Trimethylaminuria (TMAuria), the excessive urinary excretion of the odorous trimethylamine (TMA), accompanies elimination of TMA in sweat and corresponding "fish-odor" syndrome. TMA was oxidized in vitro in rat liver microsomes from male Sprague-Dawley rats to TMA N-oxide and N-demethylated to dimethylamine (DMA). Both reactions were inhibited to 1-3% of normal activity by preincubation of microsomes without NADPH-generating system at 37 degrees C for 10 minutes indicating the FAD-containing monooxygenase-catalyzed reactions. On the other hand, the reactions were not inhibited by gas phase containing up to 80% carbon monoxide/20% oxygen mixture. The results are compatible with the hypothesis that in rat liver microsomes the N-oxygenation and N-demethylation of TMA are catalyzed only or predominantly by FAD-containing monooxygenases, and the cytochrome P-450 monooxygenases play a negligible, if any, role.
- MeSH
- chromatografie plynová MeSH
- dealkylace MeSH
- flavinadenindinukleotid * metabolismus MeSH
- inbrední kmeny potkanů MeSH
- jaterní mikrozomy * enzymologie MeSH
- krysa rodu rattus MeSH
- methylaminy * metabolismus MeSH
- NAD metabolismus MeSH
- NADP metabolismus MeSH
- oxidace-redukce MeSH
- oxygenasy se smíšenou funkcí * metabolismus MeSH
- systém (enzymů) cytochromů P-450 metabolismus MeSH
- techniky in vitro MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
BACKGROUND: Flax (Linum usitatissimum L.) is grown for fiber and seed in many countries. Flax cultivars differ in the oil composition and, depending on the ratio of fatty acids, are used in pharmaceutical, food, or paint industries. It is known that genes of SAD (stearoyl-ACP desaturase) and FAD (fatty acid desaturase) families play a key role in the synthesis of fatty acids, and some alleles of these genes are associated with a certain composition of flax oil. However, data on genetic polymorphism of these genes are still insufficient. RESULTS: On the basis of the collection of the Institute for Flax (Torzhok, Russia), we formed a representative set of 84 cultivars and lines reflecting the diversity of fatty acid composition of flax oil. An approach for the determination of full-length sequences of SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes using the Illumina platform was developed and deep sequencing of the 6 genes in 84 flax samples was performed on MiSeq. The obtained high coverage (about 400x on average) enabled accurate assessment of polymorphisms in SAD1, SAD2, FAD2A, FAD2B, FAD3A, and FAD3B genes and evaluation of cultivar/line heterogeneity. The highest level of genetic diversity was observed for FAD3A and FAD3B genes - 91 and 62 polymorphisms respectively. Correlation analysis revealed associations between particular variants in SAD and FAD genes and predominantly those fatty acids whose conversion they catalyze: SAD - stearic and oleic acids, FAD2 - oleic and linoleic acids, FAD3 - linoleic and linolenic acids. All except one low-linolenic flax cultivars/lines contained both the substitution of tryptophan to stop codon in the FAD3A gene and histidine to tyrosine substitution in the FAD3B gene, while samples with only one of these polymorphisms had medium content of linolenic acid and cultivars/lines without them were high-linolenic. CONCLUSIONS: Genetic polymorphism of SAD and FAD genes was evaluated in the collection of flax cultivars and lines with diverse oil composition, and associations between particular polymorphisms and the ratio of fatty acids were revealed. The achieved results are the basis for the development of marker-assisted selection and DNA-based certification of flax cultivars.
- MeSH
- desaturasy mastných kyselin genetika MeSH
- DNA rostlinná MeSH
- genetická heterogenita MeSH
- genetická variace * MeSH
- kyselina alfa-linolenová metabolismus MeSH
- len enzymologie genetika metabolismus MeSH
- mastné kyseliny metabolismus MeSH
- oxygenasy se smíšenou funkcí genetika metabolismus MeSH
- rostlinné geny MeSH
- sekvenční analýza DNA MeSH
- substituce aminokyselin MeSH
- Publikační typ
- časopisecké články MeSH
Alzheimerova nemoc (AD) patří mezi na věku závislá neurodegenerativní onemocnění a je současně nejčastější příčinou demence u stárnoucích osob. Nejčastější podobu primární demence představují idiopatické formy AD, jejichž příznaky se objevují u osob po 65. roce věku, zatímco malá část nemocných trpí familiární formou této poruchy (FAD), s nástupem klinických příznaků před 55. rokem věku. Pokrok v oblasti lidské genetiky vztahující se k AD přinesl objev 3 genů (APP, PSENl a PSEN2), které odpovídají za autozomálně dominantní formu FAD v časným nástupem. Avšak výzkum odhalil jen jediný významný kandidátní gen (APOE) pro idiopatickou formu AD. V současnosti existují i významné doklady o existenci dalších genů majících vztah k oběma hlavním formám AD. Kromě již zmíněných genů existuje i gen MAPT pro tau protein, který se podílí na fenomenologii AD se vztahem k frontotemporální demenci S projevy začínajícími kolem 60. roku věku. Tento krátký přehled shrnuje především výsledky nedávného genetického výzkumu, které umožňují novou strategii v predikci AD, a to navzdory složitosti a genetické heterogenite této devastující neuropsychiatrické poruchy s velkým socioekonomickým dopadem.
