Disease relevance of NCP1

• Native yeast NADPH-cytochrome P450 oxidoreductase (CPR; EC 1.6.2.4) and a soluble derivative lacking 33 amino acids of the NH(2)-terminus have been overexpressed as recombinant proteins in Escherichia coli [1]
• We isolated cDNA (pgCYR, about 2.1 kb) and genomic DNA (pgGYR, about 4 kb) clones coding for NADPH-cytochrome P450 reductase by immunoscreening of yeast Saccharomyces cerevisiae cDNA and genomic DNA libraries in phage lambda gt11 [2].

High impact information on NCP1

• Phenylalanine ammonia lyase (PAL), cinnamate 4-hydroxylase (C4H), and the C4H redox partner cytochrome p450 reductase (CPR) are important in allocating significant amounts of carbon from phenylalanine into phenylpropanoid biosynthesis in plants [3].
• Purification of the cytosolic mutant CPR indicated properties identical to native CPR and an ability to reconstitute ergosterol biosynthesis when added to a cell-free system, as well as to allow reconstitution of activity with purified CYP61, sterol 22-desaturase [4].
• The disruption of Saccharomyces cerevisiae NADPH- cytochrome P450 oxidoreductase (CPR) gene resulted in a viable strain accumulating approximately 25% of the ergosterol observed in a sterol wild-type parent [4].
• The associated phenotypes could be reversed in transformants after expression of native CPR and a mutant lacking the N-terminal 33 amino acids, which localized in the cytosol [4].
• Purified recombinant cytochrome P450 52A3 and the corresponding NADPH-cytochrome P450 reductase from the alkane-assimilating yeast Candida maltosa were reconstituted into an active alkane monooxygenase system [5].

Biological context of NCP1

• This alternative catalytic cycle, where both the first and second electrons were donated via the NADH cytochrome b5 electron transport system, can account for the continued ergosterol production seen in yeast strains containing a disruption of the gene encoding CPR [6].
• A second FMN binding site in yeast NADPH-cytochrome P450 reductase suggests a mechanism of electron transfer by diflavin reductases [7].
• Two NADPH-cytochrome P450 reductase-encoding cDNAs were isolated from an Arabidopsis cDNA library by metabolic interference in a Saccharomyces cerevisiae mutant disrupted for its endogenous cpr1 gene [8].
• A cDNA coding for human oxidoreductase (NADPH-cytochrome P450 reductase) was expressed in S. cerevisiae on a high copy plasmid under control of a constitutive promoter [9].
• 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 [10].

Anatomical context of NCP1

• This enzyme activity is supported by an associated NAPDH-dependent reductase encoded by NCPR1 (NCP1), which is also associated with the endoplasmic reticulum [11].
• Cytochrome P-450 reductase (encoded by the NCP1 gene) was found to catalyse all the NADPH-dependent ferrireductase activities associated with isolated plasma membranes of the yeast Saccharomyces cerevisiae [12].
• NADPH-cytochrome P-450 reductase of yeast microsomes [13].

Associations of NCP1 with chemical compounds

• 32-Oxo-24,25-dihydrolanosterol (32-oxo-DHL) was deformylated to 4,4-dimethylcholesta-8,14-dien-3 beta-ol, the product of 14 alpha-demethylation of 24,25-dihydro-lanosterol (DHL), by the reconstituted lanosterol 14 alpha-demethylase system consisting of cytochrome P-45014DM and NADPH-cytochrome P-450 reductase of yeast [14].
• Overexpression of CYP51 and FUS produced different levels of ketoconazole resistance in wild-type cells, indicating that the availability of CPR may limit the potential of overproduction of CYP51 as a mechanism of resistance to azole antifungal agents [15].
• 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 [10].
• Selection for azole resistance following expression of a cDNA library constructed with RNA from progesterone-treated R. nigricans in the yeast Saccharomyces cerevisiae led to the identification of CPR1-FL and CYB5-1 cDNAs, which code for functionally competent NADPH-cytochrome P450 reductase and cytochrome b5, respectively [16].
• Enhanced production of ethylene from methional by iron chelates and heme containing proteins in the system consisting of quinone compounds and NADPH-cytochrome P-450 reductase [17].

Other interactions of NCP1

• Furthermore, reconstitution of the soluble enzyme with soluble yeast Ncpr1p, expressed and purified as an N-terminal deletion of 33 amino acids encompassing its membrane anchor, resulted in a fully functional and soluble eukaryotic Erg11p system [11].
• In contrast, cells that overexpressed FRE2 had maximal ferrireductase activity when NCP1 was repressed [12].
• Overexpression of FRE1 did not lead to an increased ferrireductase activity of the cells when NCP1 was repressed [12].
• Transcripts for the three poplar CPR genes were present ubiquitously in all tissues examined, but CPR2 showed highest expression in young leaf tissue [18].
• The divergent CPR isoforms (CPR1 and CPR2/3) contained entirely different N-terminal sequences, which are conserved in other plant CPRs and are diagnostic for two distinct classes of CPRs within the angiosperms [18].

Analytical, diagnostic and therapeutic context of NCP1

• Neither Southern hybridization nor Western immunoblot techniques provided evidence for a second NADPH-cytochrome P450 reductase gene, suggesting that an alternate pathway may provide for the functions of this reductase in S. cerevisiae [19].
• Probing the role of lysines and arginines in the catalytic function of cytochrome P450d by site-directed mutagenesis. Interaction with NADPH-cytochrome P450 reductase [20].
• The sequences encoding human liver cytochrome b5, the native and a modified form of the yeast NADPH-cytochrome P-450 reductase were cloned by making use of PCR techniques, over-expressed in yeast as functional forms, and characterized [21].

References