The activity of the light-oxygen-voltage/helix-turn-helix (LOV-HTH) photoreceptor EL222 is regulated through protein-protein and protein-DNA interactions, both triggered by photo-excitation of its flavin mononucleotide (FMN) cofactor. To gain molecular-level insight into the photocycle of EL222, we applied complementary methods: macromolecular X-ray crystallography (MX), nuclear magnetic resonance (NMR) spectroscopy, optical spectroscopies (infrared and UV-visible), molecular dynamics/metadynamics (MD/metaD) simulations, and protein engineering using noncanonical amino acids. Kinetic experiments provided evidence for two distinct EL222 conformations (lit1 and lit2) that become sequentially populated under illumination. These two lit states were assigned to covalently bound N5 protonated, and noncovalently bound hydroquinone forms of FMN, respectively. Only subtle structural differences were observed between the monomeric forms of all three EL222 species (dark, lit1, and lit2). While the dark state is largely monomeric, both lit states undergo monomer-dimer exchange. Furthermore, molecular modeling revealed differential dynamics and interdomain separation times arising from the three FMN states (oxidized, adduct, and reduced). Unexpectedly, all three EL222 species can associate with DNA, but only upon blue-light irradiation, a high population of stable complexes is obtained. Overall, we propose a model of EL222 activation where photoinduced changes in the FMN moiety shift the population equilibrium toward an open conformation that favors self-association and DNA-binding.

• MeSH
• bakteriální proteiny chemie   metabolismus   MeSH
• DNA vazebné proteiny chemie   metabolismus   MeSH
• DNA * chemie   metabolismus   MeSH
• flavinmononukleotid * chemie   metabolismus   MeSH
• flaviny chemie   metabolismus   MeSH
• kinetika MeSH
• konformace proteinů MeSH
• krystalografie rentgenová MeSH
• oxidace-redukce * MeSH
• simulace molekulární dynamiky MeSH
• světlo * MeSH
• Thermosynechococcus metabolismus   MeSH
• transkripční faktory metabolismus   chemie   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

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

Old Yellow Enzymes (OYEs) are NAD(P)H dehydrogenases of not fully resolved physiological roles that are widespread among bacteria, plants, and fungi and have a great potential for biotechnological applications. We determined the apo form crystal structure of a member of the OYE class, glycerol trinitrate reductase XdpB, from Agrobacterium bohemicum R89-1 at 2.1 Å resolution. In agreement with the structures of the related bacterial OYEs, the structure revealed the TIM barrel fold with an N-terminal β-hairpin lid, but surprisingly, the structure did not contain its cofactor FMN. Its putative binding site was occupied by a pentapeptide TTSDN from the C-terminus of a symmetry related molecule. Biochemical experiments confirmed a specific concentration-dependent oligomerization and a low FMN content. The blocking of the FMN binding site can exist in vivo and regulates enzyme activity. Our bioinformatic analysis indicated that a similar self-inhibition could be expected in more OYEs which we designated as subgroup OYE C1. This subgroup is widespread among G-bacteria and can be recognized by the conserved sequence GxxDYP in proximity of the C termini. In proteobacteria, the C1 subgroup OYEs are typically coded in one operon with short-chain dehydrogenase. This operon is controlled by the tetR-like transcriptional regulator. OYEs coded in these operons are unlikely to be involved in the oxidative stress response as the other known members of the OYE family because no upregulation of XdpB was observed after exposing A. bohemicum R89-1 to oxidative stress.

• MeSH
• Agrobacterium enzymologie   genetika   MeSH
• bakteriální geny MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• flavinmononukleotid metabolismus   MeSH
• katalytická doména MeSH
• kinetika MeSH
• krystalografie rentgenová MeSH
• kvarterní struktura proteinů MeSH
• molekulární modely MeSH
• NADPH-dehydrogenasa chemie   genetika   metabolismus   MeSH
• operon MeSH
• oxidační stres MeSH
• oxidoreduktasy chemie   genetika   metabolismus   MeSH
• výpočetní biologie MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH

We report a cluster of genes encoding two monooxygenases (SadA and SadB) and one FMN reductase (SadC) that enable Microbacterium sp. strain BR1 and other Actinomycetes to inactivate sulfonamide antibiotics. Our results show that SadA and SadC are responsible for the initial attack of sulfonamide molecules resulting in the release of 4-aminophenol. The latter is further transformed into 1,2,4-trihydroxybenzene by SadB and SadC prior to mineralization and concomitant production of biomass. As the degradation products lack antibiotic activity, the presence of SadA will result in an alleviated bacteriostatic effect of sulfonamides. In addition to the relief from antibiotic stress this bacterium gains access to an additional carbon source when this gene cluster is expressed. As degradation of sulfonamides was also observed when Microbacterium sp. strain BR1 was grown on artificial urine medium, colonization with such strains may impede common sulfonamide treatment during co-infections with pathogens of the urinary tract. This case of biodegradation exemplifies the evolving catabolic capacity of bacteria, given that sulfonamide bacteriostatic are purely of synthetic origin. The wide distribution of this cluster in Actinomycetes and the presence of traA encoding a relaxase in its vicinity suggest that this cluster is mobile and that is rather alarming.

