All microsomal P450s require POR (cytochrome P450 reductase) for catalytic activity. Most of the clinically used drugs are metabolized by a small number of P450s and polymorphisms in the cytochrome P450s are known to cause changes in drug metabolism. We have recently found a number of POR missense mutations in the patients with disordered steroidogenesis. Our initial report described five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in four patients. We built bacterial expression vectors for each POR variant, purified the membranes expressing normal or variant POR and characterized their activities with cytochrome c and P450c17 assays. We have recently completed an extensive study of the range of POR mutations and characterized the mutants/polymorphisms A112V, T139A, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del from sequencing of patient DNA. We also studied POR variants Y179D, P225L, R313W, G410S and G501R that were available in databases or the published literature. We analysed the mutations with a three-dimensional model of human POR that was based on an essentially similar rat POR with known crystal structure. The missense mutations found in patients with disordered steroidogenesis mapped to functionally important domains of POR and the apparent polymorphisms mapped to less crucial regions. Since a variation in POR can alter the activity of all microsomal P450s, it can also affect the drug metabolism even with a normal P450. Understanding the genetic and biochemical basis of POR-mediated drug metabolism will provide valuable information about possible differences in P450-mediated reactions among the individuals carrying a variant or polymorphic form of POR.

Introduction

There is a single copy of 50 kb POR (cytochrome P450 reductase) gene (NCBI accession no. NM_000941.2) in humans on chromosome 7 (7q11.23) and it consists of 16 exons and the exons 2–16 code for a 677-amino-acid protein (NCBI accession no. NP_000932.2). POR was initially identified by Horecker in 1950 [1] as cytochrome c reductase. Later studies by Williams and Kamin [1a] and Phillips and Langdon [2] showed that this flavoprotein was found in endoplasmic reticulum (microsomes), and although cytochrome c, a mitochondrial protein, was later found to be not the natural substrate of POR, it is still used to assay the POR activity. Subsequent studies in 1960s and 1970s linked POR to the newly discovered microsomal electron transport chains, cytochromes P450 and b5 involved in drug and steroid hydroxylations [3]. In addition to P450s, POR also supplies electrons to haem oxygenase, fatty acid desaturase and elongase, squalene mono-oxygenase, cytochrome b5 and sterol reductase. As all of these microsomal enzymes require POR for catalysis, it is expected that disruption of POR would have devastating consequences. POR knockout mice are embryonic lethal [4,5] probably due to the lack of electron transport to extrahepatic P450 enzymes, since liver-specific knockout of POR yields phenotypically and reproductively normal mice that accumulate hepatic lipids and have remarkably diminished capacity of hepatic drug metabolism [6,7]. Similarly, hypomorphic POR mice with 74–95% reduction in POR expression have reduced hepatic drug metabolism [8].

We had initially identified five missense mutations and a splicing mutation in the POR genes of four patients who had hormonal evidence for combined deficiencies of two steroidogenic cytochrome P450 enzymes: P450c17, which catalyses steroid 17α-hydroxylation and 17,20 lyase reaction and P450c21, which catalyses steroid 21-hydroxylation [911]. In a follow-up study, we have examined the POR genes in 32 additional patients [12]. Fifteen of 19 patients having abnormal genitalia and disordered steroidogenesis were homozygous or apparent compound heterozygous for POR mutations that destroyed or dramatically inhibited POR activity. The amino acid numbers in the protein are based on NCBI NP_000932.2 and correspond to the full-length, 677-amino-acid human POR sequence. The original reports describing POR mutations were based on NCBI accession number NP_000932.1, which was edited on 14 May 2006. Therefore all numbering has now been edited to correspond to the current database reference.

Population distribution of A284P and R454H mutations

Most missense mutations were found once, but A284P was found on ten alleles, and R454H was found on seven alleles. These two missense mutations accounted for 17 of 34 (50%) of the identified POR missense mutations. The A287P mutation was found only in samples from subjects described as ‘white’, ‘Caucasian’, or ‘European’. This same mutation was also found on two (of eight) alleles in our initial report [9] in individuals of European heritage. Thus A284P is a frequent mutation causing POR deficiency in this group. The R454H mutation was found in four of eight alleles from Japanese patients. This same mutation was also found on one (of two) Japanese allele in our initial report [9] and on two of four alleles [13] and ten of 16 alleles [14] in two subsequent reports of Japanese patients. Thus R457H is strongly associated with alleles of Japanese heritage.

Enzymatic activities of POR variants

Our original report described the kinetics of the five missense mutations (A284P, R454H, V489E, C566Y and V605F) identified in our initial four patients [9]. The follow-up study added the mutants or polymorphisms A112V, T139A, Q150R, M260V, Y456H, A500V, G536R, L562P, R613X, V628I and F643del [12]. In addition, we have found the mutants or polymorphisms P225L, R313W, G410S and G501R from the Bioventures POR sequencing results (http://www.bioventures.com). We built a three-dimensional model of the human POR structure based on the 95% identical structure of rat POR [15]. In general, the missense mutations found in patients mapped to functionally important domains of POR and the polymorphisms appear in less important locations. To study the enzymology of POR variants, we expressed human POR variants in bacteria in a form lacking 24 N-terminal residues (N-24 POR) [16]. Table 1 shows the Km, Vmax and kcat for the capacity of the wild-type and the variants of POR for the reduction of cytochrome c and the oxidation of NADPH. POR variants P225L, R313W, G410S, A500V and G501R, which were polymorphisms identified from databases, retained 47–100% activity in both assays, but the apparent polymorphism A112V, found in a Beare-Stevenson patient with an FGFR3 (fibroblast growth factor receptor 3) mutation, had less than 40–60% of activity, and M260V and V628I, found as heterozygotes in the sequencing of other individuals, had 23–76% of wild-type activity.

Table 1
Kinetics of POR variants using cytochrome c as the substrate

Km values are in μM (cytochrome c or NADPH); Vmax values are in nmol of cytochrome c reduced/mg of POR protein per min. kcat values were calculated based on the molar content of POR and hence the values are in min−1. WT, wild-type. Taken from Huang, N., Pandey, A.V., Agrawal, V., Reardon, W., Lapunzina, P.D., Mowat, D., Jabs, E.W., Van Vliet, G., Sack, J., Flück, C.E. and Miller, W.L. (2005) Diversity and function of mutations in P450 oxidoreductase in patients with Antley–Bixler syndrome and disordered steroidogenesis. Am. J. Hum. Genet. 76, 729–749 [12] with permission of the University of Chicago Press. © 2005 by The American Society of Human Genetics.

 Cytochrome c variable NADPH variable 
POR variant Km Vmax kcat Km Vmax kcat 
WT 1.4±0.3 91.2±5.1 388 0.22±0.05 68.4±2.9 292 
A112V 2.0±0.4 82.5±4.3 350 0.29±0.04 37.2±5.9 158 
T139A 1.6±0.2 51.3±6.3 218 0.23±0.03 41.7±4.6 178 
Q150R 3.7±0.4 21.4±5.7 90 1.2±0.09 43.2±3.1 184 
Y178D >50 − − >50 − − 
P225L 1.4±0.3 69.2±5.2 294 0.24±0.06 53.7±3.8 229 
M260V 1.9±0.2 94.5±3.7 402 0.39±0.07 69.1±2.4 294 
A284P 5.9±0.8 37.3±6.4 158 0.71±0.10 35.1±4.3 150 
R313W 1.9±0.4 75.9±8.3 322 0.29±0.07 69.3±9.7 294 
G410S 1.3±0.2 64.5±7.9 274 0.20±0.05 61.7±2.1 262 
R454H 17.2±2.5 7.5±2.1 32 >50 − − 
Y456H 22.9±6.6 6.1±0.7 26 >50 − − 
V489E 29.7±5.1 6.8±1.1 28 >50 − − 
A500V 1.8±0.2 81.3±6.6 344 0.21±0.06 55.7±1.9 238 
G501R 2.1±0.2 72.9±4.1 312 0.35±0.06 51.2±3.9 218 
G536R 6.3±1.2 37.8±6.7 160 18.3±2.1 9.3±1.2 40 
L562P 2.8±0.6 26.2±3.2 112 5.3±1.6 23.8±2.3 102 
C566Y 5.8±0.2 24.3±2.7 104 1.8±0.43 12.1±0.7 52 
V605F 5.2±1.3 26.3±5.2 112 3.3±0.7 32.7±0.6 140 
R616X >50 − − >50 − − 
V628I 1.6±0.4 77.3±8.6 328 0.47±0.06 32.8±5.3 140 
F643del 3.9±0.7 90.7±5.7 384 0.21±0.13 61.2±8.5 260 
 Cytochrome c variable NADPH variable 
POR variant Km Vmax kcat Km Vmax kcat 
WT 1.4±0.3 91.2±5.1 388 0.22±0.05 68.4±2.9 292 
A112V 2.0±0.4 82.5±4.3 350 0.29±0.04 37.2±5.9 158 
T139A 1.6±0.2 51.3±6.3 218 0.23±0.03 41.7±4.6 178 
Q150R 3.7±0.4 21.4±5.7 90 1.2±0.09 43.2±3.1 184 
Y178D >50 − − >50 − − 
P225L 1.4±0.3 69.2±5.2 294 0.24±0.06 53.7±3.8 229 
M260V 1.9±0.2 94.5±3.7 402 0.39±0.07 69.1±2.4 294 
A284P 5.9±0.8 37.3±6.4 158 0.71±0.10 35.1±4.3 150 
R313W 1.9±0.4 75.9±8.3 322 0.29±0.07 69.3±9.7 294 
G410S 1.3±0.2 64.5±7.9 274 0.20±0.05 61.7±2.1 262 
R454H 17.2±2.5 7.5±2.1 32 >50 − − 
Y456H 22.9±6.6 6.1±0.7 26 >50 − − 
V489E 29.7±5.1 6.8±1.1 28 >50 − − 
A500V 1.8±0.2 81.3±6.6 344 0.21±0.06 55.7±1.9 238 
G501R 2.1±0.2 72.9±4.1 312 0.35±0.06 51.2±3.9 218 
G536R 6.3±1.2 37.8±6.7 160 18.3±2.1 9.3±1.2 40 
L562P 2.8±0.6 26.2±3.2 112 5.3±1.6 23.8±2.3 102 
C566Y 5.8±0.2 24.3±2.7 104 1.8±0.43 12.1±0.7 52 
V605F 5.2±1.3 26.3±5.2 112 3.3±0.7 32.7±0.6 140 
R616X >50 − − >50 − − 
V628I 1.6±0.4 77.3±8.6 328 0.47±0.06 32.8±5.3 140 
F643del 3.9±0.7 90.7±5.7 384 0.21±0.13 61.2±8.5 260 