Alzheimer ́s disease (AD) is the most common age-related neurodegenerative disorder and the most frequent cause of dementia duri ng aging. Most forms of AD are idiopathic, with the onset of symptoms after 65 years of age, but a small part of patients exhibit familial form of dementia (FAD) with clinical symptoms appearing before age 55. Genetic progress in the field of AD revealed three genes (APP, PSEN1 and PSEN2) responsible for the autosomal-dominant early onset form of FAD and one late-onset candidate gene (APOE) for idiopathic AD. However, strong evidence exists suggesting the presence of additional AD genes for both forms of the disease. Besides genes for the familial and idiopathic forms, also MAPT gene for tau protein is involved in the phenomenology of AD, being related to frontotemporal dementia with the onset around age 60. This minireview is focused at recent genetic research allowing new strategies in the pre diction of AD in spite of complexity and genetic heterogeneity of this devastating disease with great social and economic impacts.
- MeSH
- Alzheimerova nemoc diagnóza etiologie genetika MeSH
- aminokyseliny, peptidy a proteiny genetika metabolismus MeSH
- amyloidový prekurzorový protein beta genetika metabolismus MeSH
- apolipoproteiny E genetika metabolismus MeSH
- demence etiologie genetika MeSH
- genetické markery genetika MeSH
- glutamátové receptory genetika metabolismus MeSH
- lidé MeSH
- přehledová literatura jako téma MeSH
- preseniliny genetika metabolismus MeSH
- Check Tag
- lidé MeSH
Cytokinins are hormones that regulate plant development and their environmental responses. Their levels are mainly controlled by the cytokinin oxidase/dehydrogenase (CKO), which oxidatively cleaves cytokinins using redox-active electron acceptors. CKO belongs to the group of flavoproteins with an 8α-N1-histidyl FAD covalent linkage. Here, we investigated the role of seven active site residues, H105, D169, E288, V378, E381, P427 and L492, in substrate binding and catalysis of the CKO1 from maize (Zea mays, ZmCKO1) combining site-directed mutagenesis with kinetics and X-ray crystallography. We identify E381 as a key residue for enzyme specificity that restricts substrate binding as well as quinone electron acceptor binding. We show that D169 is important for catalysis and that H105 covalently linked to FAD maintains the enzyme's structural integrity, stability and high rates with electron acceptors. The L492A mutation significantly modulates the cleavage of aromatic cytokinins and zeatin isomers. The high resolution X-ray structures of ZmCKO1 and the E381S variant in complex with N6-(2-isopentenyl)adenosine reveal the binding mode of cytokinin ribosides. Those of ZmCKO2 and ZmCKO4a contain a mobile domain, which might contribute to binding of the N9 substituted cytokinins.
- MeSH
- cytokininy metabolismus MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- katalytická doména MeSH
- kinetika MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- kukuřice setá enzymologie MeSH
- mutageneze cílená MeSH
- oxidoreduktasy chemie genetika metabolismus MeSH
- substrátová specifita MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Glucose oxidase (GOX) is a homodimeric glycoprotein with tightly bound one molecule of FAD cofactor per monomer of the protein. GOX has numerous applications, but the preparation of biotechnologically interesting GOX sensors requires a removal of the native FAD cofactor. This process often leads to unwanted irreversible deflavination and, as a consequence, to the low enzyme recovery. Molecular mechanisms of reversible reflavination are poorly understood; our current knowledge is based only on empiric rules, which is clearly insufficient for further development. To develop conceptual understanding of flavin-binding competent states, we studied the effect of deflavination protocols on conformational properties of GOX. After deflavination, the apoform assembles into soluble oligomers with nearly native-like holoform secondary structure but largely destabilized tertiary structure presumambly due to the packing density defects around the vacant flavin binding site. The reflavination is cooperative but not fully efficient; after the binding the flavin cofactor, the protein directly disassembles into native homodimers while the fraction of oligomers remains irreversibly inactivated. Importantly, the effect of Hofmeister salts on the conformational properties of GOX and reflavination efficiency indicates that the native-like residual tertiary structure in the molten-globule states favorably supports the reflavination and minimizes the inactivated oligomers. We interpret our results by combining the ligand-induced changes in quaternary structure with salt-sensitive, non-equilibrated conformational selection model. In summary, our work provides the very first steps toward molecular understanding the complexity of the GOX reflavination mechanism.
