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Applied and Environmental Microbiology Jul 2015In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4'-dihydroxybiphenyl, 4-hydroxy-4'-chlorobiphenyl,...
In this work, we examined the profile of metabolites produced from the doubly para-substituted biphenyl analogs 4,4'-dihydroxybiphenyl, 4-hydroxy-4'-chlorobiphenyl, 3-hydroxy-4,4'-dichlorobiphenyl, and 3,3'-dihydroxy-4,4'-chlorobiphenyl by biphenyl-induced Pandoraea pnomenusa B356 and by its biphenyl dioxygenase (BPDO). 4-Hydroxy-4'-chlorobiphenyl was hydroxylated principally through a 2,3-dioxygenation of the hydroxylated ring to generate 2,3-dihydro-2,3,4-trihydroxy-4'-chlorobiphenyl and 3,4-dihydroxy-4'-chlorobiphenyl after the removal of water. The former was further oxidized by the biphenyl dioxygenase to produce ultimately 3,4,5-trihydroxy-4'-chlorobiphenyl, a dead-end metabolite. 3-Hydroxy-4,4'-dichlorobiphenyl was oxygenated on both rings. Hydroxylation of the nonhydroxylated ring generated 2,3,3'-trihydroxy-4'-chlorobiphenyl with concomitant dechlorination, and 2,3,3'-trihydroxy-4'-chlorobiphenyl was ultimately metabolized to 2-hydroxy-4-chlorobenzoate, but hydroxylation of the hydroxylated ring generated dead-end metabolites. 3,3'-Dihydroxy-4,4'-dichlorobiphenyl was principally metabolized through a 2,3-dioxygenation to generate 2,3-dihydro-2,3,3'-trihydroxy-4,4'-dichlorobiphenyl, which was ultimately converted to 3-hydroxy-4-chlorobenzoate. Similar metabolites were produced when the biphenyl dioxygenase of Burkholderia xenovorans LB400 was used to catalyze the reactions, except that for the three substrates used, the BPDO of LB400 was less efficient than that of B356, and unlike that of B356, it was unable to further oxidize the initial reaction products. Together the data show that BPDO oxidation of doubly para-substituted hydroxychlorobiphenyls may generate nonnegligible amounts of dead-end metabolites. Therefore, biphenyl dioxygenase could produce metabolites other than those expected, corresponding to dihydrodihydroxy metabolites from initial doubly para-substituted substrates. This finding shows that a clear picture of the fate of polychlorinated biphenyls in contaminated sites will require more insights into the bacterial metabolism of hydroxychlorobiphenyls and the chemistry of the dihydrodihydroxylated metabolites derived from them.
Topics: Bacterial Proteins; Biocatalysis; Biodegradation, Environmental; Burkholderia; Burkholderiaceae; Dioxygenases; Molecular Structure; Oxidation-Reduction; Polychlorinated Biphenyls; Substrate Specificity
PubMed: 25956777
DOI: 10.1128/AEM.00786-15 -
Genome Announcements Feb 2015Pandoraea is an emerging respiratory pathogen capable of causing chronic lung infections in people with cystic fibrosis (CF), but the clinical significance of this...
Pandoraea is an emerging respiratory pathogen capable of causing chronic lung infections in people with cystic fibrosis (CF), but the clinical significance of this infection is ambiguous. We have sequenced and annotated the genomes of two multidrug-resistant Pandoraea pnomenusa isolates recovered 11 months apart from the same CF patient.
PubMed: 25657265
DOI: 10.1128/genomeA.01389-14 -
Sensors (Basel, Switzerland) Jun 2014Strain RB38 was recovered from a former dumping area in Malaysia. MALDI-TOF mass spectrometry and genomic analysis identified strain RB-38 as Pandoraea pnomenusa....
Strain RB38 was recovered from a former dumping area in Malaysia. MALDI-TOF mass spectrometry and genomic analysis identified strain RB-38 as Pandoraea pnomenusa. Various biosensors confirmed its quorum sensing properties. High resolution triple quadrupole liquid chromatography-mass spectrometry analysis was subsequently used to characterize the N-acyl homoserine lactone production profile of P. pnomenusa strain RB38, which validated that this isolate produced N-octanoyl homoserine lactone as a quorum sensing molecule. This is the first report of the production of N-octanoyl homoserine lactone by P. pnomenusa strain RB38.
Topics: Burkholderiaceae; Chromatography, Liquid; Homoserine; Lactones; Quorum Sensing; Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
PubMed: 24919016
DOI: 10.3390/s140610177 -
Genome Announcements May 2014Pandoraea pnomenusa strain 3kgm has been identified as a quorum-sensing strain isolated from soil. Here, we report the complete genome sequence of P. pnomenusa strain...
Pandoraea pnomenusa strain 3kgm has been identified as a quorum-sensing strain isolated from soil. Here, we report the complete genome sequence of P. pnomenusa strain 3kgm by using the Pacific Biosciences single-molecule real-time (PacBio RS SMRT) sequencer high-resolution technology.
PubMed: 24812228
DOI: 10.1128/genomeA.00427-14 -
Journal of Bacteriology Aug 2013In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl...
In this work, we have compared the ability of Pandoraea pnomenusa B356 and of Burkholderia xenovorans LB400 to metabolize diphenylmethane and benzophenone, two biphenyl analogs in which the phenyl rings are bonded to a single carbon. Both chemicals are of environmental concern. P. pnomenusa B356 grew well on diphenylmethane. On the basis of growth kinetics analyses, diphenylmethane and biphenyl were shown to induce the same catabolic pathway. The profile of metabolites produced during growth of strain B356 on diphenylmethane was the same as the one produced by isolated enzymes of the biphenyl catabolic pathway acting individually or in coupled reactions. The biphenyl dioxygenase oxidizes diphenylmethane to 3-benzylcyclohexa-3,5-diene-1,2-diol very efficiently, and ultimately this metabolite is transformed to phenylacetic acid, which is further metabolized by a lower pathway. Strain B356 was also able to cometabolize benzophenone through its biphenyl pathway, although in this case, this substrate was unable to induce the biphenyl catabolic pathway and the degradation was incomplete, with accumulation of 2-hydroxy-6,7-dioxo-7-phenylheptanoic acid. Unlike strain B356, B. xenovorans LB400 did not grow on diphenylmethane. Its biphenyl pathway enzymes metabolized diphenylmethane, but they poorly metabolize benzophenone. The fact that the biphenyl catabolic pathway of strain B356 metabolized diphenylmethane and benzophenone more efficiently than that of strain LB400 brings us to postulate that in strain B356, this pathway evolved divergently to serve other functions not related to biphenyl degradation.
Topics: Bacteriological Techniques; Benzhydryl Compounds; Benzophenones; Biphenyl Compounds; Burkholderiaceae; Gene Expression Regulation, Bacterial; Models, Molecular; Molecular Structure
PubMed: 23749969
DOI: 10.1128/JB.00161-13 -
PloS One 2013The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a...
The oxidative degradation of biphenyl and polychlorinated biphenyls (PCBs) is initiated in Pandoraea pnomenusa B-356 by biphenyl dioxygenase (BPDO(B356)). BPDO(B356), a heterohexameric (αβ)(3) Rieske oxygenase (RO), catalyzes the insertion of dioxygen with stereo- and regioselectivity at the 2,3-carbons of biphenyl, and can transform a broad spectrum of PCB congeners. Here we present the X-ray crystal structures of BPDO(B356) with and without its substrate biphenyl 1.6-Å resolution for both structures. In both cases, the Fe(II) has five ligands in a square pyramidal configuration: H233 Nε2, H239 Nε2, D386 Oδ1 and Oδ2, and a single water molecule. Analysis of the active sites of BPDO(B356) and related ROs revealed structural features that likely contribute to the superior PCB-degrading ability of certain BPDOs. First, the active site cavity readily accommodates biphenyl with minimal conformational rearrangement. Second, M231 was predicted to sterically interfere with binding of some PCBs, and substitution of this residue yielded variants that transform 2,2'-dichlorobiphenyl more effectively. Third, in addition to the volume and shape of the active site, residues at the active site entrance also apparently influence substrate preference. Finally, comparison of the conformation of the active site entrance loop among ROs provides a basis for a structure-based classification consistent with a phylogeny derived from amino acid sequence alignments.
