Nicotinamide Adenine Dinucleotide Phosphate Binding Research Articles

Exploring the clinical implications of novel SRD5A2 variants in 46,XY disorders of sex development.

This study was conducted retrospectively on a cohort of 68 patients with steroid 5 α-reductase 2 (SRD5A2) deficiency and 46,XY disorders of sex development (DSD). Whole-exon sequencing revealed 28 variants of SRD5A2, and further analysis identified seven novel mutants. The preponderance of variants was observed in exon 1 and exon 4, specifically within the nicotinamide adenine dinucleotide phosphate (NADPH)-binding region. Among the entire cohort, 53 patients underwent initial surgery at Sichuan Provincial People's Hospital (Chengdu, China). The external genitalia scores (EGS) of these participants varied from 2.0 to 11.0, with a mean of 6.8 (standard deviation [s.d.]: 2.5). Thirty patients consented to hormone testing. Their average testosterone-to-dihydrotestosterone (T/DHT) ratio was 49.3 (s.d.: 23.4). Genetic testing identified four patients with EGS scores between 6 and 9 as having this syndrome; and their T/DHT ratios were below the diagnostic threshold. Furthermore, assessments conducted using the crystal structure of human SRD5A2 have provided insights into the potential pathogenic mechanisms of these novel variants. These mechanisms include interference with NADPH binding (c.356G>C, c.365A>G, c.492C>G, and c.662T>G) and destabilization of the protein structure (c.727C>T). The c.446-1G>T and c.380delG variants were verified to result in large alterations in the transcripts. Seven novel variations were identified, and the variant database for the SRD5A2 gene was expanded. These findings contribute to the progress of diagnostic and therapeutic approaches for individuals with SRD5A2 deficiency.

Biochemical characterisation of Cytochrome P450 oxidoreductase from the cattle tick, Rhipicephalus microplus, highlights potential new acaricide target

Management of the cattle tick, Rhipicephalus microplus, presents a challenge because some populations of this cosmopolitan and economically important ectoparasite are resistant to multiple classes of acaricides. Cytochrome P450 oxidoreductase (CPR) is part of the cytochrome P450 (CYP450) monooxygenases that are involved in metabolic resistance by their ability to detoxify acaricides. Inhibiting CPR, the sole redox partner that transfers electrons to CYP450s, could overcome this type of metabolic resistance. This report represents the biochemical characterisation of a CPR from ticks. Recombinant CPR of R. microplus (RmCPR), minus its N-terminal transmembrane domain, was produced in a bacterial expression system and subjected to biochemical analyses. RmCPR displayed a characteristic dual flavin oxidoreductase spectrum. Incubation with nicotinamide adenine dinucleotide phosphate (NADPH) lead to an increase in absorbance between 500 and 600 nm with a corresponding appearance of a peak absorbance at 340–350 nm indicating functional transfer of electrons between NADPH and the bound flavin cofactors. Using the pseudoredox partner, kinetic parameters for both cytochrome c and NADPH binding were calculated as 26.6 ± 11.4 µM and 7.03 ± 1.8 µM, respectively. The turnover, Kcat, for RmCPR for cytochrome c was calculated as 0.08 s−1 which is significantly lower than the CPR homologues of other species. IC50 (Half maximal Inhibitory Concentration) values obtained for the adenosine analogues 2’, 5’ ADP, 2’- AMP, NADP+and the reductase inhibitor diphenyliodonium were: 140, 82.2, 24.5, and 75.3 µM, respectively. Biochemically, RmCPR resembles CPRs of hematophagous arthropods more so than mammalian CPRs. These findings highlight the potential of RmCPR as a target for the rational design of safer and potent acaricides against R. microplus.

Proteome changes of dairy calves rumen epithelium from birth to postweaning.

