Environmental microorganisms usually exhibit a high level of genomic plasticity and metabolic versatility that allow them to be well-adapted to diverse environmental challenges. This study used shotgun metagenomics to decipher the functional and metabolic attributes of an uncultured Paracoccus recovered from a polluted soil metagenome and determine whether the detected attributes are influenced by the nature of the polluted soil. Functional and metabolic attributes of the uncultured Paracoccus were elucidated via functional annotation of the open reading frames (ORFs) of its contig. Functional tools deployed for the analysis include KEGG, KEGG KofamKOALA, Clusters of Orthologous Groups of proteins (COG), Comprehensive Antibiotic Resistance Database (CARD), and the Antibiotic Resistance Gene-ANNOTation (ARG-ANNOT V6) for antibiotic resistance genes, TnCentral for transposable element, Transporter Classification Database (TCDB) for transporter genes, and FunRich for gene enrichment analysis. Analyses revealed the preponderance of ABC transporter genes responsible for the transport of oligosaccharides (malK, msmX, msmK, lacK, smoK, aglK, togA, thuK, treV, msiK), monosaccharides (glcV, malK, rbsC, rbsA, araG, ytfR, mglA), amino acids (thiQ, ynjD, thiZ, glnQ, gluA, gltL, peb1C, artP, aotP, bgtA, artQ, artR), and several others. Also detected are transporter genes for inorganic/organic nutrients like phosphate/phosphonate, nitrate/nitrite/cyanate, sulfate/sulfonate, bicarbonate, and heavy metals such as nickel/cobalt, molybdate/tungstate, and iron, among others. Antibiotic resistance genes that mediate efflux, inactivation, and target protection were detected, while transposable elements carrying resistance phenotypes for antibiotics and heavy metals were also annotated. The findings from this study have established the resilience, adaptability, and survivability of the uncultured Paracoccus in the hydrocarbon-polluted soil.

• MeSH
• ABC transportéry genetika   MeSH
• antibakteriální látky farmakologie   MeSH
• bakteriální toxiny * MeSH
• Clostridioides difficile * genetika   MeSH
• metagenom MeSH
• Paracoccus * genetika   MeSH
• půda chemie   MeSH
• těžké kovy * MeSH
• transpozibilní elementy DNA MeSH
• uhlovodíky MeSH
• Publikační typ
• časopisecké články MeSH

Whey, the main by-product obtained from the manufacture of cheese, which contains a very high organic load (mainly due to the lactose content), is not easily degradable and creates concern over environmental issues. Hydrolysis of lactose present in whey and conversion of whey lactose into valuable products such as bioethanol, sweet syrup, and animal feed offers the possibility of whey bioremediation. The increasing need for bioremediation in the dairy industry has compelled researchers to search for a novel source of β-galactosidase with diverse properties. In the present study, the bacterium Paracoccus marcusii KGP producing β-galactosidase was subjected to morphological, biochemical, and probiotic characterisation. The bacterial isolate was found to be non-pathogenic and resistant to low pH (3 and 4), bile salts (0.2%), salt (10%), pepsin (at pH 3), and pancreatin (at pH 8). Further characterisation revealed that the bacteria have a good auto-aggregation ability (40% at 24 h), higher hydrophobicity (chloroform-60%, xylene-50%, and ethyl acetate-40%) and a broad spectrum of antibiotic susceptibility. The highest growth of P. marcusii KGP was achieved at pH 7 and 28 °C, and the yeast extract, galactose, and MgSO4 were the best for the growth of the bacterial cells. The bacterium KGP was able to utilise whey as a substrate for its growth with good β-galactosidase production potential. Furthermore, the β-galactosidase extracted from the isolate KGP could hydrolyse 47% whey lactose efficiently at 50 °C. The study thus reveals the potential application of β-galactosidase from P. marcusii KGP in whey bioremediation.

• MeSH
• biodegradace MeSH
• mlékárenství MeSH
• Paracoccus * metabolismus   MeSH
• probiotika * metabolismus   MeSH
• průmyslový odpad * MeSH
• syrovátka * mikrobiologie   MeSH
• zvířata MeSH
• Check Tag
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH

A new β-galactosidase-producing bacterium KGP, isolated from the Bay of Bengal, was identified as Paracoccus marcusii through morphology, biochemistry and 16S rRNA sequencing. This study is the first report on the production of β-galactosidase from P. marcusii. The medium components for the high yield of β-galactosidase were optimised using response surface methodology (RSM). A set of 17 experiments consisting of three independent variables, viz. yeast extract, galactose and MgSO4, was employed. A second-order polynomial equation was used for the analysis of the response, and the optimum β-galactosidase yield was achieved using 12.5 g/L yeast extract, 12.5 g/L galactose and 12.5 mmol/L MgSO4. The predicted quadratic model was inferred to be significant from the F-value, P value and the lack of fit value. Optimisation of the media components resulted in a ninefold increase (560 Miller units) in β-galactosidase production. Furthermore, the hydrolysis and transgalactosylation efficiency of the crude β-galactosidase was assessed and the results showed that the lactose was successfully hydrolysed and transgalactosylated at an optimum temperature of 40 °C and 50 °C, respectively. Considering the overall yield and productivity, P. marcusii can be considered a candidate for the industrial production of β-galactosidase. This study provides an essential basis for the future production and use of the alkali-tolerant β-galactosidase from P. marcusii KGP.

• MeSH
• beta-galaktosidasa metabolismus   MeSH
• bioreaktory MeSH
• fylogeneze MeSH
• galaktosa metabolismus   MeSH
• hydrolýza MeSH
• koncentrace vodíkových iontů MeSH
• laktosa metabolismus   MeSH
• oligosacharidy biosyntéza   MeSH
• Paracoccus klasifikace   genetika   izolace a purifikace   metabolismus   MeSH
• RNA ribozomální 16S genetika   MeSH
• teplota 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

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

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

The bacterial enzyme designated QhpD belongs to the radical S-adenosyl-L-methionine (SAM) superfamily of enzymes and participates in the post-translational processing of quinohemoprotein amine dehydrogenase. QhpD is essential for the formation of intra-protein thioether bonds within the small subunit (maturated QhpC) of quinohemoprotein amine dehydrogenase. We overproduced QhpD from Paracoccus denitrificans as a stable complex with its substrate QhpC, carrying the 28-residue leader peptide that is essential for the complex formation. Absorption and electron paramagnetic resonance spectra together with the analyses of iron and sulfur contents suggested the presence of multiple (likely three) [4Fe-4S] clusters in the purified and reconstituted QhpD. In the presence of a reducing agent (sodium dithionite), QhpD catalyzed the multiple-turnover reaction of reductive cleavage of SAM into methionine and 5'-deoxyadenosine and also the single-turnover reaction of intra-protein sulfur-to-methylene carbon thioether bond formation in QhpC bound to QhpD, producing a multiknotted structure of the polypeptide chain. Homology modeling and mutagenic analysis revealed several conserved residues indispensable for both in vivo and in vitro activities of QhpD. Our findings uncover another challenging reaction catalyzed by a radical SAM enzyme acting on a ribosomally translated protein substrate.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• elektronová paramagnetická rezonance MeSH
• oxidoreduktasy chemie   genetika   metabolismus   MeSH
• Paracoccus denitrificans enzymologie   genetika   MeSH
• proteiny obsahující železo a síru chemie   genetika   metabolismus   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