Solution structure of the donor site of a trans-splicing RNA

The CYP51 gene encoding eburicol 14 alpha-demethylase (P450(14DM)) was cloned from a genomic library of the filamentous fungal plant pathogen Penicillium italicum, by heterologous hybridisation with the corresponding gene encoding lanosterol 14 alpha-demethylase from the yeast Candida tropicalis. The nucleotide sequence of a 1739-bp genomic fragment and the corresponding cDNA clone comprises an open reading frame (ORF) of 1545 bp, encoding a protein of 515 amino acids with a predicted molecular mass of 57.3 kDa. The ORF is interrupted by three introns of 60, 72 and 62 bp. The C-terminal part of the protein includes a characteristic haem-binding domain, HR2, common to all P450 genes. The deduced P. italicum P450(14DM) protein and the P450(14DM) proteins from Candida albicans, C. tropicalis and Saccharomyces cerevisiae share 47.2, 47.0 and 45.8% amino acid sequence identity. Therefore, the cloned gene is classified as a member of the CYP51 family. Multiple copies of a genomic DNA fragment of Pl italicum containing the cloned P450 gene were introduced into Aspergillus niger by transformation. Transformants were significantly less sensitive to fungicides which inhibit P450(14DM) activity, indicating that the cloned gene encodes a functional eburicol 14 alpha-demethylase.     

Structural and evolutionary studies on sterol 14-demethylase P450 (CYP51), the most conserved P450 monooxygenase: II. Evolutionary analysis of protein and gene structures

Phylogenetic analyses based on protein sequence data indicated that sterol 14-demethylase P450 (CYP51) and bacterial CYP51-like protein were joined into a distinctive evolutionary cluster, CYP51 cluster, within the CYP protein superfamily. The most probable branch topology of the CYP51 phylogenetic tree was (bacteria, (plants, (fungi, mammals))), which is comparable to the phylogeny of major kingdoms of living matter, suggesting that CYP51 has been conserved from the era of prokaryotic evolution. This may be strong evidence supporting the prokaryotic origin of P450. Structure of flanking regions and the number and insertion sites of introns are quite different between mammalian and fungal CYP51s. This fact indicates that different mechanisms are operative in evolution of protein sequences and gene structures. CYP51 is the first example violating the well-documented rule that the basic structure of a gene, including intron insertion sites, is well conserved in each P450 family. One CYP51 processed a pseudogene was found in rat genome. Nonsynonymous nucleotide divergence observed between the pseudogene and CYP51 cDNA was less than one-fifth of the synonymous divergence. This unusually low rate of nonsynonymous nucleotide changes in the pseudogene suggests that it may be derived from another CYP51, which might have been active for a significant duration in the past.     

Functional and DNA sequence divergence of the CYP71 gene family in higher plants

During ripening of avocado (Persea americana), the CYP71A1 mRNA and protein accumulate to relatively high levels. Although the CYP71A1 gene was the first plant P450 to be cloned and sequenced, the functional role of this P450 remains obscure. Substrate studies have shown that CYP71A1 will metabolize various monoterpenes (nerol and geraniol), although these have not been detected in ripening fruit. Using DNA from a conserved domain of the CYP71A1 gene, we have explored the scope of the CYP71 (or related) gene family in avocado using low stringency DNA hybridization. This analysis suggests that there are approximately 10-12 genes in the CYP71 family. An alternative approach using PCR gave essentially identical results.     

Germ line polymorphisms in cytochrome-P450 1A1 (C4887 CYP1A1) and methylenetetrahydrofolate reductase (MTHFR) genes and endometrial cancer susceptibility

We describe here a case-control study to identify associations between polymorphisms at the methylenetetrahydrofolate reductase (MTHFR) and cytochrome P-450 1A1 (CYP1A1) genes and susceptibility to endometrial cancer. Accordingly, genotype frequencies in 80 endometrial carcinoma patients were compared with frequencies in 60 controls. DNA analysis suggest a significantly increased endometrial cancer risk with an alanine to valine substitution at nucleotide 677 of MTHFR gene with an odds ratio of 2.8 (95% confidence interval: 1.36-6.14, P = 0.002). Moreover, the tumors from patients with the valine allele were more undifferentiated (P = 0.03). On the other hand, a recently described mutation in exon 7 of CYP1A1 gene (threonine exchanged to asparagine in codon 461) showed a strong association with endometrial cancer risk with an odds ratio of 6.36 (95% confidence interval: 1.99-26.5, P = 0.0004). Thus, this study suggests that polymorphisms at MTHFR and a novel CYP1A1 variant could influence susceptibility to endometrial cancer, although larger sample sizes would be required to corroborate these findings.     

