Dataset Information

A novel angular dioxygenase gene cluster encoding 3-phenoxybenzoate 1',2'-dioxygenase in Sphingobium wenxiniae JZ-1.

ABSTRACT: Sphingobium wenxiniae JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy. A mutant JZ-1 strain, MJZ-1, defective in the degradation of 3-phenoxybenzoate was obtained by successive streaking on LB agar. Comparison of the draft genomes of strains JZ-1 and MJZ-1 revealed that a 29,366-bp DNA fragment containing a putative angular dioxygenase gene cluster (pbaA1A2B) is missing in strain MJZ-1. PbaA1, PbaA2, and PbaB share 65%, 52%, and 10% identity with the corresponding ? and ? subunits and the ferredoxin component of dioxin dioxygenase from Sphingomonas wittichii RW1, respectively. Complementation of pbaA1A2B in strain MJZ-1 resulted in the active 3-phenoxybenzoate 1',2'-dioxygenase, but the enzyme activity in Escherichia coli was achieved only through the coexpression of pbaA1A2B and a glutathione reductase (GR)-type reductase gene, pbaC, indicating that the 3-phenoxybenzoate 1',2'-dioxygenase belongs to a type IV Rieske non-heme iron aromatic ring-hydroxylating oxygenase system consisting of a hetero-oligomeric oxygenase, a [2Fe-2S]-type ferredoxin, and a GR-type reductase. The pbaC gene is not located in the immediate vicinity of pbaA1A2B. 3-Phenoxybenzoate 1',2'-dioxygenase catalyzes the hydroxylation in the 1' and 2' positions of the benzene moiety of 3-phenoxybenzoate, yielding 3-hydroxybenzoate and catechol. Transcription of pbaA1A2B and pbaC was induced by 3-phenoxybenzoate, but the transcriptional level of pbaC was far less than that of pbaA1A2B, implying the possibility that PbaC may not be the only reductase that can physiologically transfer electrons to PbaA1A2B in strain JZ-1. Some GR-type reductases from other sphingomonad strains could also transfer electrons to PbaA1A2B, suggesting that PbaA1A2B has a low specificity for reductase.

SUBMITTER: Wang C

PROVIDER: S-EPMC4054222 | biostudies-literature | 2014 Jul

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

A novel angular dioxygenase gene cluster encoding 3-phenoxybenzoate 1',2'-dioxygenase in Sphingobium wenxiniae JZ-1.

Wang Chenghong C Chen Qing Q Wang Rui R Shi Chao C Yan Xin X He Jian J Hong Qing Q Li Shunpeng S

Applied and environmental microbiology 20140418 13

Sphingobium wenxiniae JZ-1 utilizes a wide range of pyrethroids and their metabolic product, 3-phenoxybenzoate, as sources of carbon and energy. A mutant JZ-1 strain, MJZ-1, defective in the degradation of 3-phenoxybenzoate was obtained by successive streaking on LB agar. Comparison of the draft genomes of strains JZ-1 and MJZ-1 revealed that a 29,366-bp DNA fragment containing a putative angular dioxygenase gene cluster (pbaA1A2B) is missing in strain MJZ-1. PbaA1, PbaA2, and PbaB share 65%, 52 ...[more]

