Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

Bohuslavek, Jan; Payne, Jason W.; Liu, Yong; Bolton, H.; Xun, Luying

doi:10.1128/aem.67.2.688-695.2001

Cited by 61 publications

(72 citation statements)

References 51 publications

(31 reference statements)

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“…Not much is known about the microbial degradation of EDTA under anaerobic conditions. Although some aerobic organisms can use EDTA for growth [14], it is not removed by microorganisms functional in conventional sewage treatment procedures [15,16]. The ferric salt is light sensitive.…”

Section: The Carbon Sources For Co and Cnmentioning

confidence: 99%

The biosynthetic routes for carbon monoxide and cyanide in the Ni–Fe active site of hydrogenases are different

et al. 2004

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The incorporation of carbon into the carbon monoxide and cyanide ligands of [NiFe]-hydrogenases has been investigated by using 13 C labelling in infrared studies of the Allochromatium vinosum enzyme and by 14 C labelling experiments with overproduced Hyp proteins from Escherichia coli. The results suggest that the biosynthetic routes of the carbon monoxide and cyanide ligands in [NiFe]-hydrogenases are different.

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Section: The Carbon Sources For Co and Cnmentioning

confidence: 99%

The biosynthetic routes for carbon monoxide and cyanide in the Ni–Fe active site of hydrogenases are different

et al. 2004

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“…Human cytochrome P450 2B6 monooxygenase oxidizes efavirenz, an HIV-1 reverse transcriptase inhibitor, to 8-hydroxyefavirenz and then to 8,14-dihydroxyefavirenz with similar kinetic parameters (26). A bacterial FMNH 2 -utilizing monooxygenase oxidizes EDTA to ethylenediaminetriacetate and then to ethylenediaminediacetate (27,28). A bacterial two-component monooxygenase of Bacillus sphaericus JS905 catalyzes the sequential conversion of p-nitrophenol to 4-nitrocatechol and then to hydroxyquinol (29).…”

Section: Fig 4 the Mass Spectra Of Acetylated 6-chlorohydroxyquinolmentioning

confidence: 99%

A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions

Xun

Webster

2004

Journal of Biological Chemistry

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Ralstonia eutropha JMP134 2,4,6-trichlorophenol (2,4,6-TCP) 4-monooxygenase catalyzes sequential dechlorinations of 2,4,6-TCP to 6-chlorohydroxyquinol. Although 2,6-dichlorohydroxyquinol is a logical metabolic intermediate, the enzyme hardly uses it as a substrate, implying it may not be a true intermediate. Evidence is provided to support the proposition that the monooxygenase oxidized 2,4,6-TCP to 2,6-dichloroquinone that remained with the enzyme and got hydrolyzed to 2-chlorohydroxyquinone, which was chemically reduced by ascorbate and NADH to 6-chlorohydroxyquinol. When the monooxygenase oxidized 2,6-dichlorophenol, the product was 2,6-dichloroquinol, which was not further converted to 6-chlorohydroxyquinol, implying that the enzyme only converts 2,6-dichloroquinone to 6-chlorohydroxyquinol. Stoichiometric analysis indicated the consumption of one O 2 molecule per 2,4,6-TCP converted to 6-chlorohydroxyquinol, ruling out the possibility of two oxidative reactions. Experiments with 18 Olabeling gave direct evidence for the incorporation of oxygen from both O 2 and H 2 O into the produced 6-chlorohydroxyquinol. A monooxygenase that catalyzes hydroxylation by both oxidative and hydrolytic reactions has not been reported to date. The ability of the enzyme to perform two types of reactions is not due to the presence of a second functional domain but rather is due to catalytic promiscuity, as a homologous monooxygenase converts 2,4,6-TCP to only 2,6-dichloroquinol. Employing both conventional catalysis and catalytic promiscuity of a single enzyme in two consecutive steps of a metabolic pathway has been unknown previously.

