scholarly journals Identification, Cloning, and Characterization of l-Phenylserine Dehydrogenase from Pseudomonas syringae NK-15

2010 ◽  
Vol 2010 ◽  
pp. 1-10 ◽  
Author(s):  
Sakuko Ueshima ◽  
Hisashi Muramatsu ◽  
Takanori Nakajima ◽  
Hiroaki Yamamoto ◽  
Shin-ichiro Kato ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Homology Search ◽  
Hydroxyl Group ◽  
Enzymatic Reactions ◽  
Reaction Products ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Single Operon

The gene encoding d-phenylserine dehydrogenase from Pseudomonas syringae NK-15 was identified, and a 9,246-bp nucleotide sequence containing the gene was sequenced. Six ORFs were confirmed in the sequenced region, four of which were predicted to form an operon. A homology search of each ORF predicted that orf3 encoded l-phenylserine dehydrogenase. Hence, orf3 was cloned and overexpressed in Escherichia coli cells and recombinant ORF3 was purified to homogeneity and characterized. The purified ORF3 enzyme showed l-phenylserine dehydrogenase activity. The enzymological properties and primary structure of l-phenylserine dehydrogenase (ORF3) were quite different from those of d-phenylserine dehydrogenase previously reported. l-Phenylserine dehydrogenase catalyzed the NAD+-dependent oxidation of the β-hydroxyl group of l-β-phenylserine. l-Phenylserine and l-threo-(2-thienyl)serine were good substrates for l-phenylserine dehydrogenase. The genes encoding l-phenylserine dehydrogenase and d-phenylserine dehydrogenase, which is induced by phenylserine, are located in a single operon. The reaction products of both enzymatic reactions were 2-aminoacetophenone and CO2.

scholarly journals Characterization of Alginate Lyase from Pseudomonas syringae pv. syringae

2000 ◽  
Vol 182 (21) ◽  
pp. 6268-6271 ◽  
Author(s):  
Lori A. Preston ◽  
T. Y. Wong ◽  
Carol L. Bender ◽  
Neal L. Schiller
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Alginate Lyase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis ◽  
Gene Encoding ◽  
Tryptophan Residues ◽  
Lyase Activity ◽  
Alginate Lyases

ABSTRACT The gene encoding alginate lyase (algL) inPseudomonas syringae pv. syringae was cloned, sequenced, and overexpressed in Escherichia coli. Alginate lyase activity was optimal when the pH was 7.0 and when assays were conducted at 42°C in the presence of 0.2 M NaCl. In substrate specificity studies, AlgL from P. syringae showed a preference for deacetylated polymannuronic acid. Sequence alignment with other alginate lyases revealed conserved regions within AlgL likely to be important for the structure and/or function of the enzyme. Site-directed mutagenesis of histidine and tryptophan residues at positions 204 and 207, respectively, indicated that these amino acids are critical for lyase activity.

scholarly journals Characterization of catechol 2,3-dioxygenase from Planococcus sp. strain S5 induced by high phenol concentration.

2012 ◽  
Vol 59 (3) ◽  
Author(s):  
Katarzyna Hupert-Kocurek ◽  
Urszula Guzik ◽  
Danuta Wojcieszyńska
Keyword(s):  
Kinetic Studies ◽  
High Reactivity ◽  
Homology Search ◽  
Nucleotide Sequencing ◽  
Positive Bacterium ◽  
Gene Encoding ◽  
Genes Encoding ◽  
High Phenol Concentration ◽  
Dioxygenase Activity

This study aimed at characterization of a new catechol 2,3-dioxygenase isolated from a Gram-positive bacterium able to utilize phenol as the sole carbon and energy source. Planococcus sp. strain S5 grown on 1 or 2 mM phenol showed activity of both a catechol 1,2- and catechol 2,3-dioxygenase while at a higher concentrations of phenol only catechol 2,3-dioxygenase activity was observed. The enzyme was optimally active at 60°C and pH 8.0. Kinetic studies showed that the K(m) and V(max) of the enzyme were 42.70 µM and 329.96 mU, respectively. The catechol 2,3-dioxygenase showed the following relative meta-cleavage activities for various catechols tested: catechol (100%), 3-methylcatechol (13.67%), 4-methylcatechol (106.33%) and 4-chlorocatechol (203.80%). The high reactivity of this enzyme towards 4-chlorocatechol is different from that observed for other catechol 2,3-dioxygenases. Nucleotide sequencing and homology search revealed that the gene encoding the S5 catechol 2,3-dioxygenase shared the greatest homology with the known genes encoding isoenzymes from Gram-negative Pseudomonas strains.