- MeSH
- Aspergillus niger enzymologie MeSH
- biokatalýza MeSH
- cirkulární dichroismus MeSH
- diferenciální skenovací kalorimetrie MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- glukosaoxidasa chemie metabolismus MeSH
- multimerizace proteinu MeSH
- protein - isoformy chemie metabolismus MeSH
- sekundární struktura proteinů MeSH
- spektrofotometrie ultrafialová MeSH
- stabilita proteinů MeSH
- teplota MeSH
- terciární struktura proteinů MeSH
- Publikační typ
- časopisecké články MeSH
The Escherichia coli protein WrbA, an FMN-dependent NAD(P)H:quinone oxidoreductase, was crystallized under new conditions in the presence of FAD or the native cofactor FMN. Slow-growing deep yellow crystals formed with FAD display the tetragonal bipyramidal shape typical for WrbA and diffract to 1.2 Å resolution, the highest yet reported. Faster-growing deep yellow crystals formed with FMN display an atypical shape, but diffract to only ∼1.6 Å resolution and are not analysed further here. The 1.2 Å resolution structure detailed here revealed only FMN in the active site and no electron density that can accommodate the missing parts of FAD. The very high resolution supports the modelling of the FMN isoalloxazine with a small but distinct propeller twist, apparently the first experimental observation of this predicted conformation, which appears to be enforced by the protein through a network of hydrogen bonds. Comparison of the electron density of the twisted isoalloxazine ring with the results of QM/MM simulations is compatible with the oxidized redox state. The very high resolution also supports the unique refinement of Met10 as the sulfoxide, confirmed by mass spectrometry. Bond lengths, intramolecular distances, and the pattern of hydrogen-bond donors and acceptors suggest the cofactor may interact with Met10. Slow incorporation of FMN, which is present as a trace contaminant in stocks of FAD, into growing crystals may be responsible for the near-atomic resolution, but a direct effect of the conformation of FMN and/or Met10 sulfoxide cannot be ruled out.
- MeSH
- difrakce rentgenového záření MeSH
- flavinadenindinukleotid chemie metabolismus MeSH
- flavinmononukleotid chemie metabolismus MeSH
- krystalizace MeSH
- krystalografie rentgenová MeSH
- NAD(P)H dehydrogenasa (chinon) chemie metabolismus MeSH
- oxidace-redukce MeSH
- proteiny z Escherichia coli chemie metabolismus MeSH
- represorové proteiny chemie metabolismus MeSH
- vazba proteinů MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. MeSH
The multifunctional nature of human flavoproteins is critically linked to their ability to populate multiple conformational states. Ligand binding, post-translational modifications and disease-associated mutations can reshape this functional landscape, although the structure-function relationships of these effects are not well understood. Herein, we characterized the structural and functional consequences of two mutations (the cancer-associated P187S and the phosphomimetic S82D) on different ligation states which are relevant to flavin binding, intracellular stability and catalysis of the disease-associated NQO1 flavoprotein. We found that these mutations affected the stability locally and their effects propagated differently through the protein structure depending both on the nature of the mutation and the ligand bound, showing directional preference from the mutated site and leading to specific phenotypic manifestations in different functional traits (FAD binding, catalysis and inhibition, intracellular stability and pharmacological response to ligands). Our study thus supports that pleitropic effects of disease-causing mutations and phosphorylation events on human flavoproteins may be caused by long-range structural propagation of stability effects to different functional sites that depend on the ligation-state and site-specific perturbations. Our approach can be of general application to investigate these pleiotropic effects at the flavoproteome scale in the absence of high-resolution structural models.
Human NAD(P)H:quinone oxidoreductase 1 (NQO1) is a multi-functional protein whose alteration is associated with cancer, Parkinson's and Alzheimer´s diseases. NQO1 displays a remarkable functional chemistry, capable of binding different functional ligands that modulate its activity, stability and interaction with proteins and nucleic acids. Our understanding of this functional chemistry is limited by the difficulty of obtaining structural and dynamic information on many of these states. Herein, we have used hydrogen/deuterium exchange monitored by mass spectrometry (HDXMS) to investigate the structural dynamics of NQO1 in three ligation states: without ligands (NQO1apo), with FAD (NQO1holo) and with FAD and the inhibitor dicoumarol (NQO1dic). We show that NQO1apo has a minimally stable folded core holding the protein dimer, with FAD and dicoumarol binding sites populating binding non-competent conformations. Binding of FAD significantly decreases protein dynamics and stabilizes the FAD and dicoumarol binding sites as well as the monomer:monomer interface. Dicoumarol binding further stabilizes all three functional sites, a result not previously anticipated by available crystallographic models. Our work provides an experimental perspective into the communication of stability effects through the NQO1 dimer, which is valuable for understanding at the molecular level the effects of disease-associated variants, post-translational modifications and ligand binding cooperativity in NQO1.
- MeSH
- Alzheimerova nemoc enzymologie MeSH
- hmotnostní spektrometrie MeSH
- konformace proteinů * MeSH
- lidé MeSH
- multimerizace proteinu genetika MeSH
- NAD(P)H dehydrogenasa (chinon) chemie genetika ultrastruktura MeSH
- nádory enzymologie MeSH
- Parkinsonova nemoc enzymologie MeSH
- stabilita enzymů genetika MeSH
- vazba proteinů genetika MeSH
- vazebná místa MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
