Sperm metabolism is fundamental to sperm motility and male fertility. Its measurement is still in its infancy, and recommendations do not exist as to whether or how to standardize laboratory procedures. Here, using the sperm of an insect, the common bedbug, Cimex lectularius, we demonstrate that standardization of sperm metabolism is required with respect to the artificial sperm storage medium and a natural medium, the seminal fluid. We used fluorescence lifetime imaging microscopy (FLIM) in combination with time-correlated single-photon counting (TCSPC) to quantify sperm metabolism based on the fluorescent properties of autofluorescent coenzymes, NAD(P)H and flavin adenine dinucleotide. Autofluorescence lifetimes (decay times) differ for the free and protein-bound state of the co-enzymes, and their relative contributions to the lifetime signal serve to characterize the metabolic state of cells. We found that artificial storage medium and seminal fluid separately, and additively, affected sperm metabolism. In a medium containing sugars and amino acids (Grace's Insect medium), sperm showed increased glycolysis compared with a commonly used storage medium, phosphate-buffered saline (PBS). Adding seminal fluid to the sperm additionally increased oxidative phosphorylation, likely reflecting increased energy production of sperm during activation. Our study provides a protocol to measure sperm metabolism independently from motility, stresses that protocol standardizations for sperm measurements should be implemented and, for the first time, demonstrates that seminal fluid alters sperm metabolism. Equivalent protocol standardizations should be imposed on metabolic investigations of human sperm samples.

• MeSH
• flavinadenindinukleotid * MeSH
• mikroskopie fluorescenční multifotonová MeSH
• motilita spermií MeSH
• NADP * MeSH
• spermie účinky léků   MeSH
• štěnice MeSH
• zvířata MeSH
• Check Tag
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

• MeSH
• Basidiomycota enzymologie   metabolismus   růst a vývoj   MeSH
• finanční podpora výzkumu jako téma MeSH
• flavinadenindinukleotid chemie   MeSH
• karbohydrátdehydrogenasy chemie   izolace a purifikace   metabolismus   MeSH
• oxidace-redukce MeSH
• vazebná místa MeSH

UNLABELLED: The Pden_2689 gene encoding FerA, an NADH:flavin oxidoreductase required for growth of Paracoccus denitrificans under iron limitation, was cloned and overexpressed as a C-terminally His6-tagged derivative. The binding of substrates and products was detected and quantified by isothermal titration calorimetry and fluorometric titration. FerA binds FMN and FAD with comparable affinity in an enthalpically driven, entropically opposed process. The reduced flavin is bound more loosely than the oxidized one, which was confirmed by a negative shift in the redox potential of FMN after addition of FerA. Initial velocity and substrate analogs inhibition studies showed that FerA follows a random-ordered sequence of substrate (NADH and FMN) binding. The primary kinetic isotope effects from stereospecifically deuterated nicotinamide nucleotides demonstrated that hydride transfer occurs from the pro-S position and contributes to rate limitation for the overall reaction. The crystal structure of FerA revealed a twisted seven-stranded antiparallel β-barrel similar to that of other short chain flavin reductases. Only minor structural changes around Arg106 took place upon FMN binding. The solution structure FerA derived from small angle X-ray scattering (SAXS) matched the dimer assembly predicted from the crystal structure. Site-directed mutagenesis pinpointed a role of Arg106 and His146 in binding of flavin and NADH, respectively. Pull down experiments performed with cytoplasmic extracts resulted in a negative outcome indicating that FerA might physiologically act without association with other proteins. Rapid kinetics experiments provided evidence for a stabilizing effect of another P. denitrificans protein, the NAD(P)H: acceptor oxidoreducase FerB, against spontaneous oxidation of the FerA-produced dihydroflavin.

