Paracoccus denitrificans ArsH is encoded by two identical genes located in two distinct putative arsenic resistance (ars) operons. Escherichia coli-produced recombinant N-His6-ArsH was characterized both structurally and kinetically. The X-ray structure of ArsH revealed a flavodoxin-like domain and motifs for the binding of flavin mononucleotide (FMN) and reduced nicotinamide adenine dinucleotide phosphate (NADPH). The protein catalyzed FMN reduction by NADPH via ternary complex mechanism. At a fixed saturating FMN concentration, it acted as an NADPH-dependent organoarsenic reductase displaying ping-pong kinetics. A 1:1 enzymatic reaction of phenylarsonic acid with the reduced form of FMN (FMNH2) and formation of phenylarsonous acid were observed. Growth experiments with P. denitrificans and E. coli revealed increased toxicity of phenylarsonic acid to cells expressing arsH, which may be related to in vivo conversion of pentavalent As to more toxic trivalent form. ArsH expression was upregulated not only by arsenite, but also by redox-active agents paraquat, tert-butyl hydroperoxide and diamide. A crucial role is played by the homodimeric transcriptional repressor ArsR, which was shown in in vitro experiments to monomerize and release from the DNA-target site. Collectively, our results establish ArsH as responsible for enhancement of organo-As(V) toxicity and demonstrate redox control of ars operon.
- 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
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
UNLABELLED: FerB is a flavin mononucleotide (FMN)-containing NAD(P)H: acceptor oxidoreductase of unknown function that is found in the cytoplasm of the bacterium Paracoccus denitrificans. Based on measurements of fluorescence anisotropy, we report here that recombinant FerB readily binds to artificial membrane vesicles. If ubiquinone is incorporated into the membrane, FerB catalyzes its conversion to ubihydroquinone, which may be followed fluorimetrically (with ferricyanide and pyranine entrapped inside the liposomes) or by HPLC. FerB also reduces exogenously added superoxide or superoxide that has been enzymatically generated by the xanthine/xanthine oxidase system or P. denitrificans membrane vesicles. In whole cells, deficiency of FerB increases sensitivity to methyl viologen, as indicated by a lower growth rate and increased production of reactive aldehydes (by-products of lipid oxidation). Taken together, these data support a role for FerB in protection of cells against lipid peroxidation-mediated oxidative stress, and suggest that FerB is a prokaryotic counterpart of mammalian NAD(P)H: quinone oxidoreductase 1.
- MeSH
- antioxidancia chemie metabolismus MeSH
- flavoproteiny chemie metabolismus MeSH
- kinetika MeSH
- membránové proteiny chemie metabolismus MeSH
- oxidace-redukce MeSH
- oxidační stres * MeSH
- Paracoccus denitrificans enzymologie MeSH
- superoxidy metabolismus MeSH
- ubichinon metabolismus MeSH
- xanthin metabolismus MeSH
- xanthinoxidasa metabolismus MeSH
- zvířata MeSH
- Check Tag
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
1. vydání 313 stran : barevné ilustrace, mapy, portréty, faksimile ; 31 cm + 1 skládanka (6 nečíslovaných stran ; 29 cm)
- MeSH
- efektivita organizační MeSH
- informační systémy MeSH
- manažerské informační systémy MeSH
- státní správa MeSH
- Geografické názvy
- Česká republika MeSH
- Konspekt
- Veřejná správa
- NLK Obory
- státní správa
- NLK Publikační typ
- kolektivní monografie
FerB is a cytoplasmic flavoprotein from the soil bacterium Paracoccus denitrificans with a putative role in defense against oxidative stress. To further explore this hypothesis, we compared protein variations upon methyl viologen treatment in wild-type and FerB mutant strains by a quantitative proteomic analysis based on iTRAQ-3DLC-MS/MS analysis. The proteins showing the most prominent increase in abundance were assigned to carbon fixation and sulfur assimilatory pathways. By employing these proteins as indirect markers, oxidative stress was found to be 15% less severe in the wild-type than in the FerB-deficient mutant cells. Oxidative stress altered the levels of proteins whose expression is dependent on the transcriptional factor FnrP. The observed down-regulation of the fnrP regulon members, most notably that of nitrous oxide reductase, was tentatively explained by an oxidative degradation of the [4Fe-4S] center of FnrP leading to a protein form which no longer activates transcription. While the level of FerB remained relatively constant, two proteins homologous to FerB accumulated during oxidative stress. When their genes were expressed in Escherichia coli, neither of the protein products contained a bound flavin, whereas they both had a high activity of flavin reductase, one preferentially utilizing NADH and the other NADPH.
- MeSH
- bakteriální proteiny biosyntéza genetika MeSH
- flavoproteiny genetika metabolismus MeSH
- mutace * MeSH
- oxidační stres účinky léků genetika MeSH
- Paracoccus denitrificans genetika metabolismus MeSH
- paraquat farmakologie MeSH
- proteomika MeSH
- regulace genové exprese u bakterií účinky léků genetika MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem 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
A comparative examination of reduced methyl [MV·](+) and benzyl [BV·](+) viologens (as artificial electron donors for quantitative estimation of the respiratory periplasmic (Nap) and membrane-embedded (Nar) nitrate reductases) using a newly constructed nap mutant strain of Paracocccus denitrificans was done. The activity with [MV·](+) was high in whole-cell assays, confirming that this compound donates electrons to Nar. Initial rates of the more lipophilic [BV·](+) were considerably lower, which was interpreted to be caused by an inhibition of the active transport of nitrate into the cells. Anionophoric activity of [BV·](+) was detectable but too low to effectively circumvent the inhibition of nitrate transporter.
- MeSH
- analýza jednotlivých buněk MeSH
- bakteriální proteiny chemie genetika metabolismus MeSH
- benzylviologen metabolismus MeSH
- dusičnany metabolismus MeSH
- kinetika MeSH
- nitrátreduktasa chemie genetika metabolismus MeSH
- oxidace-redukce MeSH
- Rhodobacteraceae chemie enzymologie genetika MeSH
- transport elektronů MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