PMID: 24747891

Similar Datasets

Project description:Carboxylesterase PytH, isolated from the pyrethroid-degrading bacterium Sphingobium faniae JZ-2, could rapidly hydrolyze the ester bond of a wide range of pyrethroid pesticides, including permethrin, fenpropathrin, cypermethrin, fenvalerate, deltamethrin, cyhalothrin, and bifenthrin. To elucidate the catalytic mechanism of PytH, we report here the crystal structures of PytH with bifenthrin (BIF) and phenylmethylsulfonyl fluoride (PMSF) and two PytH mutants. Though PytH shares low sequence identity with reported α/β-hydrolase fold proteins, the typical triad catalytic center with Ser-His-Asp triad (Ser78, His230, and Asp202) is present and vital for the hydrolase activity. However, no contact was found between Ser78 and His230 in the structures we solved, which may be due to the fact that the PytH structures we determined are in their inactive or low-activity forms. The structure of PytH is composed of a core domain and a lid domain; some hydrophobic amino acid residues surrounding the substrate from both domains form a deeper and wider hydrophobic pocket than its homologous structures. This indicates that the larger hydrophobic pocket makes PytH fit for its larger substrate binding; both lid and core domains are involved in substrate binding, and the lid domain-induced core domain movement may make the active center correctly positioned with substrates.IMPORTANCE Pyrethroid pesticides are widely applied in agriculture and household; however, extensive use of these pesticides also causes serious environmental and health problems. The hydrolysis of pyrethroids by carboxylesterases is the major pathway of microbial degradation of pyrethroids, but the structure of carboxylesterases and its catalytic mechanism are still unknown. Carboxylesterase PytH from Sphingobium faniae JZ-2 could effectively hydrolyze a wide range of pyrethroid pesticides. The crystal structures of PytH are solved in this study. This showed that PytH belongs to the α/β-hydrolase fold proteins with typical catalytic Ser-His-Asp triad, though PytH has a low sequence identity (about 20%) with them. The special large hydrophobic binding pocket enabled PytH to bind bigger pyrethroid family substrates. Our structures shed light on the substrate selectivity and the future application of PytH and deepen our understanding of α/β-hydrolase members.

Project description:Sphingobium phenoxybenzoativorans SC_3 degrades and utilizes diphenyl ether (DE) or 2-carboxy-DE as its sole carbon and energy source. In this study, we report the degradation of DE and 2-carboxy-DE initiated by a novel ring cleavage angular dioxygenase (diphenyl ether dioxygenase [Dpe]) in the strain. Dpe functions at the angular carbon and its adjacent carbon (C-1a, C-2) of a benzene ring in DE (or the 2-carboxybenzene ring in 2-carboxy-DE) and cleaves the C-1a-C-2 bond (decarboxylation occurs simultaneously for 2-carboxy-DE), yielding 2,4-hexadienal phenyl ester, which is subsequently hydrolyzed to muconic acid semialdehyde and phenol. Dpe is a type IV Rieske non-heme iron oxygenase (RHO) and consists of three components: a hetero-oligomer oxygenase, a [2Fe-2S]-type ferredoxin, and a glutathione reductase (GR)-type reductase. Genetic analyses revealed that dpeA1A2 plays an essential role in the degradation and utilization of DE and 2-carboxy-DE in S. phenoxybenzoativorans SC_3. Enzymatic study showed that transformation of 1 molecule of DE needs two molecules of oxygen and two molecules of NADH, supporting the assumption that the cleavage of DE catalyzed by Dpe is a continuous two-step dioxygenation process: DE is dioxygenated at C-1a and C-2 to form a hemiacetal-like intermediate, which is further deoxygenated, resulting in the cleavage of the C-1a-C-2 bond to form one molecule of 2,4-hexadienal phenyl ester and two molecules of H2O. This study extends our knowledge of the mode and mechanism of ring cleavage of aromatic compounds.IMPORTANCE Benzene ring cleavage, catalyzed by dioxygenase, is the key and speed-limiting step in the aerobic degradation of aromatic compounds. As previously reported, in the ring cleavage of DEs, the benzene ring needs to be first dihydroxylated at a lateral position and subsequently dehydrogenated and opened through extradiol cleavage. This process requires three enzymes (two dioxygenases and one dehydrogenase). In this study, we identified a novel angular dioxygenase (Dpe) in S. phenoxybenzoativorans SC_3. Under Dpe-mediated catalysis, the benzene ring of DE is dioxygenated at the angular position (C-1a, C-2), resulting in the cleavage of the C-1a-C-2 bond to generate a novel product, 2,4-hexadienal phenyl ester. This process needs only one angular dioxygenase, Dpe. Thus, the ring cleavage catalyzed by Dpe represents a novel mechanism of benzene ring cleavage.