• MeSH
• chromatografie afinitní MeSH
• exprese genu MeSH
• flavinadenindinukleotid metabolismus   MeSH
• flavinmononukleotid metabolismus   MeSH
• FMN-reduktasa chemie   genetika   metabolismus   MeSH
• kinetika MeSH
• klonování DNA MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• maloúhlový rozptyl MeSH
• molekulární modely MeSH
• multimerizace proteinu MeSH
• NAD metabolismus   MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• vazba proteinů MeSH
• Publikační typ
• časopisecké články MeSH

Pden_5119, annotated as an NADPH-dependent FMN reductase, shows homology to proteins assisting in utilization of alkanesulfonates in other bacteria. Here, we report that inactivation of the pden_5119 gene increased susceptibility to oxidative stress, decreased growth rate and increased growth yield; growth on lower alkanesulfonates as sulfur sources was not specifically influenced. Pden_5119 transcript rose in response to oxidative stressors, respiratory chain inhibitors and terminal oxidase downregulation. Kinetic analysis of a fusion protein suggested a sequential mechanism in which FMN binds first, followed by NADH. The affinity of flavin toward the protein decreased only slightly upon reduction. The observed strong viscosity dependence of kcat demonstrated that reduced FMN formed tends to remain bound to the enzyme where it can be re-oxidized by oxygen or, less efficiently, by various artificial electron acceptors. Stopped flow data were consistent with the enzyme-FMN complex being a functional oxidase that conducts the reduction of oxygen by NADH. Hydrogen peroxide was identified as the main product. As shown by isotope effects, hydride transfer occurs from the pro-S C4 position of the nicotinamide ring and partially limits the overall turnover rate. Collectively, our results point to a role for the Pden_5119 protein in maintaining the cellular redox state.

• MeSH
• flavinadenindinukleotid metabolismus   MeSH
• flavinmononukleotid metabolismus   MeSH
• flaviny metabolismus   MeSH
• FMN-reduktasa genetika   metabolismus   MeSH
• NADP MeSH
• NADPH-cytochrom c-reduktasa metabolismus   MeSH
• oxidace-redukce MeSH
• Paracoccus denitrificans genetika   metabolismus   MeSH
• sekvence aminokyselin genetika   MeSH
• terciární struktura proteinů MeSH
• transport elektronů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

The homodimeric flavoprotein FerB of Paracoccus denitrificans catalyzed the reduction of chromate with NADH as electron donor. When present, oxygen was reduced concomitantly with chromate. The recombinant enzyme had a maximum activity at pH 5.0. The stoichiometric ratio of NADH oxidized to chromate reduced was found to be 1.53 ± 0.09 (O(2) absent) or > 2 (O(2) present), the apparent K (M) value for chromate amounted to 70 ± 10 μM with the maximum rate of 2.9 ± 0.3 μmol NADH s(-1) (mg protein)(-1). Diode-array spectrophotometry and experiments with one-electron acceptors provided evidence for oxygen consumption being due to a flavin semiquinone, formed transiently during the interaction of FerB with chromate. At the whole-cell level, a ferB mutant strain displayed only slightly diminished rate of chromate reduction when compared to the wild-type parental strain. Anaerobically grown cells were more active than cells grown aerobically. The activity could be partly inhibited by antimycin, suggesting an involvement of the respiratory chain. Chromate concentrations above ten micromolars transiently slowed or halted culture growth, with the effect being more pronounced for the mutant strain. It appears, therefore, that, rather than directly reducing chromate, FerB confers a protection of cells against the oxidative stress accompanying chromate reduction. With a strain carrying the chromosomally integrated ferB promoter-lacZ fusion, it was shown that the ferB gene is not inducible by chromate.

• MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• chromany metabolismus   MeSH
• flavinadenindinukleotid analogy a deriváty   metabolismus   MeSH
• flavoproteiny genetika   metabolismus   MeSH
• FMN-reduktasa genetika   metabolismus   MeSH
• koncentrace vodíkových iontů MeSH
• NAD metabolismus   MeSH
• oxidace-redukce MeSH
• oxidační stres MeSH
• oxidoreduktasy genetika   metabolismus   MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• spotřeba kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem 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.