Background: Rumen epithelium plays a central role in absorbing, transporting, and metabolizing of short-chain fatty acids. For dairy calves, the growth of rumen papillae greatly enhances the rumen surface area to absorb nutrients. However, the molecular mechanism underlying dairy calves rumen postnatal development remains rarely understood. Results: Here, we firstly describe the histological change of rumen epithelium from birth to day 90 of age. Then, a shotgun approach and bioinformatics analyses were used to investigate and compare proteomic profiles of Holstein calve rumen epithelium on day 0, 30, 60 and 90 of age. A total of 4372 proteins were identified, in which we found 852, 342, 164 and 95 differentially expressed proteins between D0 and D30, between D30 and D60, between D60 and D90, respectively. Finally, Gene Ontology and Kyoto Encyclopedia of Genes and Genomes (KEGG) analyses were performed to provide a comprehensive proteomic landscape of dairy calves rumen development at tissue level. Conclusion: To conclude, our data indicated that keratinocyte differentiation, mitochondrion formation, the establishment of urea transport and innate immune system play central roles during rumen epithelium development. Tetrahydrobiopterin (BH4) presents an important role in rumen epithelial keratinization. The biological processes of BH4 biosynthesis and molecular function of nicotinamide adenine dinucleotide phosphate binding participate in mitochondrial cristae formation. The proposed datasets provide a useful basis for future studies to better comprehend dairy calves rumen epithelial development.

Identification, analysis, and modeling of the YUCCA protein family genome-wide in Coffea canephora.

Auxin is involved in almost every aspect of plant growth and development, from embryogenesis to senescence. Indole-3-acetic acid (IAA) is the main known natural auxin that is synthesized by enzymes tryptophan aminotransferase of arabidopsis (TAA) and YUCCA (YUC) of the flavin-containing monooxygenases family (FMO) from one of the tryptophan-dependent pathways. Genome-wide identification and comprehensive analysis of the YUC-protein family have been conducted in Coffea canephora in the present study. A total of 10 members CcYUC gene family were identified in C. canephora. Phylogenetic analysis revealed that the CcYUC protein family is evolutionarily conserved, and they consist of four groups. In contrast, bioinformatic analysis predicted a hydrophobic transmembrane helix (TMH) for one CcYUC (YUC10) member only. Isoelectric point (pI), molecular mass (Ms), signal peptide, subcellular localization, and phosphorylation sites were predicted for CcYUC proteins. YUC enzymes require the prosthetic group flavin adenine dinucleotide (FAD) and the cofactor nicotinamide adenine dinucleotide phosphate (NADPH) for their enzymatic activity. Therefore, we include the molecular docking for CcYUC2-FAD-NADPH-IPyA and yucasin, which is a specific inhibitor for YUC activity. The docking results showed FAD and NADPH binding at the big and small domain sites, respectively, in CcYUC2. IPyA binds very close to FAD along the big domain, and yucasin competes for the same site as IPA, blocking IAA production. Furthermore, in silico point mutations affect the stability of the CcYUC2-4 proteins.

Structural basis for substrate recognition of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis

Glucose-6-phosphate dehydrogenase is the first enzyme in the pentose phosphate pathway. The reaction catalyzed by the enzyme is considered to be the main source of reducing power for nicotinamide adenine dinucleotide phosphate (NADPH) and is a precursor of 5-carbon sugar used by cells. To uncover the structural features of the enzyme, we determined the crystal structures of glucose-6-phosphate dehydrogenase from Kluyveromyces lactis (KlG6PD) in both the apo form and a binary complex with its substrate glucose-6-phosphate. KlG6PD contains a Rossman-like domain for cofactor NADPH binding; it also presents a typical antiparallel β sheet at the C-terminal domain with relatively the same pattern as those of other homologous structures. Moreover, our structural and biochemical analyses revealed that Lys153 contributes significantly to substrate G6P recognition. This study may provide insights into the structural variation and catalytic features of the G6PD enzyme.

Chemo-enzymatic synthesis of adenine substituted nicotinic acid adenine dinucleotide phosphate (NAADP) analogs

Nicotinamide adenine dinucleotide phosphate (NADP) is an indispensable metabolic co-substrate and nicotinic acid adenine dinucleotide phosphate (NAADP) is an important Ca2+ releasing intracellular second messenger. Exploration of the NADP and NAADP interactome often requires the synthesis of NADP derivatives substituted on the adenosine nucleoside. The introduction of the 2′-phosphate of NADP makes the synthesis of substituted NADP derivatives difficult. We have employed recombinant human NAD kinase expressed in E. coli as an enzymatic reagent to convert readily available synthetic NAD derivatives to NADP analogs, which were subsequently transformed into NAADP derivatives using enzyme catalyzed pyridine base exchange. 8-Ethynyl-NADP, 8-ethynyl-NAADP and 5-N3-8-ethynyl-NAADP were synthesized starting from a protected 8-ethynyladenosine using a combination of chemical and enzymatic steps and the NAADP derivatives shown to be recognized by the sea urchin NAADP receptor at low concentration. Our methodology will enable researchers to produce mono- and bi-substituted NADP and NAADP analogs that can be applied in proteomic studies to identify NADP and NAADP binding proteins.

