NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family

Iyanagi, Takashi; Xia, Chuanwu; Kim, Jung‐Ja P.

doi:10.1016/j.abb.2012.09.002

Cited by 105 publications

(123 citation statements)

References 213 publications

(359 reference statements)

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“…Our recent HDX-MS studies provided detailed models of the iNOS output state, but, because the FAD/NADPH subdomain was not included in the truncations used in this study, the relative positioning of the entire reductase and oxidase domains could not be determined (7). Based on the crystal structure of the nNOS reductase domain (8), it has been hypothesized that the reductase domains dimerize in the NOS holoenzyme, but this hypothesis remains a point of major uncertainty and contention (6,19,20). Furthermore, despite significant homology, it is unknown whether all three NOS isoforms adopt a similar overall structure.…”

Section: Significancementioning

confidence: 99%

“…This binding is thought to release the FMN subdomain from the input state (6). Our recent studies investigating iNOS through hydrogendeuterium exchange MS (HDX-MS) suggested an intermediate state as the enzyme transitions from input to output state (7).…”

mentioning

confidence: 99%

“…Although progress has been made elucidating the interaction surfaces present in the input and output states (7)(8)(9)(10)(11)(12), the trajectory of this conformational change between the input and output is only beginning to be explored (13). Further understanding of this conformational change is particularly important as electron transfer is rate-limiting during NOS catalysis (6).…”

mentioning

confidence: 99%

“…The FMN subdomain is connected to the oxidase domain via a polypeptide linker approximately 30 aa in length, which forms a helical binding site for calmodulin (CaM) (5). During catalysis, the reductase domain passes electrons from the reduced NADPH substrate through the bound FAD and FMN cofactors and ultimately to the heme cofactor in the oxidase domain of the opposite monomer (4,6). The oxidase domain binds the arginine substrate directly above the heme and tetrahydrobiopterin cofactors.…”

mentioning

confidence: 99%

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Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

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

122

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NOSs are homodimeric multidomain enzymes responsible for producing NO. In mammals, NO acts as an intercellular messenger in a variety of signaling reactions, as well as a cytotoxin in the innate immune response. Mammals possess three NOS isoformsinducible, endothelial, and neuronal NOS-that are composed of an N-terminal oxidase domain and a C-terminal reductase domain. Calmodulin (CaM) activates NO synthesis by binding to the helical region connecting these two domains. Although crystal structures of isolated domains have been reported, no structure is available for full-length NOS. We used high-throughput single-particle EM to obtain the structures and higher-order domain organization of all three NOS holoenzymes. The structures of inducible, endothelial, and neuronal NOS with and without CaM bound are similar, consisting of a dimerized oxidase domain flanked by two separated reductase domains. NOS isoforms adopt many conformations enabled by three flexible linkers. These conformations represent snapshots of the continuous electron transfer pathway from the reductase domain to the oxidase domain, which reveal that only a single reductase domain participates in electron transfer at a time, and that CaM activates NOS by constraining rotational motions and by directly binding to the oxidase domain. Direct visualization of these large conformational changes induced during electron transfer provides significant insight into the molecular underpinnings governing NO formation.heme | flavin | electron microscopy | conformational heterogeneity N O has emerged as an integral signaling molecule in biology.NOSs are the only enzymes responsible for NO production in mammals. Disruptions in NO signaling are linked to hypertension, erectile dysfunction, neurodegeneration, stroke, and heart disease (1-3). Three NOS isoforms, which vary slightly in size and composition from 260 to 321 kDa for the NOS homodimer, are present in mammals; each is a distinct gene product differing in subcellular localization, tissue distribution, and mode of regulation. The constitutively expressed NOS isoforms are found primarily in endothelial cells [endothelial NOS (eNOS)] and neuronal cells [neuronal NOS (nNOS)], and NO produced by these isoforms initiates diverse signaling processes, including vasodilation, platelet aggregation, myocardial functions, and neurotransmission (4). The activity of the constitutive NOS isoforms is dependent on intracellular Ca 2+ concentrations. The third isoform, inducible NOS (iNOS), is transcriptionally controlled and produces cytotoxic concentrations of NO at sites of infection or inflammation. Unlike eNOS and nNOS, the activity of iNOS is independent of the intracellular Ca 2+ concentration.Mammalian NOS isoforms use a complex assembly of domains and cofactors to convert L-arginine to L-citrulline and NO, via the intermediate N-hydroxy-L-arginine. Mammalian NOS isoforms are active as homodimers and composed of two primary domains: the N-terminal oxidase domain and the C-terminal reductase domain (Fig. 1A). The re...