Cytochrome c is not a natural substrate for POR, so we also assayed the capacity of POR mutants to catalyse a cytochrome P450 enzyme. We used microsomes containing human P450c17 expressed in yeast combined with a membrane fraction containing human N-24 POR expressed in Escherichia coli. P450c17 has two different catalytic activities that depend on ratio of POR and phosphorylation state of the enzyme as well as cytochrome b5 [1721]. Table 2 shows the Km, Vmax and kcat for the 17α-hydroxylase and 17,20 lyase activities of P450c17 when catalysis is supported by different POR variants. The polymorphic A112V, P225L, R313W, G410S, A500V and G501R variants retained 41–141% of wild-type activity.

Table 2
Kinetics of P450c17 activities supported by POR variants

Km values are in μM progesterone for 17α-hydroxylase and μM 17OH-pregnenolone for 17,20 lyase; Vmax values are in pmol/μg of P450c17 per min, kcat values were calculated based on the molar content of P450c17 and hence the values are in min−1. WT, wild-type. Taken from Huang, N., Pandey, A.V., Agrawal, V., Reardon, W., Lapunzina, P.D., Mowat, D., Jabs, E.W., Van Vliet, G., Sack, J., Flück, C.E. and Miller, W.L. (2005) Diversity and function of mutations in P450 oxidoreductase in patients with Antley–Bixler syndrome and disordered steroidogenesis. Am. J. Hum. Genet. 76, 729–749 [12] with permission of the University of Chicago Press. © 2005 by The American Society of Human Genetics.