Project description:The 1,125-bp mabB gene encoding 5-aminosalicylate (5ASA) 1,2-dioxygenase, a nonheme iron dioxygenase in the bicupin family that catalyzes the cleavage of the 5ASA aromatic ring to form cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate in the biodegradation of 3-aminobenzoate, was cloned from Comamonas sp. strain QT12 and characterized. The deduced amino acid sequence of the enzyme has low sequence identity with that of other reported ring-cleaving dioxygenases. MabB was heterologously expressed in Escherichia coli cells and purified as a His-tagged enzyme. The optimum pH and temperature for MabB are 8.0 and 10°C, respectively. FeII is required for the catalytic activity of the purified enzyme. The apparent Km and Vmax values of MabB for 5ASA are 52.0 ± 5.6 μM and 850 ± 33.2 U/mg, respectively. The two oxygen atoms incorporated into the product of the MabB-catalyzed reaction are both from the dioxygen molecule. Both 5ASA and gentisate could be converted by MabB; however, the catalytic efficiency of MabB for 5ASA was much higher (∼70-fold) than that for gentisate. The mabB-disrupted mutant lost the ability to grow on 3-aminobenzoate, and mabB expression was higher when strain QT12 was cultivated in the presence of 3-aminobenzoate. Thus, 5ASA is the physiological substrate of MabB.IMPORTANCE For several decades, 5-aminosalicylate (5ASA) has been advocated as the drug mesalazine to treat human inflammatory bowel disease and considered the key intermediate in the xenobiotic degradation of many aromatic organic pollutants. 5ASA biotransformation research will help us elucidate the microbial degradation of these pollutants. Most studies have reported that gentisate 1,2-dioxygenases (GDOs) can convert 5ASA with significantly high activity; however, the catalytic efficiency of these enzymes for gentisate is much higher than that for 5ASA. This study showed that MabB can convert 5ASA to cis-4-amino-6-carboxy-2-oxohexa-3,5-dienoate, incorporating two oxygen atoms from the dioxygen molecule into the product. Unlike GDOs, MabB uses 5ASA instead of gentisate as the primary substrate. mabB is the first reported 5-aminosalicylate 1,2-dioxygenase gene.

Project description:Bifidobacteria constitute a specific group of commensal bacteria typically found in the gastrointestinal tract (GIT) of humans and other mammals. Bifidobacterium breve strains are numerically prevalent among the gut microbiota of many healthy breastfed infants. In the present study, we investigated glycosulfatase activity in a bacterial isolate from a nursling stool sample, B. breve UCC2003. Two putative sulfatases were identified on the genome of B. breve UCC2003. The sulfated monosaccharide N-acetylglucosamine-6-sulfate (GlcNAc-6-S) was shown to support the growth of B. breve UCC2003, while N-acetylglucosamine-3-sulfate, N-acetylgalactosamine-3-sulfate, and N-acetylgalactosamine-6-sulfate did not support appreciable growth. By using a combination of transcriptomic and functional genomic approaches, a gene cluster designated ats2 was shown to be specifically required for GlcNAc-6-S metabolism. Transcription of the ats2 cluster is regulated by a repressor open reading frame kinase (ROK) family transcriptional repressor. This study represents the first description of glycosulfatase activity within the Bifidobacterium genus.ImportanceBifidobacteria are saccharolytic organisms naturally found in the digestive tract of mammals and insects. Bifidobacterium breve strains utilize a variety of plant- and host-derived carbohydrates that allow them to be present as prominent members of the infant gut microbiota as well as being present in the gastrointestinal tract of adults. In this study, we introduce a previously unexplored area of carbohydrate metabolism in bifidobacteria, namely, the metabolism of sulfated carbohydrates. B. breve UCC2003 was shown to metabolize N-acetylglucosamine-6-sulfate (GlcNAc-6-S) through one of two sulfatase-encoding gene clusters identified on its genome. GlcNAc-6-S can be found in terminal or branched positions of mucin oligosaccharides, the glycoprotein component of the mucous layer that covers the digestive tract. The results of this study provide further evidence of the ability of this species to utilize mucin-derived sugars, a trait which may provide a competitive advantage in both the infant gut and adult gut.

Dataset Information

A novel angular dioxygenase gene cluster encoding 3-phenoxybenzoate 1',2'-dioxygenase in Sphingobium wenxiniae JZ-1.

Publications

A novel angular dioxygenase gene cluster encoding 3-phenoxybenzoate 1',2'-dioxygenase in Sphingobium wenxiniae JZ-1.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets