Disease relevance of POR

• The two functional domains of a cloned human fibroblast NADPH-cytochrome P450 reductase have been expressed in Escherichia coli and purified on the milligram scale for crystallization studies [1].
• Establishment of ten strains of genetically engineered Salmonella typhimurium TA1538 each co-expressing a form of human cytochrome P450 with NADPH-cytochrome P450 reductase sensitive to various promutagens [2].
• The reductive activation of the antitumor drug RH1 to its semiquinone free radical by NADPH cytochrome P450 reductase and by HCT116 human colon cancer cells [3].
• Enzyme kinetics parameters V(max) and apparent K(m), determined using pooled human liver microsomes and microsomes from baculovirus cells coexpressing human CYP2D6 and NADPH-cytochrome P450 reductase, were 245 pmol [(14)C]HCHO/min/mg protein and 11 microM, and 27 pmol [(14)C]HCHO/min/pmol and 1.6 microM, respectively [4].
• In this study, the involvement of NADPH-cytochrome P-450 reductase in the conversion of FNTZ to 7A FNTZ was investigated in two human hepatoma cell lines, human lymphoblast microsomes specifically expressing human NADPH-cytochrome P-450 reductase and purified recombinant human HADPH-cytochrome P-450 reductase [5].

Psychiatry related information on POR

• After adjusting for potential confounders (history of gastrointestinal diseases, smoking, alcohol consumption) the strong association remained (POR: 6.7; 2.7-19.2) [6].
• Pregnant women with panic disorders had a shorter (0.4 week) gestational age (adjusted t = 2.3; p = 0.02) and a larger proportion of preterm births (17.1% versus 9.1%) (adjusted POR with 95% CI = 1.9, 1.3-2.8) [7].

High impact information on POR

• With these key residues as a guide, conclusive evidence is presented that the ferredoxin reductase structure is a prototype for the nicotinamide dinucleotide and FAD binding domains of the enzymes NADPH-cytochrome P450 reductase, NADPH-sulfite reductase, NADH-cytochrome b5 reductase, and NADH-nitrate reductase [8].
• In 11 patients with decompensated cirrhosis and deteriorating renal function, the effect of the vasoconstrictor substance 8-ornithin vasopressin (ornipressin; POR 8; Sandoz, Basel, Switzerland) on renal function, hemodynamic parameters, and humoral mediators was studied [9].
• The key enzyme of chlorophyll biosynthesis in higher plants, the light-dependent NADPH:protochlorophyllide oxidoreductase (POR, EC 1.6.99.1), is a nuclear-encoded plastid protein [10].
• Our large survey of patients with ABS shows that individuals with an ABS-like phenotype and normal steroidogenesis have FGFR mutations, whereas those with ambiguous genitalia and disordered steroidogenesis should be recognized as having a distinct new disease: POR deficiency [11].
• P450 oxidoreductase (POR) is the obligatory flavoprotein intermediate that transfers electrons from reduced nicotinamide adenine dinucleotide phosphate (NADPH) to all microsomal cytochrome P450 enzymes [11].

Chemical compound and disease context of POR

• An homology model of protochlorophyllide reductase (POR) from Synechocystis sp. was constructed on a template from the tyrosine-dependent oxidoreductase family [12].
• This advance in diagnostic ability has introduced the concept of conservative management for this condition.The authors describe the conservative management of nine cases of ectopic pregnancy using intrachorionic injection of either methotrexate alone or in combination with POR 8 [13].
• Helicobacter pylori flavodoxin is the electronic acceptor of the pyruvate-oxidoreductase complex (POR) that catalyzes pyruvate oxidative decarboxilation [14].
• Local infiltration of ornithine 8-vasopressin (POR 8) as a vasoconstrictive agent in surgical pelviscopy (applied to myoma enucleation, salpingotomy in cases of tubal pregnancy and peripheral salpingostomy) [15].
• NADPH:protochlorophyllide oxidoreductase (POR) catalyzes hydrogen transfer from NADPH to protochlorophyllide (PChlide) in the course of chlorophyll biosynthesis in photosynthetic organisms and is involved in the regulation of the development of photosynthetic apparatus in higher plants, algae and cyanobacteria [16].

Biological context of POR

• To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed [17].
• A combination of calorimetry and stopped-flow spectrophotometry kinetics experiments showed that this linkage between coenzyme binding energetics and diffusional domain motion impinges directly on the molecular recognition of cytochrome c by CPR [18].
• S. cerevisiae BWG1-7alpha transformed with the expression plasmids produced the respective proteins in the expected molecular sizes reactive with both anti-hCYP3A4 immunoglobulin (Ig) and anti-hCPR Ig [19].
• BWG-CPR was further tested daily by the PCR amplification of hCPR of yeast genome, Western blot analysis, and the activity assay of hCPR of yeast microsome [19].
• Cytochrome P450s (CYPs) hold a balance in studying pharmacokinetics, toxico-kinetics, drug metabolism, and drug-drug interactions, which require association with cytochrome P450 reductase (CPR) to achieve optimal activity [19].

Anatomical context of POR

• Numerous mutations/polymorphisms of the POR gene, encoding NADPH:cytochrome P450 oxidoreductase (CYPOR), have been described in patients with Antley-Bixler syndrome (ABS), presenting with craniofacial dysmorphogenesis, and/or disordered steroidogenesis, exhibiting ambiguous genitalia [17].
• Here we report that FAC binds to NADPH cytochrome-P450 reductase (RED), a microsomal membrane protein involved in electron transfer, in both transfected COS-1 and normal murine liver cells [20].
• NADPH-cytochrome P-450 reductase in the plasma membrane modulates the activation of hypoxia-inducible factor 1 [21].
• Organization of multiple cytochrome P450s with NADPH-cytochrome P450 reductase in membranes [22].
• In cancer cell lines, MMC cytotoxicity is correlated with DTD and P450R expression levels [23].