 17α-Hydroxylase 17,20 Lyase 
POR variant Km Vmax kcat Km Vmax kcat 
WT 4.3±0.09 0.149±0.021 0.67 0.93±0.18 0.059±0.014 0.27 
A112V 3.5±0.05 0.097±0.013 0.43 0.71±0.11 0.032±0.006 0.14 
T139A 5.1±0.07 0.106±0.019 0.48 0.86±0.15 0.029±0.009 0.13 
Q150R 3.7±0.09 0.039±0.017 0.18 0.97±0.20 0.017±0.007 0.08 
Y178D >50 − − >50 − − 
P225L 3.7±0.06 0.131±0.013 0.59 1.54±0.21 0.041±0.005 0.18 
M260V 7.2±1.7 0.025±0.011 0.11 1.17±0.13 0.009±0.003 0.04 
A284P 3.5±1.0 0.049±0.010 0.22 1.34±0.09 0.018±0.002 0.08 
R313W 3.1±0.04 0.104±0.017 0.47 0.87±0.18 0.078±0.003 0.35 
G410S 5.9±1.1 0.172±0.015 0.77 1.28±0.15 0.089±0.011 0.40 
R454H 5.1±1.2 0.008±0.003 0.04 − − − 
Y456H 6.5±1.1 0.027±0.009 0.12 − − − 
V489E 7.7±1.5 0.011±0.004 0.05 − − − 
A500V 5.5±0.03 0.110±0.018 0.50 1.04±0.09 0.037±0.006 0.17 
G501R 3.9±0.07 0.124±0.020 0.56 0.85±0.12 0.056±0.009 0.25 
G536R 4.5±1.0 0.073±0.006 0.33 1.95±0.27 0.009±0.002 0.04 
L562P 5.9±0.65 0.067±0.008 0.30 1.39±0.16 0.017±0.005 0.08 
C566Y 9.3±1.9 0.094±0.009 0.42 1.22±0.15 0.010±0.012 0.05 
V605F 3.5±0.04 0.099±0.014 0.45 1.03±0.17 0.037±0.012 0.17 
R616X 4.7±0.07 0.008±0.003 0.04 − − − 
V628I 4.3±0.06 0.077±0.008 0.34 1.32±0.22 0.033±0.009 0.15 
F643del 3.5±0.07 0.118±0.011 0.53 1.77±0.27 0.051±0.006 0.23 
 17α-Hydroxylase 17,20 Lyase 
POR variant Km Vmax kcat Km Vmax kcat 
WT 4.3±0.09 0.149±0.021 0.67 0.93±0.18 0.059±0.014 0.27 
A112V 3.5±0.05 0.097±0.013 0.43 0.71±0.11 0.032±0.006 0.14 
T139A 5.1±0.07 0.106±0.019 0.48 0.86±0.15 0.029±0.009 0.13 
Q150R 3.7±0.09 0.039±0.017 0.18 0.97±0.20 0.017±0.007 0.08 
Y178D >50 − − >50 − − 
P225L 3.7±0.06 0.131±0.013 0.59 1.54±0.21 0.041±0.005 0.18 
M260V 7.2±1.7 0.025±0.011 0.11 1.17±0.13 0.009±0.003 0.04 
A284P 3.5±1.0 0.049±0.010 0.22 1.34±0.09 0.018±0.002 0.08 
R313W 3.1±0.04 0.104±0.017 0.47 0.87±0.18 0.078±0.003 0.35 
G410S 5.9±1.1 0.172±0.015 0.77 1.28±0.15 0.089±0.011 0.40 
R454H 5.1±1.2 0.008±0.003 0.04 − − − 
Y456H 6.5±1.1 0.027±0.009 0.12 − − − 
V489E 7.7±1.5 0.011±0.004 0.05 − − − 
A500V 5.5±0.03 0.110±0.018 0.50 1.04±0.09 0.037±0.006 0.17 
G501R 3.9±0.07 0.124±0.020 0.56 0.85±0.12 0.056±0.009 0.25 
G536R 4.5±1.0 0.073±0.006 0.33 1.95±0.27 0.009±0.002 0.04 
L562P 5.9±0.65 0.067±0.008 0.30 1.39±0.16 0.017±0.005 0.08 
C566Y 9.3±1.9 0.094±0.009 0.42 1.22±0.15 0.010±0.012 0.05 
V605F 3.5±0.04 0.099±0.014 0.45 1.03±0.17 0.037±0.012 0.17 
R616X 4.7±0.07 0.008±0.003 0.04 − − − 
V628I 4.3±0.06 0.077±0.008 0.34 1.32±0.22 0.033±0.009 0.15 
F643del 3.5±0.07 0.118±0.011 0.53 1.77±0.27 0.051±0.006 0.23 

