scholarly journals A ferredoxin-dependent dihydropyrimidine dehydrogenase in Clostridium chromiireducens

2020 ◽  
Vol 40 (7) ◽  
Author(s):  
Feifei Wang ◽  
Yifeng Wei ◽  
Qiang Lu ◽  
Ee Lui Ang ◽  
Huimin Zhao ◽  
...  
Keyword(s):  
Anaerobic Bacterium ◽  
Methyl Viologen ◽  
Flavin Adenine Dinucleotide ◽  
Biochemical Characterization ◽  
Dihydropyrimidine Dehydrogenase ◽  
Anaerobic Bacteria ◽  
Gene Clusters ◽  
Flavin Mononucleotide ◽  
Pyrimidine Degradation

Abstract Dihydropyrimidine dehydrogenase (PydA) catalyzes the first step of the reductive pyrimidine degradation (Pyd) pathway in bacteria and eukaryotes, enabling pyrimidines to be utilized as substrates for growth. PydA homologs studied to date catalyze the reduction of uracil to dihydrouracil, coupled to the oxidation of NAD(P)H. Uracil reduction occurs at a flavin mononucleotide (FMN) site, and NAD(P)H oxidation occurs at a flavin adenine dinucleotide (FAD) site, with two ferredoxin domains thought to mediate inter-site electron transfer. Here, we report the biochemical characterization of a Clostridial PydA homolog (PydAc) from a Pyd gene cluster in the strict anaerobic bacterium Clostridium chromiireducens. PydAc lacks the FAD domain, and instead is able to catalyze uracil reduction using reduced methyl viologen or reduced ferredoxin as the electron source. Homologs of PydAc are present in Pyd gene clusters in many strict anaerobic bacteria, which use reduced ferredoxin as an intermediate in their energy metabolism.

scholarly journals The thermochemical characterization of sodium dithionite, flavin mononucleotide, flavin-adenine dinucleotide and methyl and benzyl viologens as low-potential reductants for biological systems

1975 ◽  
Vol 152 (1) ◽  
pp. 33-37 ◽  
Author(s):  
G D Watts ◽  
A Burns
Keyword(s):  
Thermodynamic Data ◽  
Methyl Viologen ◽  
Flavin Adenine Dinucleotide ◽  
Sodium Dithionite ◽  
Flavin Mononucleotide ◽  
Heat Of Reaction ◽  
Calorimetric Data ◽  
Aqueous Buffer ◽  
Buffer Solutions

The heat of reaction (ΔH) of Fe(CN)63−, Methyl Viologen, FMN and FAD with S2O42− in aqueous buffer solutions was measured calorimetrically. In addition ΔH values for reduction of Fe(CN)63-, FMN and FAD by reduced Methyl Viologen were determined. The resulting calorimetric data and corresponding E0′ values were combined to yield thermodynamic data for these simple reducing agents in a form useful for applications to biological reactions. Thermodynamic data for the reduction of spinach ferredoxin are also presented.

Isolation and Characterization of an Anaerobic Bacterium, Eubacterium tarantellus sp.nov., Associated with Striped Mullet (Mugil cephalus) Mortality in Biscayne Bay, Florida

1977 ◽  
Vol 34 (3) ◽  
pp. 402-409 ◽  
Author(s):  
Lanny R. Udey ◽  
Elaine Young ◽  
Bennett Sallman
Keyword(s):  
Ictalurus Punctatus ◽  
Anaerobic Bacterium ◽  
Biochemical Characterization ◽  
Anaerobic Bacteria ◽  
Mugil Cephalus ◽  
Estuarine Fish ◽  
Biscayne Bay ◽  
Striped Mullet ◽  
Isolation And Characterization

An asporogenous, gram-positive, anaerobic bacterium was isolated in pure culture from the brains of numerous dead and moribund striped mullet (Mugil cephalus) from Biscayne Bay, Florida. Biochemical characterization confirmed that it was a new species and has been named Eubacterium tarantellus (ATCC 29255). All isolates produced lecithinase and deoxyribonuclease, were beta hemolytic, but only weakly fermented selected carbohydrates. This anaerobe was pathogenic for channel catfish (Ictalurus punctatus) but not for white mice or guinea pigs.More recently E. tarantellus has been isolated from 10 additional fish species. This and other anaerobic bacteria may be significant pathogens of estuarine fish. Key words: Anaerobic bacteria, Eubacterium, Mugil cephalus, and fish diseases.

