Extradiol Dioxygenases Retrieved from the Metagenome

2015 ◽  
pp. 167-171
Author(s):  
Kentaro Miyazaki ◽  
Hikaru Suenaga
Keyword(s):  
Extradiol Dioxygenases

Extradiol Dioxygenases Retrieved from the Metagenome

2013 ◽  
pp. 1-5
Author(s):  
Kentaro Miyazaki ◽  
Hikaru Suenaga
Keyword(s):  
Extradiol Dioxygenases

scholarly journals Functional Metagenomics of a Biostimulated Petroleum-Contaminated Soil Reveals an Extraordinary Diversity of Extradiol Dioxygenases

2016 ◽  
Vol 82 (8) ◽  
pp. 2467-2478 ◽  
Author(s):  
Laura Terrón-González ◽  
Guadalupe Martín-Cabello ◽  
Manuel Ferrer ◽  
Eduardo Santero
Keyword(s):  
Contaminated Soil ◽  
Phylogenetic Analyses ◽  
Metagenomic Library ◽  
Type I ◽  
Type Ii ◽  
Extradiol Dioxygenase ◽  
Metagenomic Dna ◽  
Petroleum Contaminated Soil ◽  
Definition Of ◽  
Extradiol Dioxygenases

ABSTRACTA metagenomic library of a petroleum-contaminated soil was constructed in a fosmid vector that allowed heterologous expression of metagenomic DNA. The library, consisting of 6.5 Gb of metagenomic DNA, was screened for extradiol dioxygenase (Edo) activity using catechol and 2,3-dihydroxybiphenyl as the substrates. Fifty-eight independent clones encoding extradiol dioxygenase activity were identified. Forty-one different Edo-encoding genes were identified. The population of Edo genes was not dominated by a particular gene or by highly similar genes; rather, the genes had an even distribution and high diversity. Phylogenetic analyses revealed that most of the genes could not be ascribed to previously defined subfamilies of Edos. Rather, the Edo genes led to the definition of 10 new subfamilies of type I Edos. Phylogenetic analysis of type II enzymes defined 7 families, 2 of which harbored the type II Edos that were found in this work. Particularly striking was the diversity found in family I.3 Edos; 15 out of the 17 sequences assigned to this family belonged to 7 newly defined subfamilies. A strong bias was found that depended on the substrate used for the screening: catechol mainly led to the detection of Edos belonging to the I.2 family, while 2,3-dihydroxybiphenyl led to the detection of most other Edos. Members of the I.2 family showed a clear substrate preference for monocyclic substrates, while those from the I.3 family showed a broader substrate range and high activity toward 2,3-dihydroxybiphenyl. This metagenomic analysis has substantially increased our knowledge of the existing biodiversity of Edos.

scholarly journals Characterization of the 3-O-Methylgallate Dioxygenase Gene and Evidence of Multiple 3-O-Methylgallate Catabolic Pathways in Sphingomonas paucimobilis SYK-6

2004 ◽  
Vol 186 (15) ◽  
pp. 4951-4959 ◽  
Author(s):  
Daisuke Kasai ◽  
Eiji Masai ◽  
Keisuke Miyauchi ◽  
Yoshihiro Katayama ◽  
Masao Fukuda
Keyword(s):  
Sole Source ◽  
Cell Extract ◽  
Mass Spectrometry Analysis ◽  
Gas Chromatography Mass Spectrometry ◽  
Insertion Mutant ◽  
Sphingomonas Paucimobilis ◽  
Spectrometry Analysis ◽  
Dioxygenase Gene ◽  
Transformation Activity ◽  
Extradiol Dioxygenases

ABSTRACT Sphingomonas paucimobilis SYK-6 is able to grow on various lignin-derived biaryls as the sole source of carbon and energy. These compounds are degraded to vanillate and syringate by the unique and specific enzymes in this strain. Vanillate and syringate are converted to protocatechuate (PCA) and 3-O-methylgallate (3MGA), respectively, by the tetrahydrofolate-dependent O-demethylases. Previous studies have suggested that these compounds are further degraded via the PCA 4,5-cleavage pathway. However, our subsequent analysis of the ligB insertion mutant, which encodes the β subunit of PCA 4,5-dioxygenase, suggested that at least one alternative route is involved in 3MGA degradation. In the present study, we isolated the desZ gene, which confers 3MGA degradation activity on Escherichia coli. The deduced amino acid sequence of desZ showed ca. 20 to 43% identity with the type II extradiol dioxygenases. Gas chromatography-mass spectrometry analysis suggested that DesZ catalyzes the 3,4-cleavage of 3MGA. Disruption of both desZ and ligB in SYK-6 resulted in loss of the dioxygen-dependent 3MGA transformation activity, but the resulting mutant retained the ability to grow on syringate. We found that the cell extract of the desZ ligB double mutant was able to convert 3MGA to gallate when tetrahydrofolate was added to the reaction mixture, and the cell extract of this mutant degraded gallate to the same degree as the wild type did. All these results suggest that syringate is degraded through multiple 3MGA degradation pathways in which ligAB, desZ, 3MGA O-demethylase, and gallate dioxygenase are participants.

scholarly journals Identification of an Extradiol Dioxygenase Involved in Tetralin Biodegradation: Gene Sequence Analysis and Purification and Characterization of the Gene Product

2000 ◽  
Vol 182 (3) ◽  
pp. 789-795 ◽  
Author(s):  
Eloísa Andújar ◽  
María José Hernáez ◽  
Stefan R. Kaschabek ◽  
Walter Reineke ◽  
Eduardo Santero
Keyword(s):  
Gene Product ◽  
Biochemical Properties ◽  
Genomic Region ◽  
Quinoline Derivative ◽  
Ring Cleavage ◽  
Insertion Mutant ◽  
Significant Activity ◽  
Extradiol Dioxygenase ◽  
Group 2 ◽  
Extradiol Dioxygenases

ABSTRACT A genomic region involved in tetralin biodegradation was recently identified in Sphingomonas strain TFA. We have cloned and sequenced from this region a gene designated thnC, which codes for an extradiol dioxygenase required for tetralin utilization. Comparison to similar sequences allowed us to define a subfamily of 1,2-dihydroxynaphthalene extradiol dioxygenases, which comprises two clearly different groups, and to show that ThnC clusters within group 2 of this subfamily. 1,2-Dihydroxy-5,6,7,8-tetrahydronaphthalene was found to be the metabolite accumulated by a thnC insertion mutant. The ring cleavage product of this metabolite exhibited behavior typical of a hydroxymuconic semialdehyde toward pH-dependent changes and derivatization with ammonium to give a quinoline derivative. The gene product has been purified, and its biochemical properties have been studied. The enzyme is a decamer which requires Fe(II) for activity and shows high activity toward its substrate (V max, 40.5 U mg−1;Km , 18.6 μM). The enzyme shows even higher activity with 1,2-dihydroxynaphthalene and also significant activity toward 1,2-dihydroxybiphenyl or methylated catechols. The broad substrate specificity of ThnC is consistent with that exhibited by other extradiol dioxygenases of the same group within the subfamily of 1,2-dihydroxynaphthalene dioxygenases.

scholarly journals The molecular basis for adaptive evolution in novel extradiol dioxygenases retrieved from the metagenome

2009 ◽  
Vol 69 (3) ◽  
pp. 472-480 ◽  
Author(s):  
Hikaru Suenaga ◽  
Shiori Mizuta ◽  
Kentaro Miyazaki
Keyword(s):  
Adaptive Evolution ◽  
Molecular Basis ◽  
Extradiol Dioxygenases

Genes for Mn(II)-dependent NahC and Fe(II)-dependent NahH located in close proximity in the thermophilic naphthalene and PCB degrader, Bacillus sp. JF8: cloning and characterization

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 993-1004 ◽  
Author(s):  
Daisuke Miyazawa ◽  
Gouri Mukerjee-Dhar ◽  
Minoru Shimura ◽  
Takashi Hatta ◽  
Kazuhide Kimbara
Keyword(s):  
Inductively Coupled Plasma ◽  
Metal Ion ◽  
Polychlorinated Biphenyl ◽  
Mass Spectrometry Analysis ◽  
Northern Hybridization ◽  
Active Site Residues ◽  
Extradiol Dioxygenase ◽  
Spectrometry Analysis ◽  
Dna Fragment ◽  
Extradiol Dioxygenases

