involvement of cytochrome P450 in the synthesis of sophorose lipid by the hydroxylation of long-chain fatty acids was suggested from a simultaneous increase of cellular P450 content. Hydroxylation studies indicated the existence of multiple P450 forms capable of hydroxylating not only long-chain fatty acids, but also n-alkanes. In this report, two different P450 DNA fragments amplified in a polymerase chain reaction with heterologous primers and chromosomal DNA of Candida apicola were used as homologous probes for the isolation of full-length clones from a genomic library. The open reading frames of both genes encode proteins of 519 amino acids with calculated molecular weights of 58,656 and 58,631, respectively, that contain N-terminal membrane anchor sequences and hallmark residues, in common with other eukaryotic P450s. The deduced amino acid sequences of the C. apicola P450 genes are 84·4% identical. They share 34·5 to 44·1% identity with the proteins of the yeast family CYP52 and about 25% identity with fatty acid hydroxylases of higher eukaryotes (family CYP4A) and of Bacillus megaterium (CYP102). Southern hybridization experiments revealed the existence of further P450-related genes in C. apicola. According to the P450 nomenclature system, the cloned genes were named CYP52E1 and CYP52E2, establishing a new subfamily in yeast family CYP52. The sequences were deposited in the EMBL/GenBank Library under the Accession Numbers X76225 and X87640.     

Potent inhibition of human cytochrome P450 3A4, 2D6, and 2C9 isoenzymes by grapefruit juice and its furocoumarins

ABSTRACT:  The cytochrome P450 enzyme family is the most abundant and responsible for the metabolism of more than 60% of currently marketed drugs and is considered central in many clinically important drug interactions. Seven different grapefruit and pummelo juices as well as 5 furocoumarins isolated from grapefruit juice were evaluated at different concentration on cytochrome P450 3A4 (CYP3A4), cytochrome P450 2C9 (CYP2C9), and cytochrome P450 2D6 (CYP2D6) isoenzyme activity. Grapefruit and pummelo juices were found to be potent inhibitors of cytochrome CYP3A4 and CYP2C9 isoenzymes at 25% concentration, while CYP2D6 is inhibited significantly low at all the tested concentration of juices ( P < 0.05). Among the 5 furocoumarins tested, the inhibitory potency was in the order of paradisin A > dihydroxybergamottin > bergamottin > bergaptol > geranylcoumarin at 0.1 μM to 0.1 mM concentrations. The IC₅₀ value was lowest for paradisin A for CYP3A4 with 0.11 μM followed by DHB for CYP2C9 with 1.58 μM.     

The ovarian expression of mRNAs for aromatase, IGF-I receptor, IGF-binding protein-2, -4 and -5, leptin and leptin receptor in cycling ewes after three days of leptin infusion

An experiment was carried out to determine the pattern of follicular expression of mRNAs for aromatase, IGF-I receptor (IGF-IR), IGF-binding protein (IGFBP)-2, -4 and -5, leptin and the long form of the leptin receptor (Ob-Rb) in ten ewes infused with human recombinant leptin (n = 5; 1 microg/h) or saline (n = 5) for 72 h in the luteal phase of the oestrous cycle. At the end of infusion a follicular phase was induced with a luteolytic dose of a prostaglandin F2alpha analogue and the ovaries were collected 32 h later. One ovary from each ewe was serially sectioned at 10 microm using a cryostat at -20 degrees C. All follicles >1 mm in diameter were counted and probed with specific oligoprobes for aromatase, IGF-IR and IGFBP-2, -4 and -5 and specific riboprobes for leptin and Ob-Rb. Leptin mRNA was detected in theca and granulosa cells and Ob-Rb mRNA was detected only in granulosa cells, of some, but not all antral follicles. Leptin doubled the number of follicles with a diameter >/=3.5 mm (1.0 +/- 0.36 (s.e.m.) vs 2.4 +/- 0.24; control vs leptin; P < 0.02) but had no effect on the number of >/=1 < 3.5 mm follicles. Leptin had no effect on the number of follicles expressing aromatase mRNA but it decreased significantly the number of follicles expressing mRNA for IGF-IR (10.7 +/- 0.79 vs 7.4 +/- 0.81; control vs leptin; P < 0.05), IGFBP-2 (10.0 +/- 0.82 vs 5.2 +/- 0.87; control vs leptin; P < 0.05) and IGFBP-5 (5.2 +/- 1.60 vs 1.2 +/- 0.30; control vs leptin; P < 0.05). Leptin increased the diameter of IGFBP-2 mRNA-positive follicles (1.5 +/- 0.15 vs 2.2 +/- 0.31 mm; control vs leptin; P < 0.05) and increased follicular mRNA expression for IGFBP-2 (0.30 +/- 0.021 vs 0.39 +/- 0.027 arbitrary units; control vs leptin; P < 0.05) and IGFBP-5 (0.46 +/- 0.019 vs 0.25 +/- 0.053 arbitary units; control vs leptin; P < 0.05). The mRNA for IGFBP-4 was detected in the theca of only two follicles from the control group. Leptin increased the number of follicles expressing Ob-Rb mRNA (0.25 +/- 0.25 vs 1.40 +/- 1.17; control vs leptin; P < 0.05) but had no effect on the number expressing leptin mRNA. Leptin decreased plasma concentrations of oestradiol (P < 0.05) and increased concentrations of FSH (P < 0.001) and insulin (P < 0.001), with no effect on glucose concentrations. These data show that: (i) ovine granulosa cells express mRNA for Ob-Rb and leptin and (ii) leptin increased the number of follicles >/=3.5 mm. Furthermore, the data suggest that suppression of oestradiol production by leptin is not mediated by inhibition of aromatase gene expression. Finally, the data indicate that the action of leptin in ovarian follicles is mediated by the IGF system, because leptin increased mRNA expression of IGFBP-2 and -5. Leptin also decreased the number of follicles expressing IGF-IR and IGFBP-2 and -5. We suggest that these actions of leptin on the IGF system decrease the bioavailability of IGF-I, resulting in decreased oestradiol production.     