Cloning, expression, and characterization of Baeyer–Villiger monooxygenases from eukaryotic Exophiala jeanselmei strain KUFI-6N

2021 ◽  
Author(s):  
Taisei Yamamoto ◽  
Kento Kobayashi ◽  
Yoshie Hasegawa ◽  
Hiroaki Iwaki
Keyword(s):  
Escherichia Coli ◽  
Catalytic Efficiency ◽  
Amino Acid Identity ◽  
Membered Ring ◽  
Gene Encoding ◽  
Exophiala Jeanselmei ◽  
Genes Encoding ◽  
Substrate Spectrum ◽  
Villiger Oxidation

Abstract The fungus Exophiala jeanselmei strain KUFI-6N produces a unique cycloalkanone monooxygenase (ExCAMO) that displays an uncommon substrate spectrum of Baeyer–Villiger oxidation of 4–10-membered ring ketones. In this study, we aimed to identify and sequence the gene encoding ExCAMO from KUFI-6N and overexpress the gene in Escherichia coli. We found that the primary structure of ExCAMO is most closely related to the cycloalkanone monooxygenase from Cylindrocarpon radicicola ATCC 11011, with 54.2% amino acid identity. ExCAMO was functionally expressed in Escherichia coli and its substrate spectrum and kinetic parameters investigated. Substrate profiling indicated that ExCAMO is unusual among known Baeyer–Villiger monooxygenases owing to its ability to accept a variety of substrates, including C4–C12 membered ring ketones. ExCAMO has high affinity and catalytic efficiency toward cycloalkanones, the highest being toward cyclohexanone. Five other genes encoding Baeyer–Villiger monooxygenases were also cloned and expressed in Escherichia coli.

Molecular characterization of three mutations in katG affecting the activity of hydroperoxidase I of Escherichia coli

1990 ◽  
Vol 68 (7-8) ◽  
pp. 1037-1044 ◽  
Author(s):  
Peter C. Loewen ◽  
Jacek Switala ◽  
Mark Smolenski ◽  
Barbara L. Triggs-Raine
Keyword(s):  
Escherichia Coli ◽  
Specific Activity ◽  
Polymerase Chain Reaction Amplification ◽  
Protein A ◽  
Wild Type ◽  
Wild Type Enzyme ◽  
Gene Encoding ◽  
Reaction Amplification ◽  
Mutant Genes

Hydroperoxidase I (HPI) of Escherichia coli is a bifunctional enzyme exhibiting both catalase and peroxidase activities. Mutants lacking appreciable HPI have been generated using nitrosoguanidine and the gene encoding HPI, katG, has been cloned from three of these mutants using either classical probing methods or polymerase chain reaction amplification. The mutant genes were sequenced and the changes from wild-type sequence identified. Two mutants contained G to A changes in the coding strand, resulting in glycine to aspartate changes at residues 119 (katG15) and 314 (katG16) in the deduced amino acid sequence of the protein. A third mutant contained a C to T change resulting in a leucine to phenylalanine change at residue 139 (katG14). The Phe139-, Asp119-, and Asp314-containing mutants exhibited 13, < 1, and 18%, respectively, of the wild-type catalase specific activity and 43, 4, and 45% of the wild-type peroxidase specific activity. All mutant enzymes bound less protoheme IX than the wild-type enzyme. The sensitivities of the mutant enzymes to the inhibitors hydroxylamine, azide, and cyanide and the activators imidazole and Tris were similar to those of the wild-type enzyme. The mutant enzymes were more sensitive to high temperature and to β-mercaptoethanol than the wild-type enzyme. The pH profiles of the mutant catalases were unchanged from the wild-type enzyme.Key words: catalase, hydroperoxidase I, mutants, sequence analysis.

scholarly journals Characterization of Epidemic IncI1-Iγ Plasmids Harboring Ambler Class A and C Genes in Escherichia coli and Salmonella enterica from Animals and Humans

2015 ◽  
Vol 59 (9) ◽  
pp. 5357-5365 ◽  
Author(s):  
Hilde Smith ◽  
Alex Bossers ◽  
Frank Harders ◽  
Guanghui Wu ◽  
Neil Woodford ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Salmonella Enterica ◽  
Phylogenetic Trees ◽  
Functional Study ◽  
Content Type ◽  
Gene Associations ◽  
Genes Encoding ◽  
Marked Resistance

ABSTRACTThe aim of the study was to identify the plasmid-encoded factors contributing to the emergence and spread of epidemic IncI1-Iγ plasmids obtained fromEscherichia coliandSalmonella entericaisolates from animal and human reservoirs. For this, 251 IncI1-Iγ plasmids carrying various extended-spectrum β-lactamase (ESBL) or AmpC β-lactamase genes were compared using plasmid multilocus sequence typing (pMLST). Thirty-two of these plasmids belonging to different pMLST types were sequenced using Roche 454 and Illumina platforms. Epidemic IncI1-Iγ plasmids could be assigned to various dominant clades, whereas rarely detected plasmids clustered together as a distinct clade. Similar phylogenetic trees were obtained using only the plasmid backbone sequences, showing that the differences observed between the plasmids belonging to distinct clades resulted mainly from differences between their backbone sequences. Plasmids belonging to the various clades differed particularly in the presence/absence of genes encoding partitioning and addiction systems, which contribute to stable inheritance during cell division and plasmid maintenance. Despite this, plasmids belonging to the various phylogenetic clades also showed marked resistance gene associations, indicating the circulation of successful plasmid-gene combinations. The variation intraYandexcAgenes found in IncI1-Iγ plasmids is conserved within pMLST sequence types and plays a role in incompatibility, although functional study is needed to elucidate the role of these genes in plasmid epidemiology.

scholarly journals Identification of Streptococci to Species Level by Sequencing the Gene Encoding the Manganese-Dependent Superoxide Dismutase

1998 ◽  
Vol 36 (1) ◽  
pp. 41-47 ◽  
Author(s):  
Claire Poyart ◽  
Gilles Quesne ◽  
Stephane Coulon ◽  
Patrick Berche ◽  
Patrick Trieu-Cuot
Keyword(s):  
Superoxide Dismutase ◽  
Pcr Assay ◽  
Degenerate Primers ◽  
Gene Encoding ◽  
Genes Encoding ◽  
Type Strains ◽  
Internal Fragment ◽  
Rrna Sequences ◽  
16S Rrna Sequences

We have used a PCR assay based on the use of degenerate primers in order to characterize an internal fragment (sodAint ) representing approximately 85% of the genes encoding the manganese-dependent superoxide dismutase in various streptococcal type strains (S. acidominimus,S. agalactiae, S. alactolyticus, S. anginosus, S. bovis, S. constellatus,S. canis, S. cricetus, S. downei,S. dysgalactiae, S. equi subsp.equi, S. equi subsp. zooepidemicus,S. equinus, S. gordonii, S. iniae,S. intermedius, S. mitis, S. mutans, S. oralis, S. parasanguis,S. pneumoniae, S. porcinus, S. pyogenes, S. salivarius, S. sanguis,S. sobrinus, S. suis, S. thermophilus, and S. vestibularis). Phylogenetic analysis of these sodAint fragments yields an evolutionary tree having a topology similar to that of the tree constructed with the 16S rRNA sequences. We have shown that clinical isolates could be identified by determining the positions of theirsodAint fragments on the phylogenetic tree of the sodAint fragments of the type species. We propose this method for the characterization of strains that cannot be assigned to a species on the basis of their conventional phenotypic reactions.

scholarly journals PhhB, a Pseudomonas aeruginosa Homolog of Mammalian Pterin 4a-Carbinolamine Dehydratase/DCoH, Does Not Regulate Expression of Phenylalanine Hydroxylase at the Transcriptional Level

1999 ◽  
Vol 181 (9) ◽  
pp. 2789-2796 ◽  
Author(s):  
Jian Song ◽  
Tianhui Xia ◽  
Roy A. Jensen
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Aeruginosa ◽  
Phenylalanine Hydroxylase ◽  
Transcriptional Level ◽  
Gene Encoding ◽  
Catalytic Function ◽  
Genes Encoding ◽  
Catalytic Role ◽  
Regulatory Effects

ABSTRACT Pterin 4a-carbinolamine dehydratase is bifunctional in mammals. In addition to playing a catalytic role in pterin recycling in the cytoplasm, it plays a regulatory role in the nucleus, where it acts as a dimerization-cofactor component (called DCoH) for the transcriptional activator HNF-1α. A thus far unique operon in Pseudomonas aeruginosa contains a gene encoding a homolog (PhhB) of the regulatory dehydratase, together with genes encoding phenylalanine hydroxylase (PhhA) and aromatic aminotransferase (PhhC). Using complementation of tyrosine auxotrophy in Escherichia colias a functional test, we have found that the in vivo function of PhhA requires PhhB. Strikingly, mammalian DCoH was an effective substitute for PhhB, and either one was effective in trans. Surprisingly, the required presence of PhhB for complementation did not reflect a critical positive regulatory effect of phhB onphhA expression. Rather, in the absence of PhhB, PhhA was found to be extremely toxic in E. coli, probably due to the nonenzymatic formation of 7-biopterin or a similar derivative. However, bacterial PhhB does appear to exert modest regulatory effects in addition to having a catalytic function. PhhB enhances the level of PhhA two- to threefold, as was demonstrated by gene inactivation ofphhB in P. aeruginosa and by comparison of the levels of expression of PhhA in the presence and absence of PhhB inEscherichia coli. Experiments using constructs having transcriptional and translational fusions with a lacZreporter indicated that PhhB activates PhhA at the posttranscriptional level. Regulation of PhhA and PhhB is semicoordinate; both PhhA and PhhB are induced coordinately in the presence of eitherl-tyrosine or l-phenylalanine, but PhhB exhibits a significant basal level of activity that is lacking for PhhA. Immunoprecipitation and affinity chromatography showed that PhhA and PhhB form a protein-protein complex.

scholarly journals Production of Glucaric Acid from a Synthetic Pathway in Recombinant Escherichia coli

2008 ◽  
Vol 75 (3) ◽  
pp. 589-595 ◽  
Author(s):  
Tae Seok Moon ◽  
Sang-Hwal Yoon ◽  
Amanda M. Lanza ◽  
Joseph D. Roy-Mayhew ◽  
Kristala L. Jones Prather
Keyword(s):  
Escherichia Coli ◽  
Pseudomonas Syringae ◽  
Glucuronic Acid ◽  
Value Added ◽  
Culture Broth ◽  
Glucaric Acid ◽  
Synthetic Pathway ◽  
Genes Encoding ◽  
Microbial System ◽  
Rate Limiting

ABSTRACT A synthetic pathway has been constructed for the production of glucuronic and glucaric acids from glucose in Escherichia coli. Coexpression of the genes encoding myo-inositol-1-phosphate synthase (Ino1) from Saccharomyces cerevisiae and myo-inositol oxygenase (MIOX) from mice led to production of glucuronic acid through the intermediate myo-inositol. Glucuronic acid concentrations up to 0.3 g/liter were measured in the culture broth. The activity of MIOX was rate limiting, resulting in the accumulation of both myo-inositol and glucuronic acid as final products, in approximately equal concentrations. Inclusion of a third enzyme, uronate dehydrogenase (Udh) from Pseudomonas syringae, facilitated the conversion of glucuronic acid to glucaric acid. The activity of this recombinant enzyme was more than 2 orders of magnitude higher than that of Ino1 and MIOX and increased overall flux through the pathway such that glucaric acid concentrations in excess of 1 g/liter were observed. This represents a novel microbial system for the biological production of glucaric acid, a “top value-added chemical” from biomass.

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