Associations of POR with chemical compounds

• Based on these findings, decreased FAD-binding affinity is proposed as the basis of the observed loss of CYPOR function in the Y459H and V492E POR mutations in ABS [17].
• Thus, the final electron for reducing ferric biliverdin-iron chelate to release ferrous iron and biliverdin is apparently provided by the FMN of CPR [24].
• This conclusion was supported by the observation that verdoheme dimethyl esters were accumulated in the reaction of the ferriprotoporphyrin IX dimethyl ester-rHO-1 complex with the wild-type CPR [24].
• CYP4A4-catalyzed omega-hydroxylation of prostaglandin E(1) was supported by WT CYPOR but not by either of the ABS mutants [17].
• Ferric biliverdin-iron chelate, generated with the FMN-depleted CPR, was converted to biliverdin by the addition of the wild-type CPR or desferrioxamine [24].

Physical interactions of POR

• The Reactions of Heme- and Verdoheme-Heme Oxygenase-1 Complexes with FMN-depleted NADPH-cytochrome P450 Reductase: ELECTRONS REQUIRED FOR VERDOHEME OXIDATION CAN BE TRANSFERRED THROUGH A PATHWAY NOT INVOLVING FMN [24].
• The concentration dependence of the rate of hydride transfer indicates that the second, noncatalytic NADPH-binding site present in wild-type CPR is retained in the mutant enzymes [25].
• A chimera consisting of the nNOS heme domain and FMN binding subdomain and the CYPOR FAD binding subdomain catalyzed significantly increased rates of cytochrome c reduction in the absence of CaM and of NO synthesis in its presence [26].

Enzymatic interactions of POR

• Human heme oxygenase-1 (hHO-1) catalyzes the NADPH-cytochrome P450 reductase-dependent oxidation of heme to biliverdin, CO, and free iron [27].
• The chlorophyll biosynthesis enzyme protochlorophyllide reductase (POR) catalyzes the light-dependent reduction of protochlorophyllide (Pchlide) into chlorophyllide in the presence of NADPH [28].

Regulatory relationships of POR

• We propose that Tyr275 and Lys279 participate in the proper coordination of NADPH and PChlide in the enzyme catalytic site and thereby control the efficiency of the formation of the POR photoactive state [29].
• Recombinant P450 3A7 coexpressed in bacterial membranes with NADPH-cytochrome P450 reductase showed similar levels of activity toward erythromycin (N-demethylation) and ethylmorphine (N-demethylation) to P450 3A4 and 3A5 expressed in the same system, whereas 3A7 was less active toward midazolam (1'- and 4-hydroxylation) and nifedipine (oxidation) [30].
• Microsomes containing recombinant CYP3A4-OR from these cell lines were up to 50-times more active in testosterone 6 beta-hydroxylase activity than recombinant CYP3A4 expressed alone and supplemented with purified rabbit CYPOR [31].
• The two novel CYP2J2 variants were co-expressed with NADPH-cytochrome P450 reductase in Sf9 cells and their catalytic activities were quantified [32].
• The CYP1A2-dependent metabolism of the triazine derivatives ametryne and terbutryne was moderately enhanced by increased CYPOR activity [33].

Other interactions of POR

• CYPOR is the obligate electron donor to 51 microsomal cytochromes P450 that catalyze critical steroidogenic and xenobiotic reactions, and to two heme oxygenase isoforms, among other redox partners [17].
• We speculate that Adx might serve as an alternative electron donor for CYP17 at the conditions of CPR deficiency in human [34].
• Abnormal microsomal detoxification implicated in Fanconi anemia group C by interaction of the FAC protein with NADPH cytochrome P450 reductase [20].
• A Salmonella typhimurium tester strain YG7108 2E1/OR co-expressing human CYP2E1 together with human NADPH-cytochrome P450 reductase (OR) was established [35].
• Metabolic activation of N-alkylnitrosamines in genetically engineered Salmonella typhimurium expressing CYP2E1 or CYP2A6 together with human NADPH-cytochrome P450 reductase [35].

Analytical, diagnostic and therapeutic context of POR

• The thermodynamics of coenzyme binding to human cytochrome P450 reductase (CPR) and its isolated FAD-binding domain have been studied by isothermal titration calorimetry [18].
• cDNAs for human cytochrome P450 2E1 and rat NADPH-cytochrome-P450 reductase were cloned separately and in tandem into bacterial expression vectors, and expression of the two proteins in Escherichia coli was monitored by immunoblotting, spectroscopy, and catalytic assays [36].
• The activity of NADPH-cytochrome P450 reductase decreased (by 24%) after 6 and 12 weeks, whereas after 24 weeks it remained unchanged, compared with the control group [37].
• Moreover, the presence of a chlorophyll-like molecule allows the use of EPR, electron nuclear double resonance, and Stark spectroscopies, previously used for the analysis of photosynthetic systems, to follow catalytic events in the active site of POR [38].
• Site-directed mutagenesis of the highly conserved Tyr275 (Y275F) and Lys279 (K279I and K279R) residues in the enzyme catalytic pocket demonstrated that the presence of these two amino acids in the wild-type POR considerably increases the probability of photoactive state formation following cofactor and substrate binding by the enzyme [29].

References