In silico and in vitro comparison of nicotinamide adenine dinucleotide phosphate dependent xylose reductase rossmaan fold in Debaryomycetaceae yeast family

abstract Meyerozyma caribbica is prominently used for the production of xylitol from agro waste hydrolysate of the corncob. M. caribbica was isolated from fermented apple juice, the nicotinamide adenine dinucleotide phosphate (NADP) dependent xylose reductase (XR) enzyme act as a catalyst for the production of xylitol from xylose during xylose fermentation by M. caribbica. The enzyme XR was extracted, partially purified, sequenced and molecular weight was determined by matrix-assisted laser desorption/ionization-time of flight (MALDI-TOF). This study explores the in silico and in vitro comparative analysis of the cofactor NADP binding mechanism in rossmann fold of isolated M. caribbica and other xylose fermenting Debaryomycetaceae family yeast. The rossmaan fold analysis of M. caribbica and Debaryomycetaceae family of yeast revealed, xylose fermenting organisms such as Scheffersomyces stipitis, Debaryomyces hansenii, Debaryomyces nepalensis, Candida parapsilosis, Candida maltosa have divergence structure of rosmann fold. Whereas M. caribbica and Candida tenuis has unique structure of rossmann fold. Also, the identified rossmann fold of Debaryomycetaceae family yeast and isolated M. caribbica are conserved to interact with cofactor NADP by H2 bonding, which displays the proximity evolutionary relationship of the enzyme in Debaryomycetaceae family yeast. Annotation analysis showed the isolated M. caribbica has the ability of aldopentose fermentation for xylitol production.

Positional Distributions of the Tethered Modules in Nitric Oxide Synthase: Monte Carlo Calculations and Pulsed EPR Measurements.

The nitric oxide synthase (NOS) enzyme consists of multiple domains connected by flexible random coil tethers. In a catalytic cycle, the NOS domains move within the limits determined by the length and flexibility of the interdomain tethers and form docking complexes with each other. This process represents a key component of the electron transport from the flavin adenine dinucleotide/reduced nicotinamide adenine dinucleotide phosphate binding domain to the catalytic heme centers located in the oxygenase domain. Studying the conformational behavior of NOS is therefore imperative for a full understanding of the overall catalytic mechanism. In this work, we have investigated the equilibrium positional distributions of the NOS domains and the bound calmodulin (CaM) by using Monte Carlo calculations of the NOS conformations. As a main experimental reference, we have used the magnetic dipole interaction between a bifunctional spin label attached to T34C/S38C mutant CaM and the NOS heme centers, which was measured by pulsed electron paramagnetic resonance. In general, the calculations of the conformational distributions allow one to determine the range and statistics of positions occupied by the tethered protein domains, assess the crowding effect of the multiple domains on each other, evaluate the accessibility of various potential domain docking sites, and estimate the interaction energies required to achieve target populations of the docked states. In the particular application described here, we have established the specific mechanisms by which the bound CaM facilitates the flavin mononucleotide (FMN)/heme interdomain docking in NOS. We have also shown that the intersubunit FMN/heme domain docking and electron transfer in the homodimeric NOS protein are dictated by the existing structural makeup of the protein. Finally, from comparison of the calculated and experimental docking probabilities, the characteristic stabilization energies for the CaM/heme domain and the FMN domain/heme domain docking complexes have been estimated as -4.5kT and -10.5kT, respectively.

Crystal Structure of the Apo-Form of NADPH-Dependent Thioredoxin Reductase from a Methane-Producing Archaeon.