• MeSH
• Actinobacteria účinky léků   genetika   růst a vývoj   metabolismus   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální geny MeSH
• biodegradace účinky léků   MeSH
• flavinmononukleotid metabolismus   MeSH
• fylogeneze MeSH
• hydrochinony metabolismus   MeSH
• multigenová rodina MeSH
• oxygenasy se smíšenou funkcí metabolismus   MeSH
• radioizotopy uhlíku MeSH
• sulfonamidy metabolismus   MeSH
• Publikační typ
• časopisecké články 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

WrbA is a novel multimeric flavodoxin-like protein of unknown function. A recent high-resolution X-ray crystal structure of E. coli WrbA holoprotein revealed a methionine sulfoxide residue with full occupancy in the FMN-binding site, a finding that was confirmed by mass spectrometry. In an effort to evaluate whether methionine sulfoxide may have a role in WrbA function, the present analyses were undertaken using molecular dynamics simulations in combination with further mass spectrometry of the protein. Methionine sulfoxide formation upon reconstitution of purified apoWrbA with oxidized FMN is fast as judged by kinetic mass spectrometry, being complete in ∼5 h and resulting in complete conversion at the active-site methionine with minor extents of conversion at heterogeneous second sites. Analysis of methionine oxidation states during purification of holoWrbA from bacterial cells reveals that methionine is not oxidized prior to reconstitution, indicating that methionine sulfoxide is unlikely to be relevant to the function of WrbA in vivo. Although the simulation results, the first reported for WrbA, led to no hypotheses about the role of methionine sulfoxide that could be tested experimentally, they elucidated the origins of the two major differences between apo- and holoWrbA crystal structures, an alteration of inter-subunit distance and a rotational shift within the tetrameric assembly.

• MeSH
• apoproteiny chemie   izolace a purifikace   metabolismus   MeSH
• flavinmononukleotid chemie   metabolismus   MeSH
• hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
• kinetika MeSH
• konformace proteinů MeSH
• methionin analogy a deriváty   chemie   metabolismus   MeSH
• oxidace-redukce MeSH
• proteiny z Escherichia coli chemie   izolace a purifikace   metabolismus   MeSH
• represorové proteiny chemie   izolace a purifikace   metabolismus   MeSH
• simulace molekulární dynamiky * MeSH
• stabilita proteinů MeSH
• vazba proteinů MeSH
• vazebná místa MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH

While the three-dimensional structures of heme- and flavin-binding domains of the NOS isoforms have been determined, the structures of the holoenzymes remained elusive. Application of electron cryo-microscopy and structural modeling of the bovine endothelial nitric oxide synthase (eNOS) holoenzyme produced detailed models of the intact holoenzyme in the presence and absence of Ca(2+)/calmodulin (CaM). These models accommodate the cross-electron transfer from the reductase in one monomer to the heme in the opposite monomer. The heme domain acts as the anchoring dimeric structure for the entire enzyme molecule, while the FMN domain is activated by CaM to move flexibly to bridge the distance between the reductase and oxygenase domains. Our results indicate that the key regulatory role of CaM involves the stabilization of structural intermediates and precise positioning of the pivot for the FMN domain tethered shuttling motion to accommodate efficient and rapid electron transfer in the homodimer of eNOS.

• MeSH
• alosterická regulace MeSH
• flavinmononukleotid chemie   MeSH
• hem chemie   MeSH
• holoenzymy chemie   MeSH
• kalmodulin chemie   metabolismus   MeSH
• kinetika MeSH
• oxidace-redukce MeSH
• skot MeSH
• synthasa oxidu dusnatého, typ III chemie   metabolismus   MeSH
• terciární struktura proteinů MeSH
• transport elektronů MeSH
• vápník chemie   metabolismus   MeSH
• zvířata MeSH
• Check Tag
• skot MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Research Support, N.I.H., Extramural 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

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