Satya Prakash Panda

Structural basis for human NADPH-cytochrome P450 oxidoreductase deficiency

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.

Diminished FAD Binding in the Y459H and V492E Antley-Bixler Syndrome Mutants of Human Cytochrome P450 Reductase

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. 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. To address the molecular basis of CYPOR dysfunction in ABS patients, the soluble catalytic domain of human CYPOR was bacterially expressed. WT enzyme was green, due to air-stable FMN semiquinone (blue) and oxidized FAD (yellow). The ABS mutant V492E was blue-gray. Flavin analysis indicated that WT had a protein:FAD:FMN ratio of ∼1:1:1, whereas ∼1:0.1:0.9 was observed for V492E, which retained 9% of the WT kcat/Km in NADPH:cytochrome c reductase assays. V492E was reconstituted upon addition of FAD, post-purification, as shown by flavin analysis, activity assay, and near UV-visible CD. Both Y459H and V492E were expressed as membrane anchor-containing proteins, which also exhibited FAD deficiency. CYP4A4-catalyzed ω-hydroxylation of prostaglandin E1 was supported by WT CYPOR but not by either of the ABS mutants. Hydroxylation activity was rescued for both Y459H and V492E upon addition of FAD to the reaction. 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.

IMPAIRMENT OF HUMAN CYP1A2-MEDIATED XENOBIOTIC METABOLISM BY ANTLEY-BIXLER SYNDROME VARIANTS OF CYTOCHROME P450 OXIDOREDUCTASE

Y459H and V492E mutations of cytochrome P450 reductase (CYPOR) cause Antley-Bixler syndrome due to diminished binding of the FAD cofactor. To address whether these mutations impaired the interaction with drug-metabolizing CYPs, a bacterial model of human liver expression of CYP1A2 and CYPOR was implemented. Four models were generated: POR(null), POR(wt), POR(YH), and POR(VE), for which equivalent CYP1A2 and CYPOR levels were confirmed, except for POR(null), not containing any CYPOR. The mutant CYPORs were unable to catalyze cytochrome c and MTT reduction, and were unable to support EROD and MROD activities. Activity was restored by the addition of FAD, with V492E having a higher apparent FAD affinity than Y459H. The CYP1A2-activated procarcinogens, 2-aminoanthracene, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, and 2-amino-3-methylimidazo(4,5-f)quinoline, were significantly less mutagenic in POR(YH) and POR(VE) models than in POR(wt), indicating that CYP1A2, and likely other drug-metabolizing CYPs, are impaired by ABS-related POR mutations as observed in the steroidogenic CYPs.

Human cytochrome P450 oxidoreductase deficiency caused by the Y181D mutation: molecular consequences and rescue of defect.

Patients with congenital adrenal hyperplasia, exhibiting combined CYP17 and CYP21 deficiency, were shown by Arlt et al. (2004) to harbor a 541T→G mutation in exon 5 of POR (encoding NADPH-cytochrome P450 reductase, CYPOR), which resulted in a Y181D substitution that obliterated electron transfer capacity. Using bacterial expression models, we examined catalytic and physical properties of the human CYPOR Y181D variant. As purified, Y181D lacked flavin mononucleotide (FMN) and NADPH-cytochrome c reductase (NCR) activity but retained normal flavin adenine dinucleotide binding and NADPH utilization. Titration of the purified protein with FMN restored 64 of wild-type (WT) NCR activity in Y181D with an activation constant of ∼2 μM. As determined by FMN fluorescence quenching, Y181D had KdFMN = 7.3 μM. Biplasmid coexpression of CYPOR and CYP1A2, at the physiological ratio of ∼1:10 in the engineered MK_1A2_POR Escherichia coli strain, showed the compromised capacity of Y181D to support CYP1A2-catalyzed metabolism of the procarcinogens 2-aminoanthracene, 2-amino-3-methylimidazo(4,5-f)quinoline, and 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone. Isolated MK1A2_POR membranes confirmed FMN stimulation of Y181D NCR activity with a 1.6 μM activation constant. CYP1A2 ethoxyresorufin-O-dealkylase activity of the MK1A2_PORY181D membranes, undetectable in the absence of added FMN, increased to 37% of MK1A2_PORWT membranes with a 1.2 μM FMN activation constant. Therefore, we conclude that compromised FMN binding is the specific molecular defect causing POR deficiency in patients with Y181D mutation and that this defect, in large part, can be overcome in vitro by FMN addition.

