A single amino acid substitution converts cytochrome P45014DM to an inactive form, cytochrome P450SG1: Complete primary structures deduced from cloned DNAs

Ishida, Nobuhiro; Aoyama, Yuri; Hatanaka, Ritsuko; Oyama, Yoshiaki; Imajo, Seiichi; Ishiguro, Masaji; Oshima, Takao; Nakazato, Hiroshi; Noguchi, Teruhisa; Maitra, Utpalendu S.; Mohan, V P; Sprinson, David B.; Yoshida, Yuzo

doi:10.1016/s0006-291x(88)81087-8

Cited by 64 publications

(32 citation statements)

References 20 publications

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“…Characterization of genes encoding CYP51 will contribute to better understanding of azole resistance mechanisms at the molecular level. The cyp51 genes from Saccharomyces cerevisiae, Candida tropicalis, Candida albicans, Candida glabrata, Cryptococcus neoformans, Ustilago maydis, Aspergillus nidulans, Botrytis cinerea, Penicillium italicum, Uncinula necator, and Erysiphe graminis have been cloned, and some of the proteins have been characterized to some extent (3,8,9,14,16,19,39). The availability of their sequences has facilitated the design of degenerate primers to amplify fragment homologues of fungal cyp51 in Aspergillus species.…”

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confidence: 99%

Identification of Two Different 14-α Sterol Demethylase-Related Genes ( cyp51A and cyp51B ) in Aspergillus fumigatus and Other Aspergillus species

et al. 2001

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Two cyp51-related genes (cyp51A and cyp51B) encoding 14-␣ sterol demethylase-like enzymes were identified in the opportunistic human pathogen Aspergillus fumigatus. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in A. fumigatus. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis, and Candida glabrata showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other Aspergillus spp. (A. flavus, A. nidulans, and A. terreus). Phylogenetic analysis showed that, at least in the most pathogenic species of Aspergillus, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-␣ sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-␣ sterol demethylase genes in other filamentous fungi.

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confidence: 99%

Identification of Two Different 14-α Sterol Demethylase-Related Genes ( cyp51A and cyp51B ) in Aspergillus fumigatus and Other Aspergillus species

et al. 2001

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“…The most widely used computational method for modeling in the cytochrome P450 field is homology modeling based on crystallized bacterial P450s. The initial models of the active site for CYP51 from Saccharomyces cerevisiae and an inactive mutant (Ishida et al, 1988) were constructed using alignment with P450cam. By using these, novel substrate analogs were developed as competitive inhibitors for this enzyme (Tuck et al, 1991).…”

Section: Discussionmentioning

confidence: 99%

“…However, because of the membrane-bound nature of mammalian P450s, there is as yet no crystal structure for human CYP51. Therefore, our understanding of the structural requirements of the human CYP51 active site are limited to homology models constructed using various bacterial P450s (Ishida et al, 1988;Tuck et al, 1991Tuck et al, , 1992Tafi et al, 1996;Talele et al, 1997;Holtje and Fattorusso, 1998;Lewis et al, 1999), including Mycobacterium tuberculosis CYP51 (Matsuura et al, 2005;Rupp et al, 2005) and the crystal structure of M. tuberculosis CYP51 (Podust et al, 2001). …”

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Three-Dimensional Quantitative Structure-Activity Relationship Analysis of Human CYP51 Inhibitors

Ekins¹,

Mankowski²,

Hoover³

et al. 2006

Drug Metab Dispos

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ABSTRACT:CYP51 fulfills an essential requirement for all cells, by catalyzing three sequential mono-oxidations within the cholesterol biosynthesis cascade. Inhibition of fungal CYP51 is used as a therapy for treating fungal infections, whereas inhibition of human CYP51 has been considered as a pharmacological approach to treat dyslipidemia and some forms of cancer. This study has demonstrated the potential for ligand-based computational pharmacophore modeling of human CYP51 and enables a high-throughput screening system for drug discovery and data base mining.