The redox regulation of proteins via reversible dithiol/disulfide exchange reactions involves the thioredoxin system, which is composed of a reductant, a thioredoxin reductase (TR), and thioredoxin (Trx). In the pyridine nucleotide-dependent Trx reduction pathway, reducing equivalents, typically from reduced nicotinamide adenine dinucleotide phosphate (NADPH), are transferred from NADPH-TR (NTR) to Trx and, in turn, to target proteins, thus resulting in the reversible modification of the structural and functional properties of the targets. NTR enzymes contain three functional sites: an NADPH binding pocket, a non-covalently bound flavin cofactor, and a redox-active disulfide in the form of CxxC. With the aim of increasing our knowledge of the thioredoxin system in archaea, we here report the high-resolution crystal structure of NTR from the methane-generating organism Methanosarcina mazei strain Gö1 (MmNTR) at 2.6 Å resolution. Based on the crystals presently described, MmNTR assumes an overall fold that is nearly identical to the archetypal fold of authentic NTRs; however, surprisingly, we observed no electron density for flavin adenine dinucleotide (FAD) despite the well-defined and conserved FAD-binding cavity in the folded module. Remarkably, the dimers of the apo-protein within the crystal were different from those observed by small angle X-ray scattering (SAXS) for the holo-protein, suggesting that the binding of the flavin cofactor does not require major protein structural rearrangements. Rather, binding results in the stabilization of essential parts of the structure, such as those involved in dimer stabilization. Altogether, this structure represents the example of an apo-form of an NTR that yields important insight into the effects of the cofactor on protein folding.

Anaerobic Heme Degradation: ChuY Is an Anaerobilin Reductase That Exhibits Kinetic Cooperativity.

Heme catabolism is an important biochemical process that many bacterial pathogens utilize to acquire iron. However, tetrapyrrole catabolites can be reactive and often require further processing for transport out of the cell or conversion to another useful cofactor. In previous work, we presented in vitro evidence of an anaerobic heme degradation pathway in Escherichia coli O157:H7. Consistent with reactions that have been reported for other radical S-adenosyl-l-methionine methyltransferases, ChuW transfers a methyl group to heme by a radical-mediated mechanism and catalyzes the β-scission of the porphyrin macrocycle. This facilitates iron release and the production of a new linear tetrapyrrole termed "anaerobilin". In this work, we describe the structure and function of ChuY, an enzyme expressed downstream from chuW within the same heme utilization operon. ChuY is structurally similar to biliverdin reductase and forms a dimeric complex in solution that reduces anaerobilin to the product we have termed anaerorubin. Steady state analysis of ChuY exhibits kinetic cooperativity that is best explained by a random addition mechanism with a kinetically preferred path for initial reduced nicotinamide adenine dinucleotide phosphate binding.

Insights into severe 5,10-methylenetetrahydrofolate reductase deficiency: molecular genetic and enzymatic characterization of 76 patients.

5,10-Methylenetetrahydrofolate reductase (MTHFR) deficiency is the most common inherited disorder of folate metabolism and causes severe hyperhomocysteinaemia. To better understand the relationship between mutation and function, we performed molecular genetic analysis of 76 MTHFR deficient patients, followed by extensive enzymatic characterization of fibroblasts from 72 of these. A deleterious mutation was detected on each of the 152 patient alleles, with one allele harboring two mutations. Sixty five different mutations (42 novel) were detected, including a common splicing mutation (c.1542G>A) found in 21 alleles. Using an enzyme assay in the physiological direction, we found residual activity (1.7%-42% of control) in 42 cell lines, of which 28 showed reduced affinity for nicotinamide adenine dinucleotide phosphate (NADPH), one reduced affinity for methylenetetrahydrofolate, five flavin adenine dinucleotide-responsiveness, and 24 abnormal kinetics of S-adenosylmethionine inhibition. Missense mutations causing virtually absent activity were found exclusively in the N-terminal catalytic domain, whereas missense mutations in the C-terminal regulatory domain caused decreased NADPH binding and disturbed inhibition by S-adenosylmethionine. Characterization of patients in this way provides a basis for improved diagnosis using expanded enzymatic criteria, increases understanding of the molecular basis of MTHFR dysfunction, and points to the possible role of cofactor or substrate in the treatment of patients with specific mutations.