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Section: Significancementioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

mentioning

confidence: 99%

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Molecular architecture of mammalian nitric oxide synthases

Campbell

Smith

Potter

et al. 2014

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

122

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“…CPR is important in transferring electrons from NADPH through to the CYP allowing the incorporation of one molecule of oxygen, hence a monooxygenase reaction, to various substrates (Guengerich 2007). CPR is a highly conserved diflavin protein composed of two flavin cofactors, flavin mononucleotide (FMN) and flavin adenenine dinucleotide (FAD), that transfer the electrons one at a time to a CYP (Iyanagi et al, 2012;Lian et al, 2011). Due to the nature of CYP research among mammals and insects, the CPR gene has been isolated from an array of species and been characterized in many of these (Chen and Zhang 2014;Liu et al, 2014;Zhu et al, 2012) however, CPR has not been isolated or characterized from a tick species to date, despite the importance of this protein to the functioning of the monoxygenase complex and subsequent detoxification of xenobiotics.…”

Section: Introductionmentioning

confidence: 99%

Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus – clues to understanding acaricide resistance

Graham¹,

Sparagano²,

Finn³

2016

Ticks and Tick-borne Diseases

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This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. A c c e p t e d M a n u s c r i p t AbstractThe monooxygenase complex is composed of three key proteins, a cytochrome P450 (CYP), the cytochrome P450 oxidoreductase (CPR) and cytochrome b 5 and plays a key role in the metabolism and detoxification of xenobiotic substances, including pesticides. In addition, overexpression of these components has been linked to pesticide resistance in several important vectors of disease. Despite this, the monooxygenase complex has not been isolated from the Southern cattle tick Rhipicephalus (Boophilus) microplus, a major disease vector in livestock.Using bioinformatics 115 transcriptomic sequences were analyzed to identify putative pesticide metabolizing CYPs. RACE-PCR was used to amplify the full length sequence of one CYP; CYP3006G8 which displays a high degree of homology to members of the CYP6 and 9 subfamilies, known to metabolize pyrethroids. mRNA expression levels of CYP3006G8 were investigated in 11 strains of R. microplus with differing resistance profiles by qPCR, the results of which indicated a correlation with pyrethroid metabolic resistance.In addition to this gene, the sequences for CPR and cytochrome b 5 were also identified and subsequently isolated from R. microplus using PCR.CYP3006G8 is only the third CYP gene isolated from R. microplus and the first to putatively metabolize pesticides. The initial results of expression analysis suggest that CYP3006G8 metabolizes pyrethroids but further biochemical characterization is required to confirm this.Differences in the kinetic parameters of human and mosquito CPR in terms of NADPH binding have been demonstrated and could potentially be used to design species specific pesticides. Similar differences in the tick CPR would confirm that this is a characteristic of heamatophagous arthropods.

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Crystal structure of a NADPH‐cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP⁺ binding

et al. 2019

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Heme oxygenase‐1 (HMOX1) catalyzes heme degradation utilizing reducing equivalents supplied from NADPH‐cytochrome P450 reductase (CYPOR). Recently, we determined the complex structure of NADP+‐bound open‐conformation stabilized CYPOR and heme‐HMOX1, but the resolution was limited to 4.3 Å. Here, we determined the crystal structure of the fusion protein of open‐conformation stabilized CYPOR and heme‐HMOX1 at 3.25 Å resolution. Unexpectedly, no NADP+ was bound to this fusion protein in the crystal. Structural comparison of the NADP+‐bound complex and the NADP+‐free fusion protein suggests that NADP+ binding regulates the conformational change in the FAD‐binding domain of CYPOR. As a result of this change, the FMN‐binding domain of CYPOR approaches heme‐bound HMOX1 upon NADP+ binding to enhance the electron‐transfer efficiency from FMN to heme.

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NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family

Cited by 105 publications

References 213 publications

Molecular architecture of mammalian nitric oxide synthases

Molecular architecture of mammalian nitric oxide synthases

Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus – clues to understanding acaricide resistance

Crystal structure of a NADPH‐cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP⁺ binding

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NADPH–cytochrome P450 oxidoreductase: Prototypic member of the diflavin reductase family

Cited by 105 publications

References 213 publications

Molecular architecture of mammalian nitric oxide synthases

Molecular architecture of mammalian nitric oxide synthases

Isolation of the monooxygenase complex from Rhipicephalus (Boophilus) microplus – clues to understanding acaricide resistance

Crystal structure of a NADPH‐cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP+ binding

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Crystal structure of a NADPH‐cytochrome P450 oxidoreductase (CYPOR) and heme oxygenase 1 fusion protein implies a conformational change in CYPOR upon NADPH/NADP⁺ binding