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

Ferric reductase B (FerB) is a flavin mononucleotide (FMN)-containing NAD(P)H:acceptor oxidoreductase structurally close to the Gluconacetobacter hansenii chromate reductase (ChrR). The crystal structure of ChrR was previously determined with a chloride bound proximal to FMN in the vicinity of Arg101, and the authors suggested that the anionic electron acceptors, chromate and uranyl tricarbonate, bind similarly. Here, we identify the corresponding arginine residue in FerB (Arg95) as being important for the reaction of FerB with superoxide. Four mutants at position 95 were prepared and found kinetically to have impaired capacity for superoxide binding. Stopped-flow data for the flavin cofactor showed that the oxidative step is rate limiting for catalytic turnover. The findings are consistent with a role for FerB as a superoxide scavenging contributor.

• MeSH
• arginin genetika   MeSH
• flavinmononukleotid chemie   genetika   MeSH
• flaviny genetika   metabolismus   MeSH
• FMN-reduktasa chemie   genetika   MeSH
• katalytická doména genetika   MeSH
• kinetika MeSH
• konformace proteinů * MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce MeSH
• oxidoreduktasy chemie   genetika   MeSH
• Paracoccus denitrificans chemie   enzymologie   MeSH
• sekvence aminokyselin genetika   MeSH
• superoxidy metabolismus   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 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