With regard to "The role of air plethysmography in monitoring results of venous surgery"

By homology to the mgt gene (encoding a macrolide glycosyltransferase) from Streptomyces lividans, a 3.3-kb DNA fragment from the oleandomycin producer, Streptomyces antibioticus, was cloned and sequenced. Analysis of the sequence revealed the presence of the 3' end of a gene (ORF1) and two complete ORFs (ORF2 and oleD), all of them translationally coupled. The deduced product of the sequenced region of ORF1 contained the typical signature of integral membrane proteins responsible for the translocation of substrates across the membrane. The ORF2 product did not show significant similarity with proteins in databases, but contains an N-terminus leader peptide region characteristic of secreted proteins, and a lipid attachment site motif characteristic of membrane lipoproteins synthesized with a precursor signal peptide. The oleD product showed clear similarity with several UDP-glucuronosyl- and UDP-glycosyl-transferases from different origins and particularly with the mgt gene from S. lividans, and might encode a glycosyltransferase activity capable of inactivating macrolides. It is proposed that these three genes could participate in the intracellular glycosylation of oleandomycin and its secretion during antibiotic production.     

Spatially distinct expression of two new cytochrome P450s in leaves of Nepeta racemosa: identification of a trichome-specific isoform

Using a PCR-based approach, two novel cytochrome P450 cDNAs were isolated from a catmint (Nepeta racemosa) leaf cDNA library. The cDNAs (pBSK3C7 and pBSK4C3) were 76.9% identical in their nucleotide sequences, indicating that they are the products of two closely-related genes. A comparison of the sequence of these cDNAs with database sequences indicated that they represent new members of the CYP71 gene family of plant cytochrome P450s. Clone pBSK3C7 contains the full-length coding sequence of a cytochrome P450, whilst pBSK4C3 lacks ca. 6 codons at the 5' end. The cytochromes P450 encoded by these clones were designated CYP71A5 and CYP71A6 (pBSK3C7 and pBSK4C3, respectively). Southern blot analysis indicated that the corresponding genes were present as single copies in the genome of N. racemosa. Northern blot analysis showed that a gene homologous with CYP71A5 was expressed in the related species N. cataria, but no homologue of CYP71A6 was detected in this species. Expression of CYP71A5 in N. racemosa was maximal in flowers, tissues within the apical bud, and young expanded leaves. That of CYP71A6 was maximal in older leaves. Expression of CYP71A5 occurred exclusively in trichomes present on the leaf surfaces, in contrast to that of CYP71A6, which occurred predominantly within the leaf blade tissues.     

18-FDG imaging in breast cancer

A previously isolated parsley (Petroselinum crispum) cDNA with high sequence similarity to cinnamate 4-hydroxylase (C4H) cDNAs from several plant sources was expressed in yeast (Saccharomyces cerevisiae) containing a plant NADPH:cytochrome P450 oxidoreductase and verified as encoding a functional C4H (CYP73A10). Low genomic complexity and the occurrence of a single type of cDNA suggest the existence of only one C4H gene in parsley. The encoded mRNA and protein, in contrast to those of a functionally related NADPH:cytochrome P450 oxidoreductase, were strictly coregulated with phenylalanine ammonia-lyase mRNA and protein, respectively, as demonstrated by coinduction under various conditions and colocalization in situ in cross-sections from several different parsley tissues. These results support the hypothesis that the genes encoding the core reactions of phenylpropanoid metabolism form a tight regulatory unit.     

