Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

Marohnic, Christopher C.; Panda, Satya Prakash; McCammon, Karen; Rueff, José; Masters, Bettie Sue Siler; Kranendonk, Michel

doi:10.1124/dmd.109.030445

Cited by 46 publications

(46 citation statements)

References 39 publications

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“…At this juncture, the only human mutations that have been shown to be reversible at the molecular level, when examined by addressing the purified, homogeneous enzymes with biophysical techniques, have been those exhibiting flavin deficiencies (22,25,26,38). However, it has become apparent from this work that certain missense mutations could lead to conformational alterations resulting in unstable or more readily degradable proteins, thereby leading to diminished activity, but not necessarily directly affecting flavin or NADPH binding.…”

Section: Discussionmentioning

confidence: 84%

“…and Purification Conditions-We have expressed full-length WT human P450 reductase and 11 POR mutants in E. coli and purified them to homogeneity (22,(25)(26)(27). 10 Our initial purification of the full-length A287P variant followed our established protocol, but the yield was extremely low and irreproducible as compared with WT.…”

Section: A287p Variant Preparation Necessitates Modified Expressionmentioning

confidence: 99%

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Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

McCammon

Panda

Xia

et al. 2016

Journal of Biological Chemistry

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Human NADPH-cytochrome P450 oxidoreductase (POR) gene mutations are associated with severe skeletal deformities and disordered steroidogenesis. The human POR mutation A287P presents with disordered sexual development and skeletal malformations. Difficult recombinant expression and purification of this POR mutant suggested that the protein was less stable than WT. The activities of cytochrome P450 17A1, 19A1, and 21A2, critical in steroidogenesis, were similar using our purified, full-length, unmodified A287P or WT POR, as were those of several xenobiotic-metabolizing cytochromes P450, indicating that the A287P protein is functionally competent in vitro, despite its functionally deficient phenotypic behavior in vivo. Differential scanning calorimetry and limited trypsinolysis studies revealed a relatively unstable A287P compared with WT protein, leading to the hypothesis that the syndrome observed in vivo results from altered POR protein stability. The crystal structures of the soluble domains of WT and A287P reveal only subtle differences between them, but these differences are consistent with the differential scanning calorimetry results as well as the differential susceptibility of A287P and WT observed with trypsinolysis. The relative in vivo stabilities of WT and A287P proteins were also examined in an osteoblast cell line by treatment with cycloheximide, a protein synthesis inhibitor, showing that the level of A287P protein post-inhibition is lower than WT and suggesting that A287P may be degraded at a higher rate. Current studies demonstrate that, unlike previously described mutations, A287P causes POR deficiency disorder due to conformational instability leading to proteolytic susceptibility in vivo, rather than through an inherent flavin-binding defect.

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Section: Discussionmentioning

confidence: 84%

Section: A287p Variant Preparation Necessitates Modified Expressionmentioning

confidence: 99%

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

McCammon

Panda

Xia

et al. 2016

Journal of Biological Chemistry

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“…Retardation of somite and limb bud formation, observed previously in embryonically lethal CYPOR knockout mice (10,11), supports this hypothesis. Subsequent work by these investigators (4,12,13) and our laboratories has supported this hypothesis by demonstrating the impaired activities of these and other mutant CYPOR preparations in purified, reconstituted systems (14) and engineered bacterial membrane systems (15,16).…”

mentioning

confidence: 80%

“…1); however, each of these variant structures reveals the structural basis for protein instability and/or abnormality in cofactor binding, as well as for the phenotypic expression of CYPOR deficiency. The implications of these findings in understanding the intricacies of protein folding/unfolding (present study), the possibility of riboflavin therapy for treatment of patients with these enzyme deficiencies (14)(15)(16), and the importance of understanding the actual structural aberrations resulting in catalytic and, thus, phenotypic outcomes has become even more critical. (4), which stated that V492E exhibited 7% of wild-type cytochrome c reduction with a K m NADPH of >50 μM and that R457H has only 8% of the wild-type V max for cytochrome c reductions with a K m NADPH > 50 μM.…”

mentioning

confidence: 99%

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

Xia

Panda

Marohnic

et al. 2011

Proc. Natl. Acad. Sci. U.S.A.