A 10 kb DNA fragment was isolated using a DNA probe derived from the N-terminal amino acid sequence of the extradiol dioxygenase purified from naphthalene-grown Bacillus sp. JF8, a thermophilic naphthalene and polychlorinated biphenyl degrader. The cloned DNA fragment had six open reading frames, designated nahHLOMmocBnahC based on sequence homology, of which the products NahH_JF8 and NahC_JF8 were extradiol dioxygenases. Although NahC_JF8 and NahH_JF8 exhibit low homology to known extradiol dioxygenases, the active-site residues and metal ion ligands are conserved. The presence of Mn(II) in culture medium was found to be essential for production of active recombinant NahC_JF8, while Fe(II) was necessary for active recombinant NahH_JF8. Inductively coupled plasma mass spectrometry analysis of active NahC_JF8 identified the cofactor to be manganese, indicating a Mn(II)-dependent extradiol dioxygenase. NahC_JF8 exhibited K m values of 32±5 μM for 1,2-dihydroxynaphthalene and 510±90 μM for 2,3-dihydroxybiphenyl at 60 °C. In cell-free extracts, NahH_JF8 exhibited a broad substrate range for 2,3-dihydroxybiphenyl, catechol, and 3- and 4-methylcatechol at 25 °C. Stability studies on the Mn(II)-dependent NahC_JF8 indicated that it was thermostable, retaining 50 % activity after incubation at 80 °C for 20 min, and it exhibited resistance to EDTA and H2O2. Northern hybridization studies clarified that both NahC_JF8 and NahH_JF8 were induced by naphthalene; RT-PCR showed that nahHLOMmocBnahC is expressed as a single transcript.

Site-directed mutagenesis of an extradiol dioxygenase involved in tetralin biodegradation identifies residues important for activity or substrate specificity

Microbiology ◽  
2003 ◽  
Vol 149 (6) ◽  
pp. 1559-1567 ◽  
Author(s):  
Eloísa Andújar ◽  
Eduardo Santero
Keyword(s):  
Substrate Specificity ◽  
Binding Site ◽  
Mutational Analysis ◽  
Structural Information ◽  
Substrate Inhibition ◽  
Site Directed Mutagenesis ◽  
Functional Importance ◽  
Extradiol Dioxygenase ◽  
Polycyclic Compounds ◽  
Extradiol Dioxygenases

The sequence of the extradiol dioxygenase ThnC, involved in tetralin biodegradation, was aligned with other extradiol dioxygenases involved in biodegradation of polycyclic compounds, and a three-dimensional model of ThnC, based on the structure of the previously crystallized 2,3-dihydroxybiphenyl dioxygenase from Burkholderia fungorum LB400, was built. In order to assess the functional importance of some non-active-site residues whose relevance could not be established by structural information, a number of positions surrounding the substrate-binding site were mutated in ThnC. Ten mutant proteins were purified and their activity towards 1,2-dihydroxytetralin, 1,2-dihydroxynaphthalene and 2,3-dihydroxybiphenyl was characterized. N213H, Q198H, G206M, A282R and A282G mutants increased k cat/K m at least twofold using 1,2-dihydroxytetralin as the substrate, thus showing that activity of ThnC is not maximized for this substrate. N213H and Q198H mutants increased k cat/K m using any of the substrates tested, thus showing the relevance for activity of these two histidines, which are highly conserved in dihydroxybiphenyl dioxygenases, but not present in dihydroxynaphthalene dioxygenases. Different substitutions in position 282 had different effects on general activity or substrate specificity, thus showing the functional importance of the most C-terminal β-sheet of the protein. A251M and G206M mutants showed increased activity specifically for a particular substrate. N213H, G206M, A282R, A282G and Y177I substitutions resulted in enzymes more tolerant to acidic pH, the most striking effect being observed in mutant Y177I, which showed maximal activity at pH 5·5. In addition, Q198D and V175D mutants, which had altered K m, also showed altered sensitivity to substrate inhibition, thus indicating that inhibition is exerted through the same binding site. This mutational analysis, therefore, identified conserved residues important for activity or substrate specificity, and also shed some light on the mechanism of substrate inhibition exhibited by extradiol dioxygenases.

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