In the reconstituted systems, P450 proteins form a complex with POR with a Km (app) of approx. 0.2 μM. The P450–POR interaction is influenced by many factors including type of P450, availability of substrates and ionic strength of the system. The interaction of POR with cytochrome P450 and other electron acceptor proteins is based primarily on electrostatic charge pairing, although there is evidence for an additional hydrophobic component. Chemical cross-linking and modification studies have shown that POR contains multiple carboxy groups, presumably contributed by the acidic amino acids aspartate and glutamate. These charge groups pair with basic amino acids (lysine and arginine residues) on the various electron acceptor proteins. In addition, cytochrome P450 forms a dipole across the molecule, with the positive charge at the proximal face of the protein where the haem makes its closest approach to the surface. This is thought to be the surface most suitable for electron transfer from POR. While electrostatic forces may serve to connect and orient the pair, hydrophobic forces contributed by non-polar amino acids (leucine, tryptophan, valine etc.) may be responsible for bringing the two proteins close together for electron transfer. Other electron acceptor proteins, such as cytochrome b5, haem oxygenase and squalene mono-oxygenase, probably interact by the same mechanism. The surface of the electron-donating (FMN) domain of POR is characterized by acidic residues [15,22,23], whereas the redox-partner binding site of microsomal P450 enzymes is typically characterized by basic residues [2426]. Thus electrostatic interactions that pair charges are obviously important in governing the association of POR with a P450 enzyme. Chemical modification of acidic residues in the FMN site of POR reduces the activity of P450 [22,27,28]. In addition, hydrophobic interactions between POR and P450 also play a role [29]. The activities assessed in different studies are a function of the specific P450 enzyme used as the read-out of the POR activity, but are also a function of the form of POR used. Both full-length POR that was used in our first report and the N-24 human POR used in our bacterial expression system remain associated with membranes and are able to support the reduction of a cytochrome P450. By contrast, when rat or human POR is rendered wholly soluble by deleting 54 N-terminal residues, it can still reduce cytochrome c but cannot reduce a P450 [3,30].

The large number of mutations we have studied provide details into the structural features of POR. Human POR shares highly conserved binding domains for FMN, FAD and NADPH with the PORs of other species (Figure 1) and the FAD- and NADPH-binding domains are also similar to the FAD- and NADPH-binding domains of ferredoxin reductase [31,32] and nitric oxide synthase [33]. The mutations described here are present in different domains of the POR. Mutations that alter the amino acids on/near FMN/FAD-binding sites resulted in severe effects on the POR activity. Mutations R454H, Y456H and V489E had almost no activity in 17,20 lyase assays, which are most sensitive to the changes in POR–P450 interactions and relies on cytochrome b5 to facilitate electron transfer from POR to P450c17. In comparison, mutations near NADPH-binding sites, C566Y, V605F and V628I, resulted in relatively milder effects on POR activity both in cytochrome c and P450c17 assays. Apparent polymorphisms P225L, R313W, G410S, A500V and G501R had 50–100% of normal activities. Mutations A112V, Q150R and Q260V are in the FMN-binding domain of POR and show a varied pattern of activity, apparently due to changes in tertiary structure induced by changes that may interfere with cofactor binding or electron transfer.

A ribbon model of human POR showing the location of the mutants

Figure 1
A ribbon model of human POR showing the location of the mutants

The model is based on the X-ray crystal structure of rat POR and is displayed using the programs Pymol and POVRAY. The missense residues identified by sequencing are depicted by spheres; mutations retaining >50% of activity are shown in green, those retaining 25–50% are shown in magenta and those retaining less than 25% are shown in red. Sphere models are used to represent the FMN (yellow), FAD (yellow) and NADPH (blue) residues.

Figure 1
A ribbon model of human POR showing the location of the mutants

The model is based on the X-ray crystal structure of rat POR and is displayed using the programs Pymol and POVRAY. The missense residues identified by sequencing are depicted by spheres; mutations retaining >50% of activity are shown in green, those retaining 25–50% are shown in magenta and those retaining less than 25% are shown in red. Sphere models are used to represent the FMN (yellow), FAD (yellow) and NADPH (blue) residues.

Mutations in the FMN-binding domain

By analogy with the crystallographically determined structure of rat POR, residues 140–144 and 170–182 of human POR participate in binding the isoalloxazine ring of FMN, with the side chain of Tyr178 stabilizing the interaction with the flavin. Replacing the Tyr178 with aspartic residue eliminated 99% of its activity to reduce cytochrome c [34]. The mutation Q150R, which is near the FMN-binding site, reduced POR activity to approx. 30% (Table 1). The highly conserved domain that interacts with and donates electrons to the cytochrome P450 comprises residues 191–228, especially residues 175–182 and 207–211 (DDDGN) [15]. We found no mutations in this domain; the closest mutations were M260V, which lost approx. 90% of activity, and A284P, the mutation commonly found in patients of European ancestry, which lost 60–80% of activity (Table 2). The RY sequence of residues 313–314 is conserved from yeast to human POR, suggesting an important function, yet the non-conservative mutant R316W was found as a polymorphism in a database and retained normal activity, as did the conservative polymorphism G410S (Table 2). Thus the activities of each mutant or polymorphism must be tested experimentally to determine the degree of impairment.