Topics: Biphenyl Compounds; Burkholderiaceae; Catalytic Domain; Crystallography, X-Ray; Dioxygenases; Models, Molecular; Mutagenesis; Phylogeny; Polychlorinated Biphenyls; Protein Conformation; Protein Subunits; Substrate Specificity
PubMed: 23308114
DOI: 10.1371/journal.pone.0052550 -
Remarkable ability of Pandoraea pnomenusa B356 biphenyl dioxygenase to metabolize simple flavonoids.Applied and Environmental Microbiology May 2012Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to...
Many investigations have provided evidence that plant secondary metabolites, especially flavonoids, may serve as signal molecules to trigger the abilities of bacteria to degrade chlorobiphenyls in soil. However, the bases for this interaction are largely unknown. In this work, we found that BphAE(B356), the biphenyl/chlorobiphenyl dioxygenase from Pandoraea pnomenusa B356, is significantly better fitted to metabolize flavone, isoflavone, and flavanone than BphAE(LB400) from Burkholderia xenovorans LB400. Unlike those of BphAE(LB400), the kinetic parameters of BphAE(B356) toward these flavonoids were in the same range as for biphenyl. In addition, remarkably, the biphenyl catabolic pathway of strain B356 was strongly induced by isoflavone, whereas none of the three flavonoids induced the catabolic pathway of strain LB400. Docking experiments that replaced biphenyl in the biphenyl-bound form of the enzymes with flavone, isoflavone, or flavanone showed that the superior ability of BphAE(B356) over BphAE(LB400) is principally attributable to the replacement of Phe336 of BphAE(LB400) by Ile334 and of Thr335 of BphAE(LB400) by Gly333 of BphAE(B356). However, biochemical and structural comparison of BphAE(B356) with BphAE(p4), a mutant of BphAE(LB400) which was obtained in a previous work by the double substitution Phe336Met Thr335Ala of BphAE(LB400), provided evidence that other residues or structural features of BphAE(B356) whose precise identification the docking experiment did not allow are also responsible for the superior catalytic abilities of BphAE(B356). Together, these data provide supporting evidence that the biphenyl catabolic pathways have evolved divergently among proteobacteria, where some of them may serve ecological functions related to the metabolism of plant secondary metabolites in soil.
Topics: Amino Acid Substitution; Biphenyl Compounds; Burkholderiaceae; Dioxygenases; Flavonoids; Gene Expression Regulation, Bacterial; Kinetics; Metabolic Networks and Pathways; Protein Conformation
PubMed: 22427498
DOI: 10.1128/AEM.00225-12 -
The Journal of Biological Chemistry Oct 2011Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in...