RNA interference of NADPH-cytochrome P450 reductase of the rice brown planthopper,Nilaparvata lugens,increases susceptibility to insecticides

NADPH-cytochrome P450 reductase (CPR) is essential for numerous biological reactions catalysed by microsomal cytochrome P450 monooxygenases (P450s). Knockdown of CPR in several insects leads to developmental defects and increased susceptibility to insecticides. However, information about the role of CPR in the brown planthopper, Nilaparvata lugens, is still unavailable. A full-length cDNA encoding CPR was cloned from N. lugens (NlCPR). The deduced amino acid sequence showed marked features of classical CPRs, such as an N-terminus membrane anchor, conserved domains for flavin mononucleotide, flavin adenine dinucleotide (FAD) and nicotinamide adenine dinucleotide phosphate binding, as well as an FAD-binding motif and catalytic residues. Phylogenetic analysis revealed that NlCPR was located in a branch along with bed bug and pea aphid hemipteran insects. NlCPR mRNA was detectable in all tissues and developmental stages of N. lugens, as determined by real-time quantitative PCR. NlCPR transcripts were most abundant in the abdomen in adults, and in first-instar nymphs. Injection of N. lugens with double-strand RNA (dsRNA) against NlCPR significantly reduced the transcription level of the mRNA, and silencing of NlCPR resulted in increased susceptibility in N. lugens to beta-cypermethrin and imidacloprid. The results provide first evidence that NlCPR contributes to the susceptibility to beta-cypermethrin and imidacloprid in N. lugens.

Structure of conjugated polyketone reductase from Candida parapsilosis IFO 0708 reveals conformational changes for substrate recognition upon NADPH binding

Conjugated polyketone reductase C2 (CPR-C2) from Candida parapsilosis IFO 0708, identified as a nicotinamide adenine dinucleotide phosphate (NADPH)-dependent ketopantoyl lactone reductase, belongs to the aldo-keto reductase superfamily. This enzyme reduces ketopantoyl lactone to D-pantoyl lactone in a strictly stereospecific manner. To elucidate the structural basis of the substrate specificity, we determined the crystal structures of the apo CPR-C2 and CPR-C2/NADPH complex at 1.70 and 1.80 Å resolutions, respectively. CPR-C2 adopted a triose-phosphate isomerase barrel fold at the core of the structure. Binding with the cofactor NADPH induced conformational changes in which Thr27 and Lys28 moved 15 and 5.0 Å, respectively, in the close vicinity of the adenosine 2'-phosphate group of NADPH to form hydrogen bonds. Based on the comparison of the CPR-C2/NADPH structure with 3-α-hydroxysteroid dehydrogenase and mutation analyses, we constructed substrate binding models with ketopantoyl lactone, which provided insight into the substrate specificity by the cofactor-induced structure. The results will be useful for the rational design of CPR-C2 mutants targeted for use in the industrial manufacture of ketopantoyl lactone.

Structural insights into cofactor recognition of yeast mitochondria 3-oxoacyl-ACP reductase OAR1

3-Oxoacyl-(acyl-carrier-protein) reductase (OAR1 or FabG, EC.1.1.1.100) is responsible for the first reductive step in fatty acid biosynthesis using Nicotinamide Adenine Dinucleotide Phosphate (NADPH) as a cofactor. Recent studies suggest there is a fatty acid synthetase II pathway that consists of a series of separate enzymes in yeast mitochondrion. Here, we present the crystal structure of the yeast mitochondria OAR1 (ymtOAR1) alone in apo-form at 2.60 Å and complexed with NADPH at 2.10 Å resolution. Unlike the reported tetrameric OARs, ymtOAR1 forms a homodimer due to the different fold. The enzyme generates conformational changes upon NADPH binding to the active site. Moreover, two different cofactor-binding patterns are observed from two forms of complex crystals, and structural analysis implies the adenine end of cofactor may recognize enzyme prior to nicotinaminde end. Additionally, biochemical studies suggest Arg14 is important for cofactor recognition of ymtOAR1.

Recombinant Nox4 cytosolic domain produced by a cell or cell-free base systems exhibits constitutive diaphorase activity

The membrane protein NADPH (nicotinamide adenine dinucleotide phosphate) oxidase Nox4 constitutively generates reactive oxygen species differing from other NADPH oxidases activity, particularly in Nox2 which needs a stimulus to be active. Although the precise mechanism of production of reactive oxygen species by Nox2 is well characterized, the electronic transfer throughout Nox4 remains unclear. Our study aims to investigate the initial electronic transfer step (diaphorase activity) of the cytosolic tail of Nox4. For this purpose, we developed two different approaches to produce soluble and active truncated Nox4 proteins. We synthesized soluble recombinant proteins either by in vitro translation or by bacteria induction. While proteins obtained by bacteria induction demonstrate an activity of 4.4±1.7nmol/min/nmol when measured against iodonitro tetrazolium chloride and 20.5±2.8nmol/min/nmol with cytochrome c, the soluble proteins produced by cell-free expression system exhibit a diaphorase activity with a turn-over of 26±2.6nmol/min/nmol when measured against iodonitro tetrazolium chloride and 48±20.2nmol/min/nmol with cytochrome c. Furthermore, the activity of the soluble proteins is constitutive and does not need any stimulus. We also show that the cytosolic tail of the isoform Nox4B lacking the first NADPH binding site is unable to demonstrate any diaphorase activity pointing out the importance of this domain.

Reduced Nicotinamide Adenine Dinucleotide Phosphate (NADPH) Blocks Transfer of [2Fe-2S] Clusters from MitoNEET to an Apo-Acceptor Protein

MitoNEET is the founding member of the recently discovered CDGSH family of [2Fe-2S] proteins. Its structure contains a unique homodimeric fold with each monomer containing one [2Fe-2S] cluster (1). MitoNEET is a target of the thiazolidinedione (TZD) class of anti-diabetes drugs (2). Recently, it was reported that the [2Fe-2S] cluster in mitoNEET is destabilized upon binding of NADPH/NADP+ (3). As mitoNEET is capable of transferring its [2Fe-2S] clusters to an apo-acceptor protein (4) and an obvious question to ask is whether NADPH binding accelerates or abrogates cluster transfer. We show that NADPH blocks transfer of mitoNEET's [2Fe-2S] cluster to an apo-acceptor protein (see Figure). This suggests a likely functional role for the mitoNEET:NADPH/NADP+ interaction.(1) Paddock ML et al. (2007) PNAS 104 14342-7(2) Colca JR et al. (2004) Am J Physiol Endocrinol Metab 286 E252-60(3) Zhou et al. (2010) Biochemistry 49 9604-12(4) Zuris JA et al. (2011) PNAS 108 13047-52∗Supported by the Heme and Blood Proteins Training Grant 5T32DK007233-34View Large Image | View Hi-Res Image | Download PowerPoint Slide

Molecular cloning and characterization of the NADPH oxidase from the kuruma shrimp, Marsupenaeus japonicus: Early gene up-regulation after Vibrio penaeicida and poly(I:C) stimulations in vitro

Free radicals such as nitric oxide (NO) and reactive oxygen species (ROS) are involved in many physiological processes. In humans, there are 5 homologs of nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (Noxes) that generate superoxide (O 2 −), which can dismute to produce ROS, and play significant roles in innate immunity and cell proliferation. Though Noxes have been identified in vertebrates (humans and fishes) and some insects, there are very few reports investigating Noxes in crustaceans. In the present study, we describe the entire cDNA sequence (4216 bp) of Marsupenaeus japonicus (kuruma shrimp) Nox ( MjNox) generated using reverse transcriptase-polymerase chain reaction (RT-PCR) and random amplification of cDNA ends (RACE). The open reading frame of MjNox encodes a protein of 1280 amino acids with an estimated mass of 146 kDa that has 46.8% sequence homology with the Nox gene of the fruit fly, Drosophila melanogaster. Highly conserved amino acid sequences were observed in the NADPH binding domain. Transcriptional analysis revealed that MjNox mRNA is highly expressed in the lymphoid organ, hepatopancreas and hemocytes of the healthy kuruma shrimp. In the hemocytes, MjNox expression reached its peak 4 h after stimulation with either Vibrio penaeicida or poly(I:C) and decreased to its normal level after 12 h.This study is the first to identify and clone a Nox family member ( MjNox) from a crustacean species.