• MeSH
• chinony MeSH
• flavinadenindinukleotid metabolismus   MeSH
• lidé MeSH
• missense mutace * MeSH
• NAD(P)H dehydrogenasa (chinon) * genetika   metabolismus   MeSH
• NAD MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

Hemoglobin Haná [β63(E7) His-Asn] is an unstable hemoglobin variant that was described in a Czech proband and her sister with Heinz body hemolytic anemia. The mother bearing the same mutation was asymptomatic; nevertheless, all three carriers had the same proportion of the mutant globin chains. Assessment of several erythrocyte antioxidant parameters revealed that both symptomatic children, unlike their asymptomatic mother, had significantly decreased glutathione reductase (GR) activity. Their GR activities were restorable in vitro by flavin adenine dinucleotide. The riboflavin supplementation improved their glutathione metabolism and ameliorated their hemolysis. Pre- and post-treatment assessment of the B(2) vitamers indicated suboptimal pre-treatment vitamin B(2) status in both children. This study provides evidence that partial GR deficiency may alter the clinical manifestation of an unstable hemoglobinopathy.

• MeSH
• dospělí MeSH
• flavinadenindinukleotid farmakologie   MeSH
• glutathion metabolismus   MeSH
• glutathionreduktasa genetika   metabolismus   MeSH
• Heinzova tělíska MeSH
• hemoglobinopatie krev   farmakoterapie   genetika   MeSH
• hemoglobiny abnormální genetika   MeSH
• hemolytické anemie krev   farmakoterapie   genetika   MeSH
• lidé MeSH
• missense mutace MeSH
• mladiství MeSH
• riboflavin aplikace a dávkování   MeSH
• rodina MeSH
• substituce aminokyselin MeSH
• vitamin B komplex aplikace a dávkování   MeSH
• Check Tag
• dospělí MeSH
• lidé MeSH
• mladiství MeSH
• mužské pohlaví MeSH
• ženské pohlaví MeSH
• Publikační typ
• časopisecké články MeSH
• kazuistiky MeSH
• práce podpořená grantem 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

FerB from Paracoccus denitrificans is a soluble cytoplasmic flavoprotein that accepts redox equivalents from NADH or NADPH and transfers them to various acceptors such as quinones, ferric complexes and chromate. The crystal structure and small-angle X-ray scattering measurements in solution reported here reveal a head-to-tail dimer with two flavin mononucleotide groups bound at the opposite sides of the subunit interface. The dimers tend to self-associate to a tetrameric form at higher protein concentrations. Amino acid residues important for the binding of FMN and NADH and for the catalytic activity are identified and verified by site-directed mutagenesis. In particular, we show that Glu77 anchors a conserved water molecule in close proximity to the O2 of FMN, with the probable role of facilitating flavin reduction. Hydride transfer is shown to occur from the 4-pro-S position of NADH to the solvent-accessible si side of the flavin ring. When using deuterated NADH, this process exhibits a kinetic isotope effect of about 6 just as does the NADH-dependent quinone reductase activity of FerB; the first, reductive half-reaction of flavin cofactor is thus rate-limiting. Replacing the bulky Arg95 in the vicinity of the active site with alanine substantially enhances the activity towards external flavins that obeys the standard bi-bi ping-pong reaction mechanism. The new evidence for a cryptic flavin reductase activity of FerB justifies the previous inclusion of this enzyme in the protein family of NADPH-dependent FMN reductases.

• MeSH
• aminokyseliny chemie   genetika   metabolismus   MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• biokatalýza MeSH
• difrakce rentgenového záření MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• flavoproteiny chemie   genetika   metabolismus   MeSH
• katalytická doména genetika   MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• maloúhlový rozptyl MeSH
• molekulární modely MeSH
• molekulární sekvence - údaje MeSH
• multimerizace proteinu MeSH
• mutageneze cílená MeSH
• NADH, NADPH oxidoreduktasy chemie   klasifikace   metabolismus   MeSH
• NADP chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• terciární struktura proteinů * MeSH
• vazba proteinů MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

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