Iron uptake by diatoms is a biochemical process with global biogeochemical implications. In large regions of the surface ocean diatoms are both responsible for the majority of primary production and frequently experiencing iron limitation of growth. The strategies used by these phytoplankton to extract iron from seawater constrain carbon flux into higher trophic levels and sequestration into sediments. In this study we use reverse genetic techniques to target putative iron-acquisition genes in the model pennate diatom Phaeodactylum tricornutum We describe components of a reduction-dependent siderophore acquisition pathway that relies on a bacterial-derived receptor protein and provides a viable alternative to inorganic iron uptake under certain conditions. This form of iron uptake entails a close association between diatoms and siderophore-producing organisms during low-iron conditions. Homologs of these proteins are found distributed across diatom lineages, suggesting the significance of siderophore utilization by diatoms in the marine environment. Evaluation of specific proteins enables us to confirm independent iron-acquisition pathways in diatoms and characterize their preferred substrates. These findings refine our mechanistic understanding of the multiple iron-uptake systems used by diatoms and help us better predict the influence of iron speciation on taxa-specific iron bioavailability.
- MeSH
- biologická dostupnost MeSH
- biologický transport MeSH
- CRISPR-Cas systémy MeSH
- druhová specificita MeSH
- FMN-reduktasa genetika metabolismus MeSH
- fylogeneze MeSH
- galium metabolismus MeSH
- genový knockout MeSH
- klimatické změny MeSH
- membránové transportní proteiny genetika metabolismus MeSH
- mikrobiota MeSH
- mořská voda chemie MeSH
- oxidace-redukce MeSH
- proteiny vnější bakteriální membrány metabolismus MeSH
- receptory buněčného povrchu metabolismus MeSH
- rekombinantní fúzní proteiny metabolismus MeSH
- rozsivky genetika růst a vývoj metabolismus MeSH
- siderofory metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Research Support, U.S. Gov't, Non-P.H.S. 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
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
The bloodstream form of Trypanosoma brucei acquires iron from transferrin by receptor-mediated endocytosis. However, it is unknown how procyclic forms that cannot bind transferrin acquire iron. Here, we show that the procyclic form of T. brucei efficiently takes up iron from ferric complexes via a reductive mechanism and that iron obtained using this mechanism is transported to, and used in, the mitochondria. The affinity of the transport system is comparable to that of Saccharomyces cerevisiae , with an apparent K(m) of 0.85 μM.
- MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- FMN-reduktasa metabolismus MeSH
- interakce hostitele a parazita MeSH
- mitochondrie metabolismus MeSH
- oxidace-redukce MeSH
- Trypanosoma brucei brucei metabolismus MeSH
- železité sloučeniny metabolismus MeSH
- železnaté sloučeniny metabolismus MeSH
- železo metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Medicago truncatula represents a model plant species for understanding legume-bacteria interactions. M. truncatula roots form a specific root-nodule symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti. Symbiotic nitrogen fixation generates high iron (Fe) demands for bacterial nitrogenase holoenzyme and plant leghemoglobin proteins. Leguminous plants acquire Fe via "Strategy I," which includes mechanisms such as rhizosphere acidification and enhanced ferric reductase activity. In the present work, we analyzed the effect of S. meliloti volatile organic compounds (VOCs) on the Fe-uptake mechanisms of M. truncatula seedlings under Fe-deficient and Fe-rich conditions. Axenic cultures showed that both plant and bacterium modified VOC synthesis in the presence of the respective symbiotic partner. Importantly, in both Fe-rich and -deficient experiments, bacterial VOCs increased the generation of plant biomass, rhizosphere acidification, ferric reductase activity, and chlorophyll content in plants. On the basis of our results, we propose that M. truncatula perceives its symbiont through VOC emissions, and in response, increases Fe-uptake mechanisms to facilitate symbiosis.