Rainbow trout cytochrome P450s: purification, molecular aspects, metabolic activity, induction and role in environmental monitoring

Cytochromes P450 (P450s or CYPs) constitute a superfamily of heme-thiolate proteins that play important roles in oxidative metabolism of endogenous and exogenous compounds. This review provides some limited history but addresses mainly the research progress on the cytochrome P450s in rainbow trout (Oncorhynchus mykiss), their purification, structures at the primary level, role in metabolism, responses to chemicals and environmental pollutants, application to biomonitoring and the effect of various factors on their expression or activities. Information obtained to date suggests that the rainbow trout P450 systems are as complex as those seen in mammals. Fourteen P450s have been purified from liver or trunk kidney to relatively high specific content. cDNAs belonging to seven different P450 families have been documented from trout liver, kidney and ovary. Two CYP1A genes, nine cDNAs containing open reading frames, and a cDNA fragment were entered into GenBank. Among them, CYP2K1, CYP2K3, CYP2K4, CYP2M1, CYP3A27 and CYP4T1 are the most recently described forms. CYP2K1, CYP2M1 and CYP4T1 represent newly identified P450 subfamilies first described in the rainbow trout. In many cases, the cloned rainbow trout P450s have subsequently been expressed in heterologous expressions systems such as COS-7 cells, yeast and baculovirus infected insect cells. Some of the overexpressed P450 isoforms have been partially characterized. Potential future research directions are discussed.     

ACTH receptor, CYP11A1, CYP17 and CYP21A2 genes are expressed in skin

Evidence is provided that mRNA for ACTH (MC-2) receptor and mRNAs for three obligatory enzymes of steroid synthesis including cytochromes P450scc, P450c17 and P450c21 are expressed in normal and pathologic human skin. Thus, molecular elements of the distal loop of the "pituitary-adrenal axis" such as the MC-2, CYP11A1, CYP17 and CYP21A2 genes are expressed in the skin.     

The TRI11 gene of Fusarium sporotrichioides encodes a cytochrome P-450 monooxygenase required for C-15 hydroxylation in trichothecene biosynthesis

Several genes in the trichothecene biosynthetic pathway of Fusarium sporotrichioides have been shown to reside in a gene cluster. Sequence analysis of a cloned DNA fragment located 3.8 kb downstream from TRI5 has led to the identification of the TRI11 gene. The nucleotide sequence of TRI11 predicts a polypeptide of 492 residues (Mr = 55,579) with significant similarity to members of the cytochrome P-450 superfamily. TRI11 is most similar to several fungal cytochromes P-450 (23 to 27% identity) but is sufficiently distinct to define a new cytochrome P-450 gene family, designated CYP65A1. Disruption of TRI11 results in an altered trichothecene production phenotype characterized by the accumulation of isotrichodermin, a trichothecene pathway intermediate. The evidence suggests that TRI11 encodes a C-15 hydroxylase involved in trichothecene biosynthesis.     

A general strategy for the expression of recombinant human cytochrome P450s in Escherichia coli using bacterial signal peptides: expression of CYP3A4, CYP2A6, and CYP2E1

Heterologous expression of unmodified recombinant human cytochrome P450 enzymes (P450s) in Escherichia coli has proved to be extremely difficult. To date, high-level expression has only been achieved after altering the 5'-end of the native cDNA, resulting in amino acid changes within the P450 protein chain. We have devised a strategy whereby unmodified P450s can be expressed to high levels in E. coli, by making NH2-terminal translational fusions to bacterial leader sequences. Using this approach, we initially tested two leader sequences, pelB and ompA, fused to CYP3A4. These were compared with an expression construct producing a conventional NH2-terminally modified CYP3A4 (17alpha-3A4). Both leader constructs produced spectrally active, functional protein. Furthermore, the ompA-3A4 fusion gave higher levels of expression, and a marked improvement in the recovery of active P450 in bacterial membrane fractions, when compared with 17alpha-3A4. We then tested the ompA leader with CYP2A6 and CYP2E1, again comparing with the conventional (17alpha-) approach. As before, the leader construct produced active enzyme, and, for CYP2E1 at least, gave a higher level of expression than the 17alpha-construct. The ompA fusion strategy thus appears to represent a significant advance for the expression of P450s in E. coli, circumventing the previous need for individual optimization of P450 sequences for expression.     