Mutations in FAD-binding domain

The FAD-binding domain (residues 450–492) is highly conserved and the sequence RYYSI (454–458) is invariant among human, rat and yeast PORs. The three-dimensional structure of POR shows that Arg454 forms a hydrogen bond with the pyrophosphate group of FAD, and Tyr456 contacts the FAD isoalloxazine ring and hydrogen-bonds with the ribityl 3′-hydroxy group [15,35]. Mutation of each of these residues abolishes all measurable activity. Similar results were obtained when these mutations were studied in rat POR [3537]. Residues 485–491, which lie in helix N, form hydrogen bonds with the FAD pyrophosphate. The mutation V489E that is predicted to disrupt hydrogen bonds with FAD has no activity but the variations A500V and the G501R that are located in an unstructured loop were identified as polymorphisms and retain >50% activity.

Mutations in NADPH-binding domain

The NADPH-binding domain comprising the C-terminus of the protein is highly conserved, especially the sequence GTGVAP (residues 534–539), which contains the consensus GXG sequence typical of NADP+-binding proteins [31,38]. The mutation G536R lost >90% activity in the 17,20 lyase assay, but retained 46% activity in the 17α-hydroxylase assay, indicating that the mutant could still bind some NADPH. Consistent with this, the cytochrome c assays showed that the greatest effect was on NADPH oxidation where the Km was increased approx. 100-fold (Table 1). Studies in which Cys566 of human POR was alkylated with iodoacetic acid eliminated activity, implicating a role for cysteine in binding of NADPH to human POR [39] but mutagenesis of the Cys566 to serine in rat POR suggested that this residue was not essential, despite a 4.6-fold higher Km value for NADPH [40] and alkylation of pig POR did not change activity [41]. The mutation C566Y in human POR, which we previously found in a patient with disordered steroidogenesis but without ABS [9], also had a high Km for NADPH (Table 1), but retained 13% of 17,20 lyase activity and 28% of 17α-hydroxylase activity (Table 2). The C566Y mutation was found as a compound heterozygote with V605F [9] which retained 57% of 17,20 lyase activity and 80% of 17α-hydroxylase activity, thus explaining that individual's hormonal profile resembling isolated 17,20 lyase deficiency. The crystallographic data indicated that Trp677 directly participates in interactions with both FMN and NADPH, suggesting that any C-terminal truncation will have severe consequences. Consistent with this, the premature chain truncation mutant R613X was devoid of activity. The mutations A112V and V628I were only detected as heterozygotes in individuals with normal steroid profile and thus may be polymorphisms. A112V is a conservative change in a region without apparent function and retained full activity with P450c17. Although the conservative mutant V628I lies in a highly conserved segment of the NADPH-binding domain, it retained normal activity in the cytochrome c assays (Table 1) and 40–50% of activity in the P450c17 assays. In-frame deletion of Phe643 had no effect on 17α-hydroxylase activity but a 54% reduction in 17,20 lyase activity.

POR interacts with all 50 human microsomal P450 enzymes, and, as the interactions with different P450 enzymes will vary with the geometry of the redox-partner binding site of the P450, no assay based on a single P450 enzyme will reliably forecast all the consequences of a specific POR mutant. Since all 50 microsomal P450 enzymes depend on POR for electron supply, we are putting forward the hypothesis that defects in POR could cause disorders in hepatic drug and xenobiotic metabolism as well as affect the functions of all steroid metabolizing microsomal P450 enzymes. Different P450 enzymes have variable affinities for POR and mutations/polymorphisms in POR could have different effects on different P450 enzymes. A change in activities of P450 by a variant POR will lead to changes in effective/toxic dosages of drugs.

8th International Symposium on Cytochrome P450 Biodiversity and Biotechnology: Independent Meeting held at Swansea Medical School, Swansea, Wales, U.K., 23–27 July 2006. Organized and Edited by D. Kelly, D. Lamb and S. Kelly (Swansea, U.K.).