Oxygen Metabolism by Endothelial Nitric-oxide Synthase

Nitric-oxide synthase (NOS) catalyzes both coupled and uncoupled reactions that generate nitric oxide and reactive oxygen species. Oxygen is often the overlooked substrate, and the oxygen metabolism catalyzed by NOS has been poorly defined. In this paper we focus on the oxygen stoichiometry and effects of substrate/cofactor binding on the endothelial NOS isoform (eNOS). In the presence of both l-arginine and tetrahydrobiopterin, eNOS is highly coupled (>90%), and the measured stoichiometry of O2/NADPH is very close to the theoretical value. We report for the first time that the presence of l-arginine stimulates oxygen uptake by eNOS. The fact that nonhydrolyzable l-arginine analogs are not stimulatory indicates that the occurrence of the coupled reaction, rather than the accelerated uncoupled reaction, is responsible for the l-arginine-dependent stimulation. The presence of 5,6,7,8-tetrahydrobiopterin quenched the uncoupled reactions and resulted in much less reactive oxygen species formation, whereas the presence of redox-incompetent 7,8-dihydrobiopterin demonstrates little quenching effect. These results reveal different mechanisms for oxygen metabolism for eNOS as opposed to nNOS and, perhaps, partially explain their functional differences.

Oxygen metabolism by neuronal nitric-oxide synthase

Nitric-oxide synthases (NOS) catalyze nitric oxide (NO) formation from the amino acid l-arginine. NOS is known to catalyze more than one reaction: the NO-producing reaction is considered to be the coupled reaction, and the uncoupled reactions are those that produce reactive (reduced) oxygen species (ROS), such as superoxide anion (\batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document}) and/or hydrogen peroxide (H2O2). As an oxygenase, NOS has been known for more than two decades, yet there is no complete description of oxygen stoichiometry. The present paper is focused on oxygen stoichiometry and the effects of cofactor binding on the neuronal isoform (nNOS) on oxygen uptake and product formation. Products of the uncoupled reactions are analyzed using diacetyldeuteroheme-substituted horseradish peroxidase as a trapping agent for both \batchmode \documentclass[fleqn,10pt,legalpaper]{article} \usepackage{amssymb} \usepackage{amsfonts} \usepackage{amsmath} \pagestyle{empty} \begin{document} \(\mathrm{O}_{2}^{{\bar{{\cdot}}}}\) \end{document} and H2O2. The addition of calmodulin not only stimulated the oxygen uptake rate but also changed the product of the uncoupled reaction, supporting the possibility of two different sites for electron leakage to molecular oxygen. Quantitative analysis of the uncoupled (substrate-free) reaction revealed a stoichiometry close to the theoretical value, and adding l-arginine not only initiates the coupled reaction, but also inhibits oxygen uptake. The presence of tetrahydrobiopterin affects oxygen metabolism by lowering the apparent Km value of nNOS for oxygen in the uncoupled reaction.

The stimulatory effects of Hofmeister ions on the activities of neuronal nitric-oxide synthase. Apparent substrate inhibition by L-arginine is overcome in the presence of protein-destabilizing agents

A variety of monovalent anions and cations were effective in stimulating both calcium ion/calmodulin (Ca2+/CaM)-independent NADPH-cytochrome creductase activity of, and Ca2+/CaM-dependent nitric oxide (NO⋅) synthesis by, neuronal nitric oxide synthase (nNOS). The efficacy of the ions in stimulating both activities could be correlated, in general, with their efficacy in precipitating or stabilizing certain proteins, an order referred to as the Hofmeister ion series. In the hemoglobin capture assay, used for measurement of NO⋅ production, apparent substrate inhibition byl-arginine was almost completely reversed by the addition of sodium perchlorate (NaClO4), one of the more effective protein-destabilizing agents tested. Examination of this phenomenon by the assay of l-arginine conversion tol-citrulline revealed that the stimulatory effect of NaClO4 on the reaction was observed only in the presence of oxyhemoglobin or superoxide anion (generated by xanthine and xanthine oxidase), both scavengers of NO⋅. Spectrophotometric examination of nNOS revealed that the addition of NaClO4 and a superoxide-generating system, but neither alone, prevented the increase of heme absorption at 436 nm, which has been attributed to the nitrosyl complex. The data are consistent with the release of autoinhibitory NO⋅ coordinated to the prosthetic group of nNOS, which, in conjunction with an NO⋅ scavenger, causes stimulation of the reaction.