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“…Only a few plant Cyt P-450 enzymes have been cloned (Bozak et al, 1990;Meijer et al, 1993). In a mutant strain of Saccharomyces cerevisiae, inactivation of a Cyt P-450 involved in demethylation of lanosterol was found to be due to substitution of a single amino acid (Ishida et al, 1988). Since EMS treatment causes mainly point mutations (Haughn and Somerville, 1987), it is possible that a putative BPT-demethylase also was inactivated by an amino acid substitution resulting from a point mutation.…”

Section: Discussionmentioning

confidence: 99%

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta

Jacobs

Arroo²,

Koning³

et al. 1995

Plant Physiol.

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Two mutants of Tagetes erecfa displaying aberrant thiophene composition were identified by screening more than 300 plants from a mutagenized M, population using high-performance liquid chromatography analysis of root extracts. Both mutants, which may have originated from the same mutational event, contained high amounts of the C,, monothiophene 2-(but-3-en-I -ynyl)-5-(penta-1,3-diynyl)-thiophene that was previously not found in T. erecfa and also high amounts of two C,, bithienyls that were absent or present at low concentrations in the wild type. The mutant phenotype was also expressed in 21 Agrobacterium rhizogenes transformed root clones derived from both mutants. Feeding experiments with root cultures derived from one mutant and from the wild type indicated that the monothiophene accumulating in the mutant is the common precursor for all bithienyl thiophenes in wild-type and mutant Tagefes erecfa. These experiments also showed that one mutant is deficient in demethylation of the monothiophene.Although mutants have been widely applied to investigate metabolic pathways in microorganisms and fungi, they have been used relatively rarely for this purpose in plants. Most metabolic mutants of plants affect primary metabolic processes, such as carbon assimilation (Somerville and Ogren, 1979) and metabolism of starch (Caspar et al., 1985), lipids (Browse et al., 1985;Kunst et al., 1989), and amino acids (Haughn and Somerville, 1986;Kreps and Town, 1991;Frankard et al., 1992;Wright et al., 1992;Wu and King, 1994), whereas only a few mutants of secondary metabolic pathways are known (Haughn et al., 1991;Chapple et al., 1992; Rathjen and Robinson, 1992). This situation exists in spite of the fact that chemically induced loss-of-function mutations can be obtained relatively easily (Haughn and Somerville, 1987).Tugetes species (marigolds) produce, mainly in the roots, aromatic sulfur-containing compounds known as thiophenes, which are toxic to nematodes when ingested Bijloo, 1958, 1959). Co-occurrence patterns and precursor-feeding experiments in related species have led to a generally accepted biogenetic scheme, as depicted in Figure 1 (Bohlmann et al., 1973;Bohlmann and Zdero, 1985). Oleic acid is converted into PYE via repeated steps of desaturation and chain shortening. PYE is then converted into the thiophenes that accumulate in Tagetes * Corresponding author; fax 31-80-553450.species. (Bohlmann and Berger, 1965;Bohlmann and Hinz, 1965;Schulte et al., 1965;Bohlmann et al., 1966). The key step in this conversion is the addition of H,S or its biochemical equivalent to conjugated triple bonds and subsequent ring formation, which is probably a two-step reaction (Bohlmann et al., 1973). In addition to the formation of two or three thiophene rings, remova1 of a terminal methyl group and modification of a vinyl group are necessary to obtain the various thiophenes that finally accumulate (Fig. 1).Not much is known about the order in which these reactions take place in Tugetes or about the specificity of the enzymes involve...

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A single amino acid substitution converts cytochrome P45014DM to an inactive form, cytochrome P450SG1: Complete primary structures deduced from cloned DNAs

Cited by 64 publications

References 20 publications

Identification of Two Different 14-α Sterol Demethylase-Related Genes ( cyp51A and cyp51B ) in Aspergillus fumigatus and Other Aspergillus species

Identification of Two Different 14-α Sterol Demethylase-Related Genes ( cyp51A and cyp51B ) in Aspergillus fumigatus and Other Aspergillus species

Three-Dimensional Quantitative Structure-Activity Relationship Analysis of Human CYP51 Inhibitors

Isolation and Characterization of Mutants of Thiophene Synthesis in Tagetes erecta

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