CYP86A1 from Arabidopsis thaliana encodes a cytochrome P450-dependent fatty acid omega-hydroxylase

The A. thaliana EST database was screened using consensus motifs derived from P450 families CYP52 and CYP4 catalyzing the omega-hydroxylation of fatty acids and alkanes in Candida and in mammals. One EST cDNA fragment was detected in this way and the corresponding full-length cDNA was cloned from a cDNA library of A. thaliana. This cDNA coded the first member of a new plant P450 family and was termed CYP86A1. The deduced peptide sequence showed highest homology with P450s from families 4 and 52. To confirm the catalytic function, CYP86A1 was expressed in a yeast overexpressing its own NADPH-P450 reductase. Efficient expression was evidenced by spectrophotometry, SDS-PAGE and catalytic activity. CYP86A1 was found to catalyze the omega-hydroxylation of saturated and unsaturated fatty acids with chain lengths from C12 to C18 but not of hexadecane. Genomic organization analyzed by Southern blot suggested a single gene encoding CYP86A1 in A. thaliana.     

Second-site suppressors of the bacteriophage P1 virs mutant reveal the interdependence of the c4, icd, and ant genes in the P1 immI operon

The immI operon of phage P1 contains the genes c4, icd, and ant, which are transcribed in that order from the same constitutive promoter, P51b. The gene c4 encodes an antisense RNA which inhibits the synthesis of an antirepressor by acting on a target ant mRNA. Interaction depends on the complementarity of two pairs of short sequences encompassing virs+ and the ribosome-binding site involved in ant expression. Accordingly, in a P1 virs mutant phage, antirepressor is synthesized constitutively. We have isolated lysogen-proficient, second-site suppressors of P1 virs in order to evaluate the interdependence of the immI-specific genes. From a total of 17 suppressors analyzed, 15 were found to be located in the icd gene. They were identified as frameshift mutations, containing base insertions or deletions in tandem repeats of a single base pair. One suppressor was identified as a P51b promoter-down mutation; the second site of another suppressor was found to be located in the c4 gene. Furthermore, it was shown that virs cannot be suppressed by ant (icd+) suppressors. The results confirm the model that the immI operon is transcribed as a unit, that the icd and ant genes are translationally coupled, and that the constitutive synthesis of Icd protein alone is lethal to the bacterial cell. The existence of a c4 suppressor of virs, whose effect is not yet known, points to a still more complex regulation of antirepressor synthesis than was anticipated from the model.     

Identification of stsC, the gene encoding the L-glutamine:scyllo-inosose aminotransferase from streptomycin-producing Streptomycetes

Eight new genes, strO-stsABCDEFG, were identified by sequencing DNA in the gene cluster that encodes proteins for streptomycin production of Streptomyces griseus N2-3-11. The StsA (calculated molecular mass 43.5 kDa) and StsC (45.5 kDa) proteins - together with another gene product, StrS (39.8 kDa), encoded in another operon of the same gene cluster - show significant sequence identity and are members of a new class of pyridoxal-phosphate-dependent aminotransferases that have been observed mainly in the biosynthetic pathways for secondary metabolites. The aminotransferase activity was demonstrated for the first time by identification of the overproduced and purified StsC protein as the L-glutamine:scyllo-inosose aminotransferase, which catalyzes the first amino transfer in the biosynthesis of the streptidine subunit of streptomycin. The stsC and stsA genes each hybridized specifically to distinct fragments in the genomic DNA of most actinomycetes tested that produce diaminocyclitolaminoglycosides. In contrast, only stsC, but not stsA, hybridized to the DNA of Streptomyces hygroscopicus ssp. glebosus, which produces the monoaminocyclitol antibiotic bluensomycin; this suggests that both genes are specifically used in the first and second steps of the cyclitol transamination reactions. Sequence comparison studies performed with the deduced polypeptides of the genes adjacent to stsC suggest that the enzymes encoded by some of these genes [strO (putative phosphatase gene), stsB (putative oxidoreductase gene), and stsE (putative phosphotransferase gene)] also could be involved in (di-)aminocyclitol synthesis.