Altered Human CYP3A4 Activity Caused by Antley-Bixler Syndrome-related Variants of NADPH-cytochrome P450 Oxidoreductase Measured in a Robust in vitro System

NADPH-cytochrome P450 oxidoreductase (CYPOR) variants have been described in patients with perturbed steroidogenesis and sexual differentiation, related to Antley-Bixler syndrome (ABS). It is important to determine the effect of these variants on CYP3A4, the major drug-metabolizing cytochrome P450 (P450) in humans. In this study, 12 CYPOR_ABS variants were separately coexpressed with CYP3A4 in a robust in vitro system to evaluate the effects of these variants on CYP3A4 activity in a milieu that recapitulates the stoichiometry of the mammalian systems. Full-length CYPOR variants were coexpressed with CYP3A4, resulting in relative expression levels comparable to those found in hepatic tissue. Dibenzylfluorescein (DBF), a CYP3A-specific reporter substrate (Biopharm Drug Dispos 24:375–384, 2003), was used to compare the variants and wild-type (WT) CYPOR activities with that of human liver microsomes. CYP3A4, combined with WT CYPOR, demonstrated kinetic parameters (kcat and Km) equal to those for pooled human liver microsomes. CYPOR variants Y181D, Y459H, V492E, L565P, and R616X all demonstrated maximal loss of CYP3A4 catalytic efficiency, whereas R457H and G539R retained ∼10 and 30% activities, respectively. Conversely, variants P228L, M263V, A287P, and G413S each showed WT-like capacity (kcat/Km), with the A287P variant being formerly reported to exhibit substantially lower catalytic efficiency. In addition, Q153R exhibited 60% of WT CYPOR capacity to support the DBF O-debenzylation reaction, contradicting increased catalytic efficiency (kcat/Km) relative to that for the WT, reported previously. Our data indicate the importance of use of simulated, validated in vitro systems, employing full-length proteins with appropriate stoichiometric incorporation of protein partners, when pharmacogenetic predictions are to be made for P450-mediated biotransformation.

The Role of a Conserved Serine Residue within Hydrogen Bonding Distance of FAD in Redox Properties and the Modulation of Catalysis by Ca2+/Calmodulin of Constitutive Nitric-oxide Synthases

The crystal structure of the neuronal nitric-oxide synthase (nNOS) NADPH/FAD binding domain indicated that Ser-1176 is within hydrogen bonding distance of Asp-1393 and the O4 atom of FAD and is also near the N5 atom of FAD (3.7Å). This serine residue is conserved in most of the ferredoxin-NADP+ reductase family of proteins and is important in electron transfer. In the present study, the homologous serines of both nNOS (Ser-1176) and endothelial nitric-oxide synthase (eNOS) (Ser-942) were mutated to threonine and alanine. Both substitutions yielded proteins that exhibited decreased rates of electron transfer through the flavin domains, in the presence and absence of Ca2+/CaM, as measured by reduction of potassium ferricyanide and cytochrome c. Rapid kinetics measurements of flavin reduction of all the mutants also showed a decrease in the rate of flavin reduction, in the absence and presence of Ca2+/CaM, as compared with the wild type proteins. The serine to alanine substitution caused both nNOS and eNOS to synthesize NO more slowly; however, the threonine mutants gave equal or slightly higher rates of NO production compared with the wild type enzymes. The midpoint redox potential measurements of all the redox centers revealed that wild type and threonine mutants of both nNOS and eNOS are very similar. However, the redox potentials of the FMN/FMNH· couple for alanine substitutions of both nNOS and eNOS are >100 mV higher than those of wild type proteins and are positive. These data presented here suggest that hydrogen bonding of the hydroxyl group of serine or threonine with the isoalloxazine ring of FAD and with the amino acids in its immediate milieu, particularly nNOS Asp-1393, affects the redox potentials of various flavin states, influencing the rate of electron transfer.

Nitric oxide synthases activation and inhibition by metallacarborane- cluster-based isoform-specific affectors

A small library of boron-cluster- and metallacarborane-cluster-based ligands was designed, prepared, and tested for isoform-selective activation or inhibition of the three nitric oxide synthase isoforms. On the basis of the concept of creating a hydrophobic analogue of a natural substrate, a stable and nontoxic basic boron cluster system, previously used for boron neutron capture therapy, was modified by the addition of positively charged moieties to its periphery, providing hydrophobic and nonclassical hydrogen bonding interactions with the protein. Several of these compounds show efficacy for inhibition of NO synthesis with differential effects on the various nitric oxide synthase isoforms.