- MeSH
- biomasa MeSH
- chlorofyl analýza MeSH
- FMN-reduktasa metabolismus MeSH
- koncentrace vodíkových iontů MeSH
- kořenové hlízky rostlin mikrobiologie MeSH
- Medicago truncatula chemie růst a vývoj metabolismus mikrobiologie MeSH
- půda chemie MeSH
- Sinorhizobium meliloti metabolismus MeSH
- těkavé organické sloučeniny metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We investigated iron uptake mechanisms in five marine microalgae from different ecologically important phyla: the diatoms Phaeodactylum tricornutum and Thalassiosira pseudonana, the prasinophyceae Ostreococcus tauri and Micromonas pusilla, and the coccolithophore Emiliania huxleyi. Among these species, only the two diatoms were clearly able to reduce iron, via an inducible (P. tricornutum) or constitutive (T. pseudonana) ferrireductase system displaying characteristics similar to the yeast (Saccharomyces cerevisiae) flavohemoproteins proteins. Iron uptake mechanisms probably involve very different components according to the species, but the species we studied shared common features. Regardless of the presence and/or induction of a ferrireductase system, all the species were able to take up both ferric and ferrous iron, and iron reduction was not a prerequisite for uptake. Iron uptake decreased with increasing the affinity constants of iron-ligand complexes and with increasing ligand-iron ratios. Therefore, at least one step of the iron uptake mechanism involves a thermodynamically controlled process. Another step escapes to simple thermodynamic rules and involves specific and strong binding of ferric as well as ferrous iron at the cell surface before uptake of iron. Binding was paradoxically increased in iron-rich conditions, whereas uptake per se was induced in all species only after prolonged iron deprivation. We sought cell proteins loaded with iron following iron uptake. One such protein in O. tauri may be ferritin, and in P. tricornutum, Isip1 may be involved. We conclude that the species we studied have uptake systems for both ferric and ferrous iron, both involving specific iron binding at the cell surface.
- MeSH
- autoradiografie MeSH
- biologické modely MeSH
- buněčná membrána účinky léků metabolismus MeSH
- chelátory železa farmakologie MeSH
- FMN-reduktasa metabolismus MeSH
- fylogeneze MeSH
- kinetika MeSH
- ligandy MeSH
- mikrořasy účinky léků enzymologie růst a vývoj metabolismus MeSH
- oxidace-redukce účinky léků MeSH
- Saccharomyces cerevisiae účinky léků metabolismus MeSH
- transport elektronů účinky léků MeSH
- vodní organismy růst a vývoj metabolismus MeSH
- železo metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
OBJECTIVES: Alzheimer's disease (AD) is a neurodegenerative disorder. Symptomatic treatment is available by inhibitors of acetylcholinesterase (AChE) such as rivastigmine, galantamine and donepezil. As huperzine is a promising compound for AD treatment, our study was aimed at evaluating its pertinent implications in oxidative stress. METHODS: Laboratory guinea pigs were exposed to huperzine A at doses of 0, 5, 25, 125 and 625 µg/kg. The animals were observed for cognitive disorders and sacrificed one hour after exposure. Tonic-clonic seizures were noticed, but only in highly dosed animals. Ferric reducing antioxidant power (FRAP), thiobarbituric acid reactive substances (TBARS), glutathione reductase and glutathione S-transferase were assessed in frontal, temporal and parietal lobes, the cerebellum, liver, spleen and kidney. RESULTS: Only minimal changes in enzymatic markers were recognized. Huperzine was not implicated in oxidative stress enhancement as the TBARS values remained quite stable. Surprisingly, antioxidants accumulated in the examined brain compartments as the FRAP value was significantly elevated following all doses of huperzine. CONCLUSIONS: We discuss the potency of huperzine in enhancing the antioxidant capacity of the central nervous system. Huperzine is probably implicated in more processes than cholinesterase inhibition only.
- MeSH
- alkaloidy farmakologie MeSH
- antioxidancia metabolismus MeSH
- biologické modely MeSH
- FMN-reduktasa metabolismus MeSH
- glutathionreduktasa metabolismus MeSH
- homeostáza účinky léků MeSH
- modely u zvířat MeSH
- morčata MeSH
- neuroprotektivní látky farmakologie MeSH
- oxidace-redukce účinky léků MeSH
- oxidační stres účinky léků MeSH
- preklinické hodnocení léčiv MeSH
- seskviterpeny farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- zvířata MeSH
- Check Tag
- morčata MeSH
- zvířata 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
Flavins contained in flavoenzymes are versatile oxidizing and reducing agents. This fact inspired many researchers to test flavin derivatives as oxidation or reduction catalysts in organic synthesis. In this article, flavinbased catalytic and biocatalytic systems are reviewed. Relevant flavin properties are discussed in the context with their possible catalytic applications.