Abbreviations

     
  • POR

    cytochrome P450 reductase

Most of the work described in this review was done in the laboratory of Professor Walter L. Miller at the University of California San Francisco (San Francisco, CA, U.S.A.). I am grateful to the members of Miller laboratory and Dr Christa E. Flück (University Children's Hospital, Bern, Switzerland) for contributions to the characterization of POR variants.

References

References
1
Horecher
B.L.
J. Biol. Chem.
1950
, vol. 
183
 (pg. 
593
-
605
)
1a
Williams
C.H.
Jr
Kamin
H.
J. Biol. Chem.
1962
, vol. 
237
 (pg. 
587
-
595
)
2
Phillips
A.H.
Langdon
R.G.
J. Biol. Chem.
1962
, vol. 
237
 (pg. 
2652
-
2660
)
3
Lu
A.Y.
Junk
K.W.
Coon
M.J.
J. Biol. Chem.
1969
, vol. 
244
 (pg. 
3714
-
3721
)
4
Shen
A.L.
O'Leary
K.A.
Kasper
C.B.
J. Biol. Chem.
2002
, vol. 
277
 (pg. 
6536
-
6541
)
5
Otto
D.M.
Henderson
C.J.
Carrie
D.
Davey
M.
Gundersen
T.E.
Blomhoff
R.
Adams
R.H.
Tickle
C.
Wolf
C.R.
Mol. Cell. Biol.
2003
, vol. 
23
 (pg. 
6103
-
6116
)
6
Gu
J.
Weng
Y.
Zhang
Q.Y.
Cui
H.
Behr
M.
Wu
L.
Yang
W.
Zhang
L.
Ding
X.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
25895
-
25901
)
7
Henderson
C.J.
Otto
D.M.
Carrie
D.
Magnuson
M.A.
McLaren
A.W.
Rosewell
I.
Wolf
C.R.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
13480
-
13486
)
8
Wu
L.
Gu
J.
Cui
H.
Zhang
Q.Y.
Behr
M.
Fang
C.
Weng
Y.
Kluetzman
K.
Swiatek
P.J.
Yang
W.
, et al. 
J. Pharmacol. Exp. Ther.
2005
, vol. 
312
 (pg. 
35
-
43
)
9
Flück
C.E.
Tajima
T.
Pandey
A.V.
Arlt
W.
Okuhara
K.
Verge
C.F.
Jabs
E.W.
Mendonca
B.B.
Fujieda
K.
Miller
W.L.
Nat. Genet.
2004
, vol. 
36
 (pg. 
228
-
230
)
10
Pandey
A.V.
Flück
C.E.
Huang
N.
Tajima
T.
Fujieda
K.
Miller
W.L.
Endocr. Res.
2004
, vol. 
30
 (pg. 
881
-
888
)
11
Miller
W.L.
Huang
N.
Flück
C.E.
Pandey
A.V.
Lancet
2004
, vol. 
364
 pg. 
1663
 