Functional analysis of six genes from chromosomes XIV and XV of Saccharomyces cerevisiae reveals YOR145c as an essential gene and YNL059c/ARP5 as a strain-dependent essential gene encoding nuclear proteins

We report here basic functional analysis of strains deleted for six open reading frames (ORFs), YNL059c and YNL148c from chromosome XIV and YOR145c, YOR152c, YOR161c and YOR162c from chromosome XV of Saccharomyces cerevisiae. ORFs were replaced with the KanMX4 resistance marker using a long flanking homology PCR strategy in FY1679 and W303 diploid strains. Replacement cassettes were constructed in plasmid pUG7 and the cognate wild-type genes were recovered by gap repair. Sporulation and tetrad analysis revealed that deletion of YNL059c/ARP5 was lethal for vegetative growth in strain W303 and caused severe growth defects in strain FY1679 while YOR145c was essential for growth in both strains. Fusion of the green fluorescent protein (GFP) gene to the 3' ends of the YNL059c/ARP5 and YOR145c coding sequences created functional chimeric genes at the respective chromosomal loci. Both Arp5-GFP and Yor145-GFP localized to the nucleus, Yor145-GFP concentrating in the nucleolus. The vectors containing the deletion cassettes and the cognate wild-type genes, the oligonucleotides, and the deletant strains are available from the EUROFAN resource centre EUROSCARF (Frankfurt).     

Monofunctional catalase P of Paracoccidioides brasiliensis: identification, characterization, molecular cloning and expression analysis

Within the context of studies on genes from Paracoccidioides brasiliensis (Pb) potentially associated with fungus-host interaction, we isolated a 61 kDa protein, pI 6.2, that was reactive with sera of patients with paracoccidioidomycosis. This protein was identified as a peroxisomal catalase. A complete cDNA encoding this catalase was isolated from a Pb cDNA library and was designated PbcatP. The cDNA contained a 1509 bp ORF containing 502 amino acids, whose molecular mass was 57 kDa, with a pI of 6.5. The translated protein PbCATP revealed canonical motifs of monofunctional typical small subunit catalases and the peroxisome-PTS-1-targeting signal. The deduced and the native PbCATP demonstrated amino acid sequence homology to known monofunctional catalases and was most closely related to catalases from other fungi. The protein and mRNA were diminished in the mycelial saprobic phase compared to the yeast phase of infection. Protein synthesis and mRNA levels increased during the transition from mycelium to yeast. In addition, the catalase protein was induced when cells were exposed to hydrogen peroxide. The identification and characterization of the PbCATP and cloning and characterization of the cDNA are essential steps for investigating the role of catalase as a defence of P. brasiliensis against oxygen-dependent killing mechanisms. These results suggest that this protein exerts an influence in the virulence of P. brasiliensis.     

Candida maltosa NADPH-cytochrome P450 reductase: Cloning of a full-length cDNA,Heterologous expression in Saccharomyces cerevisiae and function of the N-terminal region for membrane anchoring and proliferation of the endoplasmic reticulum

A full-length cDNA for NADPH-cytochrome P450 reductase from Candida maltosa was cloned and sequenced. The derived amino acid sequence showed a high similarity to the reductases from other eukaryotes. Expression in Saccharomyces cerevisiae under control of the GAL10 promoter resulted in an approximately 70-fold increase in NADPH-cytochrome c reductase activity in the microsomal fraction. The functional integrity of the heterologously expressed reductase as an electron transfer component for alkane hydroxylating cytochrome P450 from C. maltosa was shown in a reconstituted system containing both enzymes in a highly purified state. The signal-anchor sequence of the reductase was identified within the N-terminal region of the protein by means of constructing and expressing fusion proteins with the cytosolic form of yeast invertase. The first 33 amino acids turned out to be sufficient for stable membrane insertion, wild-type membrane orientation and retention in the endoplasmic reticulum. As shown by immunoelectron microscopy, the heterologously expressed reductase was integrated into the endoplasmic reticulum of the host organism. It triggered a strong proliferation of the membrane system. This membrane-inducing property of the reductase was transferable to the cytosolic reporter protein with the same N-terminal sequences that confer membrane insertion. The nucleotide sequence of the cDNA of NADPH-cytochrome P450 reductase from C. maltosa is available from the EMBL data library under Accession Number X76226.