scholarly journals Identification and characterization of cytochrome P450 1232A24 and 1232F1 from Arthrobacter sp. and their role in the metabolic pathway of papaverine

2019 ◽  
Vol 166 (1) ◽  
pp. 51-66 ◽  
Author(s):  
Jan M Klenk ◽  
Max-Philipp Fischer ◽  
Paulina Dubiel ◽  
Mahima Sharma ◽  
Benjamin Rowlinson ◽  
...  
Keyword(s):  
Cytochrome P450 ◽  
Metabolic Pathway ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Cytochrome P450 Monooxygenases ◽  
Dihydroxyphenylacetic Acid ◽  
Meta Position ◽  
Redox Partners

AbstractCytochrome P450 monooxygenases (P450s) play crucial roles in the cell metabolism and provide an unsurpassed diversity of catalysed reactions. Here, we report the identification and biochemical characterization of two P450s from Arthrobacter sp., a Gram-positive organism known to degrade the opium alkaloid papaverine. Combining phylogenetic and genomic analysis suggested physiological roles for P450s in metabolism and revealed potential gene clusters with redox partners facilitating the reconstitution of the P450 activities in vitro. CYP1232F1 catalyses the para demethylation of 3,4-dimethoxyphenylacetic acid to homovanillic acid while CYP1232A24 continues demethylation to 3,4-dihydroxyphenylacetic acid. Interestingly, the latter enzyme is also able to perform both demethylation steps with preference for the meta position. The crystal structure of CYP1232A24, which shares only 29% identity to previous published structures of P450s helped to rationalize the preferred demethylation specificity for the meta position and also the broader substrate specificity profile. In addition to the detailed characterization of the two P450s using their physiological redox partners, we report the construction of a highly active whole-cell Escherichia coli biocatalyst expressing CYP1232A24, which formed up to 1.77 g l−1 3,4-dihydroxyphenylacetic acid. Our results revealed the P450s’ role in the metabolic pathway of papaverine enabling further investigation and application of these biocatalysts.

scholarly journals Structure elucidation and biosynthesis of fuscachelins, peptide siderophores from the moderate thermophileThermobifida fusca

2008 ◽  
Vol 105 (40) ◽  
pp. 15311-15316 ◽  
Author(s):  
Eric J. Dimise ◽  
Paul F. Widboom ◽  
Steven D. Bruner
Keyword(s):  
Natural Products ◽  
Natural Product ◽  
Gene Cluster ◽  
Structure Elucidation ◽  
Biochemical Characterization ◽  
Gene Clusters ◽  
Biologically Active ◽  
Biosynthetic Gene ◽  
Nonribosomal Peptide

Bacteria belonging to the order Actinomycetales have proven to be an important source of biologically active and often therapeutically useful natural products. The characterization of orphan biosynthetic gene clusters is an emerging and valuable approach to the discovery of novel small molecules. Analysis of the recently sequenced genome of the thermophilic actinomyceteThermobifida fuscarevealed an orphan nonribosomal peptide biosynthetic gene cluster coding for an unknown siderophore natural product.T. fuscais a model organism for the study of thermostable cellulases and is a major degrader of plant cell walls. Here, we report the isolation and structure elucidation of the fuscachelins, siderophore natural products produced byT. fusca. In addition, we report the purification and biochemical characterization of the termination module of the nonribosomal peptide synthetase. Biochemical analysis of adenylation domain specificity supports the assignment of this gene cluster as the producer of the fuscachelin siderophores. The proposed nonribosomal peptide biosynthetic pathway exhibits several atypical features, including a macrocyclizing thioesterase that produces a 10-membered cyclic depsipeptide and a nonlinear assembly line, resulting in the unique heterodimeric architecture of the siderophore natural product.

scholarly journals vanD and vanG-Like Gene Clusters in a Ruminococcus Species Isolated from Human Bowel Flora

2007 ◽  
Vol 51 (11) ◽  
pp. 4111-4117 ◽  
Author(s):  
M.-C. Domingo ◽  
A. Huletsky ◽  
R. Giroux ◽  
F. J. Picard ◽  
M. G. Bergeron
Keyword(s):  
Gene Cluster ◽  
Anaerobic Bacterium ◽  
Anaerobic Bacteria ◽  
Gene Clusters ◽  
Surveillance Program ◽  
Rrna Gene ◽  
Genes Encoding ◽  
Bacterium Strain ◽  
Vancomycin Resistant ◽  
First Time

ABSTRACT A vancomycin-resistant, anaerobic, gram-positive coccus containing the vanD and vanG-like genes (strain CCRI-16110) was isolated from a human fecal specimen during a hospital surveillance program to detect carriers of vancomycin-resistant enterococci. Comparison of the 16S rRNA gene sequence of strain CCRI-16110 with databases revealed a potentially novel Ruminococcus species that was most similar (<94% identity) to Clostridium and Ruminococcus species. Strain CCRI-16110 was highly resistant to vancomycin and teicoplanin (MICs of >256 μg/ml). The complete DNA sequence of the vanD cluster was most similar (98.2% identity) to that of Enterococcus faecium BM4339, containing the vanD1 allele. An intD gene with 99% identity with that of this E. faecium strain was found to be associated with the vanD gene cluster of this novel anaerobic bacterium. Strain CCRI-16110 also harbors genes encoding putative VanSG, VanG, and VanTG proteins displaying 56, 73.6, and 55% amino acid sequence identity, respectively, compared to the corresponding proteins encoded by the vanG1 and vanG2 operons of Enterococcus faecalis BM4518 and N03-0233. This study reports for the first time an anaerobic bacterium containing the vanD gene cluster. This strain also harbors a partial vanG-like gene cluster. The presence of vanD- and vanG-containing anaerobic bacteria in the human bowel flora suggests that these bacteria may serve as a reservoir for the vanD and vanG vancomycin resistance genes.

scholarly journals Biochemical Characterization of Anaerobic Bacteria by an Impregnated Disk Method

1977 ◽  
Vol 5 (6) ◽  
pp. 673-675
Author(s):  
Jere M. Boyer
Keyword(s):  
Biochemical Characterization ◽  
Anaerobic Bacteria ◽  
Clinical Isolates ◽  
Disk Method

An impregnated disk method was used to characterize 20 clinical isolates of anaerobic bacteria by utilization of substrate. The procedure was easy to perform, reliable, and economical.

scholarly journals Characterization of l-2-keto-3-deoxyfuconate aldolases in a nonphosphorylating l-fucose metabolism pathway in anaerobic bacteria

2019 ◽  
Vol 295 (5) ◽  
pp. 1338-1349 ◽  
Author(s):  
Seiya Watanabe
Keyword(s):  
Gene Cluster ◽  
Biochemical Characterization ◽  
Aldol Condensation ◽  
Condensation Reaction ◽  
Anaerobic Bacteria ◽  
Site Directed Mutagenesis ◽  
Strictly Anaerobic ◽  
Dihydrodipicolinate Synthase ◽  
Equivalent Position

The genetic context in bacterial genomes and screening for potential substrates can help identify the biochemical functions of bacterial enzymes. The Gram-negative, strictly anaerobic bacterium Veillonella ratti possesses a gene cluster that appears to be related to l-fucose metabolism and contains a putative dihydrodipicolinate synthase/N-acetylneuraminate lyase protein (FucH). Here, screening of a library of 2-keto-3-deoxysugar acids with this protein and biochemical characterization of neighboring genes revealed that this gene cluster encodes enzymes in a previously unknown “route I” nonphosphorylating l-fucose pathway. Previous studies of other aldolases in the dihydrodipicolinate synthase/N-acetylneuraminate lyase protein superfamily used only limited numbers of compounds, and the approach reported here enabled elucidation of the substrate specificities and stereochemical selectivities of these aldolases and comparison of them with those of FucH. According to the aldol cleavage reaction, the aldolases were specific for (R)- and (S)-stereospecific groups at the C4 position of 2-keto-3-deoxysugar acid but had no structural specificity or preference of methyl groups at the C5 and C6 positions, respectively. This categorization corresponded to the (Re)- or (Si)-facial selectivity of the pyruvate enamine on the (glycer)aldehyde carbonyl in the aldol-condensation reaction. These properties are commonly determined by whether a serine or threonine residue is positioned at the equivalent position close to the active site(s), and site-directed mutagenesis markedly modified C4-OH preference and selective formation of a diastereomer. I propose that substrate specificity of 2-keto-3-deoxysugar acid aldolases was convergently acquired during evolution and report the discovery of another l-2-keto-3-deoxyfuconate aldolase involved in the same nonphosphorylating l-fucose pathway in Campylobacter jejuni.

scholarly journals Identification and characterization of a new sulfoacetaldehyde reductase from the human gut bacterium Bifidobacterium kashiwanohense

2019 ◽  
Vol 39 (6) ◽  
Author(s):  
Yan Zhou ◽  
Yifeng Wei ◽  
Ankanahalli N. Nanjaraj Urs ◽  
Lianyun Lin ◽  
Tong Xu ◽  
...  
Keyword(s):  
Nitrogen Assimilation ◽  
Biochemical Characterization ◽  
Anaerobic Bacteria ◽  
Binary Complex ◽  
Active Site Residues ◽  
Human Gut ◽  
Mammalian Tissues ◽  
Environmental Bacteria ◽  
Identification And Characterization

AbstractHydroxyethylsulfonate (isethionate (Ise)) present in mammalian tissues is thought to be derived from aminoethylsulfonate (taurine), as a byproduct of taurine nitrogen assimilation by certain anaerobic bacteria inhabiting the taurine-rich mammalian gut. In previously studied pathways occurring in environmental bacteria, isethionate is generated by the enzyme sulfoacetaldehyde reductase IsfD, belonging to the short-chain dehydrogenase/reductase (SDR) family. An unrelated sulfoacetaldehyde reductase SarD, belonging to the metal-dependent alcohol dehydrogenase superfamily (M-ADH), was recently discovered in the human gut sulfite-reducing bacterium Bilophila wadsworthia (BwSarD). Here we report the structural and biochemical characterization of a sulfoacetaldehyde reductase from the human gut fermenting bacterium Bifidobacterium kashiwanohense (BkTauF). BkTauF belongs to the M-ADH family, but is distantly related to BwSarD (28% sequence identity). The crystal structures of BkTauF in the apo form and in a binary complex with NAD+ were determined at 1.9 and 3.0 Å resolution, respectively. Mutagenesis studies were carried out to investigate the involvement of active site residues in binding the sulfonate substrate. Our studies demonstrate the presence of sulfoacetaldehyde reductase in Bifidobacteria, with a possible role in isethionate production as a byproduct of taurine nitrogen assimilation.

scholarly journals Identification and Characterization of Catabolic para-Nitrophenol 4-Monooxygenase and para-Benzoquinone Reductase from Pseudomonas sp. Strain WBC-3

2009 ◽  
Vol 191 (8) ◽  
pp. 2703-2710 ◽  
Author(s):  
Jun-Jie Zhang ◽  
Hong Liu ◽  
Yi Xiao ◽  
Xian-En Zhang ◽  
Ning-Yi Zhou
Keyword(s):  
Gene Cluster ◽  
Flavin Adenine Dinucleotide ◽  
Gel Filtration ◽  
Sole Source ◽  
Open Reading Frames ◽  
Flavin Mononucleotide ◽  
Large Component ◽  
Identification And Characterization ◽  
Reading Frames

ABSTRACT Pseudomonas sp. strain WBC-3 utilizes para-nitrophenol (PNP) as a sole source of carbon, nitrogen, and energy. In order to identify the genes involved in this utilization, we cloned and sequenced a 12.7-kb fragment containing a conserved region of NAD(P)H:quinone oxidoreductase genes. Of the products of the 13 open reading frames deduced from this fragment, PnpA shares 24% identity to the large component of a 3-hydroxyphenylacetate hydroxylase from Pseudomonas putida U and PnpB is 58% identical to an NAD(P)H:quinone oxidoreductase from Escherichia coli. Both PnpA and PnpB were purified to homogeneity as His-tagged proteins, and they were considered to be a monomer and a dimer, respectively, as determined by gel filtration. PnpA is a flavin adenine dinucleotide-dependent single-component PNP 4-monooxygenase that converts PNP to para-benzoquinone in the presence of NADPH. PnpB is a flavin mononucleotide-and NADPH-dependent p-benzoquinone reductase that catalyzes the reduction of p-benzoquinone to hydroquinone. PnpB could enhance PnpA activity, and genetic analyses indicated that both pnpA and pnpB play essential roles in PNP mineralization in strain WBC-3. Furthermore, the pnpCDEF gene cluster next to pnpAB shares significant similarities with and has the same organization as a gene cluster responsible for hydroquinone degradation (hapCDEF) in Pseudomonas fluorescens ACB (M. J. Moonen, N. M. Kamerbeek, A. H. Westphal, S. A. Boeren, D. B. Janssen, M. W. Fraaije, and W. J. van Berkel, J. Bacteriol. 190:5190-5198, 2008), suggesting that the genes involved in PNP degradation are physically linked.

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