Binding of Reduced Nicotinamide Adenine Dinucleotide Phosphate Destabilizes the Iron−Sulfur Clusters of Human MitoNEET

The outer mitochondrial membrane protein mitoNEET is a cellular target of the antidiabetic drug pioglitazone. Binding of pioglitazone stabilizes the protein against [2Fe-2S] cluster release. Here, we report that reduced nicotinamide adenine dinucleotide phosphate (NADPH) can bind to homodimeric mitoNEET, influencing the stability of the [2Fe-2S] cluster that is bound within a loop region (Y71−H87) in each subunit. Nuclear magnetic resonance (NMR) and isothermal titration calorimetry experiments demonstrated that NADPH binds weakly to mitoNEET(44−108), a soluble domain of mitoNEET containing residues 44−108. Visible−UV absorption measurements revealed the destabilizing effect of NADP binding on the [2Fe-2S] clusters. Disruption of the three-dimensional structure of mitoNEET(44−108) as a result of decomposition of the iron−sulfur clusters was observed by NMR and circular dichroism experiments. Binding of NADPH facilitated release of the iron−sulfur clusters from the protein at pH≤7.0. Residues K55 and H58 of each subunit of mitoNEET were shown to be involved in NADPH binding. NADPH binding may perturb the interactions of K55 and H58 from one subunit with H87′ and R73′, respectively, from the other subunit, thereby interfering with [2Fe-2S] cluster binding. This may account for the destabilization effect of NADPH binding on the [2Fe-2S] clusters.

Effect of ligand binding on the intraminimum dynamics of proteins

Effects of ligand binding on protein dynamics are studied via molecular dynamics (MD) simulations on two different enzymes, dihydrofolate reductase (DHFR) and triosephosphate isomerase (TIM), in their unliganded (free) and liganded states. Domain motions in MD trajectories are analyzed by collectivities and rotation angles along the principal components (PCs). DHFR in the free state has well-defined domain rotations, whereas rotations are slightly damped in the binary complex with nicotinamide adenine dinucleotide phosphate (NADPH), and remarkably distorted in the presence of NADP(+) , showing that NADP(+) is solely responsible for the loss of correlation of the domains in DHFR. Although mean square fluctuations of MD simulations in the same PC subspaces are similar for different ligation states, linear stochastic time series models show that backbone flexibility along the first five PCs is decreased upon NADPH and NADP(+) binding in subpicosecond scale. This shows that mobility of the protein along the PCs is closely related with intraminimum dynamics, and alterations in ligation states may change the intraminimum dynamics significantly. Low vibrational frequencies of the alpha-carbon atoms of DHFR are determined from the time series models of a larger number of low indexed PCs, and it is found that number of modes in the lowest frequencies is reduced upon ligand binding. A similar result is obtained for TIM in the unliganded and dihydroxyacetone phosphate bound states. We suggest that stochastic time series modeling is a promising method to be used in determining subtle perturbations in protein dynamics.

The C-terminal flavin domain of gp91phox bound to plasma membranes of granulocyte-like X-CGD PLB-985 cells is sufficient to anchor cytosolic oxidase components and support NADPH oxidase-associated diaphorase activity independent of cytosolic phospholipase A2 regulation

We have previously established a model of cytosolic phospholipase A(2) (cPLA(2))-deficient PLB-985 cells and demonstrated that cPLA(2)-generated arachidonic acid (AA) is essential for reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activation and NADPH-dependent diaphorase activity. The present study focuses on the C-terminal cytoplasmic domain of gp91(phox) (residues 283-570), which contains the NADPH binding and flavin adenine dinucleotide-reducing center, to determine if this portion is regulated by AA. The gp91(phox) C-terminal reductase domain was expressed in X-CGD PLB-985 cells lacking normal gp91(phox) (X-CGD PLB 91CT cells) and was detected in the plasma membrane. It appears to be bound electrostatically to the plasma membrane, as it is eluted by high salt. Permeabilized, granulocyte-like X-CGD PLB 91CT cells lacking cPLA(2) protein and activity, as well as AA release after stimulation, supported NADPH-dependent diaphorase activity after stimulation, similar to granulocyte-like X-CGD PLB 91CT cells. Normal translocation of p47(phox) and p67(phox) to the membrane fractions of both stimulated cell types indicated that the gp91(phox) C-terminal region is sufficient to anchor the cytosolic oxidase components to the membranes. cPLA(2) translocated to membranes and bound the assembled oxidase in granulocyte-like X-CGD PLB 91CT cells after stimulation. Therefore, the assembled membrane-bound oxidase complex encompassing the flavin domain of gp91(phox) provides a docking site for cPLA(2) but is not the site of AA-based regulation of oxidase activity.