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“…strain IGTS8 (17), organosulfur monooxygenases (SsuD) in Pseudomonas putida S-313 (18), and EDTA monooxygenase in Mesorhizobium sp. bacterium BNC1 (6). Furthermore, reduced flavin adenine dinucleotide (FADH 2 )-dependent monooxygenases have been (20), and 2,4,5-trichlorophenol 4-monooxygenase of Burkholderia cepacia AC1100 (21).…”

mentioning

confidence: 99%

“…However, another HpaC from Thermus thermophilus HB8 and EmoB from Mesorhizobium sp. BCN1 have weakly bound FAD and FMN, respectively (6,25), but their reaction mechanisms are unknown so far. Crystal structures for some of these smallcomponent reductases have been reported, such as PheA2 (26), HpaC St (27), and HpaC Tt (25).…”

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Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

Nissen

Youn²,

Knowles

et al. 2008

Journal of Biological Chemistry

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EDTA has become a major organic pollutant in the environment because of its extreme usage and resistance to biodegradation. Recently, two critical enzymes, EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB), belonging to the newly established two-component flavin-diffusible monooxygenase family, were identified in the EDTA degradation pathway in Mesorhizobium sp. BNC1. EmoA is an FMNH 2 -dependent enzyme that requires EmoB to provide FMNH 2 for the conversion of EDTA to ethylenediaminediacetate. To understand the molecular basis of this FMN-mediated reaction, the crystal structures of the apo-form, FMN⅐FMN complex, and FMN⅐NADH complex of EmoB were determined at 2.5 Å resolution. The structure of EmoB is a homotetramer consisting of four ␣/␤-single-domain monomers of five parallel ␤-strands flanked by five ␣-helices, which is quite different from those of other known two-component flavin-diffusible monooxygenase family members, such as PheA2 and HpaC, in terms of both tertiary and quaternary structures. For the first time, the crystal structures of both the FMN⅐FMN and FMN⅐NADH complexes of an NADH:FMN oxidoreductase were determined. Two stacked isoalloxazine rings and nicotinamide/isoalloxazine rings were at a proper distance for hydride transfer. The structures indicated a ping-pong reaction mechanism, which was confirmed by activity assays. Thus, the structural data offer detailed mechanistic information for hydride transfer between NADH to an enzyme-bound FMN and between the bound FMNH 2 and a diffusible FMN.EDTA has quietly become a major organic pollutant, currently present in the environment at higher concentrations than any other organic pollutant (1). A high level of EDTA in natural waters is due to its extensive usage, such as in industrial cleaning to remove calcium deposits, in detergent as a sequestering agent, in phytoremediation to mobilize heavy metals, and in scientific laboratories as a chelating agent (2, 3). EDTA is recalcitrant to biodegradation and exists mainly in metal⅐EDTA complexes, many of which are toxic (4, 5). In addition, the codisposal of EDTA with radionuclides has led to the enhanced mobilization of radionuclides in groundwater, rapidly spreading radioactive contamination (3, 6 -8). Concerns over EDTA recalcitrance and the potential mobilization of heavy metals and radionuclides have led the European Union, Australia, and some parts of the United States to ban EDTA in detergent. It is now also being carefully controlled in many other products to reduce contamination of water resources.Several bacteria that can degrade EDTA and the related compound, nitrilotriacetate, and use them as a sole source of carbon and energy have been isolated (9 -12). They are phylogenetically related to Mesorhizobium and Agrobacterium species (11), likely forming a new branch within the Phyllobacteriaceae, the "Mesorhizobia" family (13). In these bacteria, reduced flavin mononucleotide (FMNH 2 )-dependent EDTA monooxygenase (EmoA) and NADH:FMN oxidoreductase (EmoB) together oxidize EDTA to e...

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Cloning, Sequencing, and Characterization of a Gene Cluster Involved in EDTA Degradation from the Bacterium BNC1

Cited by 61 publications

References 51 publications

The biosynthetic routes for carbon monoxide and cyanide in the Ni–Fe active site of hydrogenases are different

The biosynthetic routes for carbon monoxide and cyanide in the Ni–Fe active site of hydrogenases are different

A Monooxygenase Catalyzes Sequential Dechlorinations of 2,4,6-Trichlorophenol by Oxidative and Hydrolytic Reactions

Crystal Structures of NADH:FMN Oxidoreductase (EmoB) at Different Stages of Catalysis

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