12
Huang
N.
Pandey
A.V.
Agrawal
V.
Reardon
W.
Lapunzina
P.D.
Mowat
D.
Jabs
E.W.
Van Vliet
G.
Sack
J.
Flück
C.E.
Miller
W.L.
Am. J. Hum. Genet.
2005
, vol. 
76
 (pg. 
729
-
749
)
13
Adachi
M.
Tachibana
K.
Asakura
Y.
Yamamoto
T.
Hanaki
K.
Oka
A.
Am. J. Med. Genet. A
2004
, vol. 
128
 (pg. 
333
-
339
)
14
Fukami
M.
Horikawa
R.
Nagai
T.
Tanaka
T.
Naiki
Y.
Sato
N.
Okuyama
T.
Nakai
H.
Soneda
S.
Tachibana
K.
, et al. 
J. Clin. Endocrinol. Metab.
2004
, vol. 
90
 (pg. 
414
-
426
)
15
Wang
M.
Roberts
D.L.
Paschke
R.
Shea
T.M.
Masters
B.S.
Kim
J.J.
Proc. Natl. Acad. Sci. U.S.A.
1997
, vol. 
94
 (pg. 
8411
-
8416
)
16
Dierks
E.A.
Davis
S.C.
Ortiz de Montellano
P.R.
Biochemistry
1998
, vol. 
37
 (pg. 
1839
-
1847
)
17
Pandey
A.V.
Mellon
S.H.
Miller
W.L.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
2837
-
2844
)
18
Pandey
A.V.
Miller
W.L.
J. Biol. Chem.
2005
, vol. 
280
 (pg. 
13265
-
13271
)
19
Auchus
R.J.
Lee
T.C.
Miller
W.L.
J. Biol. Chem.
1998
, vol. 
273
 (pg. 
3158
-
3165
)
20
Zhang
L.H.
Rodriguez
H.
Ohno
S.
Miller
W.L.
Proc. Natl. Acad. Sci. U.S.A.
1995
, vol. 
92
 (pg. 
10619
-
10623
)
21
Yanagibashi
K.
Hall
P.F.
J. Biol. Chem.
1986
, vol. 
261
 (pg. 
8429
-
8433
)
22
Shen
A.L.
Kasper
C.B.
J. Biol. Chem.
1995
, vol. 
270
 (pg. 
27475
-
27480
)
23
Estabrook
R.W.
Shet
M.S.
Fisher
C.W.
Jenkins
C.M.
Waterman
M.R.
Arch. Biochem. Biophys.
1996
, vol. 
333
 (pg. 
308
-
315
)
24
Hasemann
C.A.
Kurumbail
R.G.
Boddupalli
S.S.
Peterson
J.A.
Deisenhofer
J.
Structure
1995
, vol. 
3
 (pg. 
41
-
62
)
25
Fisher
C.W.
Shet
M.S.
Estabrook
R.W.
Methods Enzymol.
1996
, vol. 
272
 (pg. 
15
-
25
)
26
Davydov
D.R.
Kariakin
A.A.
Petushkova
N.A.
Peterson
J.A.
Biochemistry
2000
, vol. 
39
 (pg. 
6489
-
6497
)
27
Nadler
S.G.
Strobel
H.W.
Arch. Biochem. Biophys.
1991
, vol. 
290
 (pg. 
277
-
284
)
28
Nadler
S.G.
Strobel
H.W.
Arch. Biochem. Biophys.
1988
, vol. 
261
 (pg. 
418
-
429
)
29
Strobel
H.W.
Nadler
S.G.
Nelson
D.R.
Drug Metab. Rev.
1989
, vol. 
20
 (pg. 
519
-
533
)
30
Roman
L.J.
McLain
J.
Masters
B.S.S.
J. Biol. Chem.
2003
, vol. 
278
 (pg. 
25700
-
25707
)
31
Hanukoglu
I.
Gutfinger
T.
Eur. J. Biochem.
1989
, vol. 
180
 (pg. 
479
-
484
)
32
Karplus
P.A.
Daniels
M.J.
Herriott
J.R.
Science
1991
, vol. 
251
 (pg. 
60
-
66
)
33
Bredt
D.S.
Hwang
P.M.
Glatt
C.E.
Lowenstein
C.
Reed
R.R.
Snyder
S.H.
Nature
1991
, vol. 
351
 (pg. 
714
-
718
)
34
Shen
A.L.
Porter
T.D.
Wilson
T.E.
Kasper
C.B.
J. Biol. Chem.
1989
, vol. 
264
 (pg. 
7584
-
7589
)
35
Shen
A.L.
Kasper
C.B.
J. Biol. Chem.
2000
, vol. 
275
 (pg. 
41087
-
41091
)
36
Shen
A.L.
Kasper
C.B.
Biochemistry
1996
, vol. 
35
 (pg. 
9451
-
9459
)
37
Shen
A.L.
Sem
D.S.
Kasper
C.B.
J. Biol. Chem.
1999
, vol. 
274
 (pg. 
5391
-
5398
)
38
Scrutton
N.S.
Berry
A.
Perham
R.N.
Nature
1990
, vol. 
343
 (pg. 
38
-
43
)
39
Haniu
M.
McManus
M.E.
Birkett
D.J.
Lee
T.D.
Shively
J.E.
Biochemistry
1989
, vol. 
28
 (pg. 
8639
-
8645
)
40
Shen
A.L.
Christensen
M.J.
Kasper
C.B.
J. Biol. Chem.
1991
, vol. 
266
 (pg. 
19976
-
19980
)
41
Haniu
M.
Iyanagi
T.
Legesse
K.
Shively
J.E.
J. Biol. Chem.
1984
, vol. 
259
 (pg. 
13703
-
13711
)