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106

135

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NADPH-cytochrome P450 oxidoreductase (CYPOR) is essential for electron donation to microsomal cytochrome P450-mediated monooxygenation in such diverse physiological processes as drug metabolism (approximately 85–90% of therapeutic drugs), steroid biosynthesis, and bioactive metabolite production (vitamin D and retinoic acid metabolites). Expressed by a single gene, CYPOR’s role with these multiple redox partners renders it a model for understanding protein–protein interactions at the structural level. Polymorphisms in human CYPOR have been shown to lead to defects in bone development and steroidogenesis, resulting in sexual dimorphisms, the severity of which differs significantly depending on the degree of CYPOR impairment. The atomic structure of human CYPOR is presented, with structures of two naturally occurring missense mutations, V492E and R457H. The overall structures of these CYPOR variants are similar to wild type. However, in both variants, local disruption of H bonding and salt bridging, involving the FAD pyrophosphate moiety, leads to weaker FAD binding, unstable protein, and loss of catalytic activity, which can be rescued by cofactor addition. The modes of polypeptide unfolding in these two variants differ significantly, as revealed by limited trypsin digestion: V492E is less stable but unfolds locally and gradually, whereas R457H is more stable but unfolds globally. FAD addition to either variant prevents trypsin digestion, supporting the role of the cofactor in conferring stability to CYPOR structure. Thus, CYPOR dysfunction in patients harboring these particular mutations may possibly be prevented by riboflavin therapy in utero, if predicted prenatally, or rescued postnatally in less severe cases.

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“…Interindividual variations in POR activity (at least 4-to 5-fold differences) have been found in human liver microsomes (Kaminsky et al, 1984;Hart et al, 2008). The notion that variations in POR expression or activity influence the rates of P450-mediated drug metabolism in patients is supported by several lines of data, including the impact of Por gene knockout on drug clearance in mouse models (Gu et al, 2003;Henderson et al, 2003;Zhang et al, 2009), positive correlations between POR activity and P450-mediated drug metabolism activities in human liver microsomes (Kaminsky et al, 1984;Hart et al, 2008), and the impact of many POR single nucleotide polymorphisms (SNPs) on P450 activities toward drugs and other xenobiotics in reconstituted enzyme systems Flück et al, 2010;Marohnic et al, 2010;Nicolo et al, 2010;Sandee et al, 2010;Miller et al, 2011).…”

Section: Introductionmentioning

confidence: 94%

Identification of Cytochrome P450 Oxidoreductase Gene Variants That Are Significantly Associated with the Interindividual Variations in Warfarin Maintenance Dose

Zhang¹,

Li²,

Ding³

et al. 2011

Drug Metab Dispos

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ABSTRACT:Cytochrome P450 oxidoreductase (POR) is required for drug metabolism by all microsomal cytochrome P450 enzymes. The aim of this study was to investigate whether any of the common single nucleotide polymorphisms (SNPs) in the POR gene and its flanking intergenic sequences correlate with interindividual variations in the warfarin maintenance dose (which is determined partly by rates of warfarin metabolism) in patients undergoing anticoagulation therapy. Warfarin dose and patients' demographic and clinical information were collected from 124 patients, who had attained a stable warfarin dose while receiving treatment at the Stratton VA Medical Center. Genomic DNAs were isolated from blood samples and were genotyped for 15 SNPs (including 10 SNPs on the POR gene). Association analysis was performed on 122 male patients by linear regression. Simple regression analysis revealed that vitamin K epoxide reductase complex subunit 1 (VKORC1) ؊1639A>G, CYP2C9*2, CYP2C9*3, age, and chronic aspirin therapy were significantly associated with warfarin dose. In contrast, multiple regression analysis revealed that, in addition to several known factors contributing to the variations in warfarin maintenance dose (VKORC1 ؊1639A>G, CYP2C9*2, CYP2C9*3, CYP4F2 rs2108622, and chronic aspirin therapy), three common POR SNPs (؊173C>A, ؊208C>T, and rs2868177) were also significantly associated with variations in warfarin maintenance dose. These results indicate, for the first time, that three common SNPs in the POR gene may contribute to the interindividual variability in warfarin maintenance dose. Further studies on functional characterization of the POR SNPs identified, including their impact on the in vivo metabolism of additional drugs, are needed.

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Human Cytochrome P450 Oxidoreductase Deficiency Caused by the Y181D Mutation: Molecular Consequences and Rescue of Defect

Cited by 46 publications

References 39 publications

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Instability of the Human Cytochrome P450 Reductase A287P Variant Is the Major Contributor to Its Antley-Bixler Syndrome-like Phenotype

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

Identification of Cytochrome P450 Oxidoreductase Gene Variants That Are Significantly Associated with the Interindividual Variations in Warfarin Maintenance Dose

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