Biphenyl dehydrogenase, a member of short-chain dehydrogenase/reductase enzymes, catalyzes the second step of the biphenyl/polychlorinated biphenyls catabolic pathway in bacteria. To understand the molecular basis for the broad substrate specificity of Pandoraea pnomenusa strain B-356 biphenyl dehydrogenase (BphB(B-356)), the crystal structures of the apo-enzyme, the binary complex with NAD(+), and the ternary complexes with NAD(+)-2,3-dihydroxybiphenyl and NAD(+)-4,4'-dihydroxybiphenyl were determined at 2.2-, 2.5-, 2.4-, and 2.1-Å resolutions, respectively. A crystal structure representing an intermediate state of the enzyme was also obtained in which the substrate binding loop was ordered as compared with the apo and binary forms but it was displaced significantly with respect to the ternary structures. These five structures reveal that the substrate binding loop is highly mobile and that its conformation changes during ligand binding, starting from a disorganized loop in the apo state to a well organized loop structure in the ligand-bound form. Conformational changes are induced during ligand binding; forming a well defined cavity to accommodate a wide variety of substrates. This explains the biochemical data that shows BphB(B-356) converts the dihydrodiol metabolites of 3,3'-dichlorobiphenyl, 2,4,4'-trichlorobiphenyl, and 2,6-dichlorobiphenyl to their respective dihydroxy metabolites. For the first time, a combination of structural, biochemical, and molecular docking studies of BphB(B-356) elucidate the unique ability of the enzyme to transform the cis-dihydrodiols of double meta-, para-, and ortho-substituted chlorobiphenyls.
Topics: Bacterial Proteins; Burkholderiaceae; Chlorophenols; Crystallography, X-Ray; Ligands; Oxidoreductases; Protein Structure, Quaternary; Protein Structure, Secondary; Protein Structure, Tertiary; Structure-Activity Relationship; Substrate Specificity
PubMed: 21880718
DOI: 10.1074/jbc.M111.291013 -
Acta Crystallographica. Section F,... Nov 2010cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa...
cis-Biphenyl-2,3-dihydrodiol-2,3-dehydrogenase (BphB) is involved in the aerobic biodegradation of biphenyl and polychlorinated biphenyls. BphB from Pandoraea pnomenusa strain B-356 was overexpressed in Escherichia coli, purified to homogeneity and crystallized. Crystals were obtained by the sitting-drop vapour-diffusion method using polyethylene glycol 3350 and 0.2 M sodium malonate. A BphB crystal diffracted to 2.8 Å resolution and belonged to space group P4(3)2(1)2, with unit-cell parameters a = b = 75.2, c = 180.4 Å. Preliminary crystallographic analysis indicated the presence of two molecules in the asymmetric unit, giving a Matthews coefficient of 2.2 Å(3) Da(-1) and a solvent content of 44%.
Topics: Burkholderiaceae; Crystallization; Crystallography, X-Ray; Gene Expression; Oxidoreductases
PubMed: 21045310
DOI: 10.1107/S1744309110036894 -
Journal of Clinical Microbiology Nov 2009The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of...
The identification of microbial species from respiratory specimens and their susceptibility to antimicrobial agents are among the most important diagnostic measures of care for patients with cystic fibrosis (CF). Under the umbrella of EuroCareCF, two quality assurance trials of CF microbiology were performed in 2007 and 2008. Nine formulations with CF bacterial isolates were dispatched. A total of 31/37 laboratories from 18/21 European countries participated in the 2007 and 2008 trials. The common CF pathogens Pseudomonas aeruginosa and Staphylococcus aureus were correctly identified by almost all participants in both trials, even if the strains presented uncommon phenotypes. Burkholderia cenocepacia IIIB and Burkholderia vietnamensis CF isolates, however, were correctly assigned to the species level by only 26% and 27% of the laboratories, respectively. Emerging pathogens such as Achromobacter xylosoxidans, Inquilinus limosus, and Pandoraea pnomenusa were also not detected or were misclassified by many laboratories. One participant correctly identified all CF isolates in both trials. The percentages of correct classifications (susceptible, intermediate, resistant) by antimicrobial susceptibility testing ranged from 55 to 100% (median, 96%) per isolate and drug. The shortcomings in the diagnostics of rare and emerging pathogens point to the need for continuing education in CF microbiology and suggest the establishment of CF microbiology reference laboratories.
Topics: Bacteria; Bacterial Infections; Bacteriological Techniques; Bronchopneumonia; Cystic Fibrosis; Diagnostic Errors; Europe; Health Services Research; Humans; Microbial Sensitivity Tests; Quality Control
PubMed: 19741077
DOI: 10.1128/JCM.01182-09