Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium <i>pseudomonas</i> C12B

Cloves, J M; Dodgson, K. S.; White, Graham F.; Fitzgerald, John W.

doi:10.1042/bj1850023

Cited by 36 publications

(40 citation statements)

References 36 publications

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“…Activity measurements were based on the amount of nitrite liberated. very significant component in the cellular protein, and this situation is similar to that obtained for other inducible enzymes (5,20,31).…”

Section: Discussionsupporting

confidence: 68%

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

et al. 1997

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Glycerol trinitrate (GTN) reductase, which enables Agrobacterium radiobacter to utilize GTN and related explosives as sources of nitrogen for growth, was purified and characterized, and its gene was cloned and sequenced. The enzyme was a 39-kDa monomeric protein which catalyzed the NADH-dependent reductive scission of GTN (K m ‫؍‬ 23 M) to glycerol dinitrates (mainly the 1,3-isomer) with a pH optimum of 6.5, a temperature optimum of 35°C, and no dependence on metal ions for activity. It was also active on pentaerythritol tetranitrate (PETN), on isosorbide dinitrate, and, very weakly, on ethyleneglycol dinitrate, but it was inactive on isopropyl nitrate, hexahydro-1,3,5-trinitro-1,3,5-triazine, 2,4,6-trinitrotoluene, ammonium ions, nitrate, or nitrite. The amino acid sequence deduced from the DNA sequence was homologous (42 to 51% identity and 61 to 69% similarity) to those of PETN reductase from Enterobacter cloacae, N-ethylmaleimide reductase from Escherichia coli, morphinone reductase from Pseudomonas putida, and old yellow enzyme from Saccharomyces cerevisiae, placing the GTN reductase in the ␣/␤ barrel flavoprotein group of proteins. GTN reductase and PETN reductase were very similar in many respects except in their distinct preferences for NADH and NADPH cofactors, respectively.Nitrate esters such as glycerol trinitrate (GTN) and pentaerythritol tetranitrate (PETN) are widely used both as explosives and as vasodilators in the treatment of angina. During their long history of exploitation in these applications (24, 34), there has been ample opportunity during production, storage, and use for significant contamination of land sites and water courses to occur, so that site remediation of explosive residues is now an urgent issue worldwide. Moreover, the current acceleration in demilitarization of ordnance and rocket formulations will produce further waste material, raising new environmental concerns. The environmental issues, together with the rarity of naturally occurring analogs (14), make the discovery of microorganisms which can degrade such compounds and thus influence their environmental fate of particular interest. Earlier interest in the elimination of nitrate ester explosives in wastewater treatment plants (35,36) and in their metabolism in fungal organisms (6,7,29,30) led to the first report (21) of denitration of GTN in pure bacterial cultures of Bacillus thuringiensis plus B. cereus and Enterobacter agglomerans, apparently occurring via a hydrolytic pathway, although formation of nitrate was not demonstrated. In contrast, White et al. showed unequivocally that assimilation of nitrogen from GTN in pure cultures of a Pseudomonas sp. (37) and Agrobacterium radiobacter (38) occurred via nitrite (not nitrate) with the concomitant formation of mainly glycerol 1,3-dinitrate (1,3-GDN) and small amounts of the corresponding 1,2-isomer. Cells were able to denitrate both of the dinitrates to mononitrates but not beyond, and they also converted PETN to its tri-and dinitrates. The enzyme responsible was identif...

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Section: Discussionsupporting

confidence: 68%

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

et al. 1997

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“…The resulting parent alcohol is further degraded (1) or incorporated into cellular lipids (2). Cleavage of the sulfate moiety has been studied in some detail, and several alkylsulfatase enzymes have been purified from cell extracts (3)(4)(5)(6)(7).…”

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confidence: 99%

Characterization of a Sulfur-regulated Oxygenative Alkylsulfatase from Pseudomonas putida S-313

Kahnert

Kertesz

2000

Journal of Biological Chemistry

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The atsK gene of Pseudomonas putida S-313 was required for growth with alkyl sulfate esters as sulfur source. The AtsK protein was overexpressed in Escherichia coli and purified to homogeneity. Sequence analysis revealed that AtsK was closely related to E. coli taurine dioxygenase (38% amino acid identity). The AtsK protein catalyzed the ␣-ketoglutarate-dependent cleavage of a range of alkyl sulfate esters, with chain lengths ranging from C 4 to C 12 , required oxygen and Fe 2؉ for activity and released succinate, sulfate, and the corresponding aldehyde as products. Enzyme activity was optimal at pH 7 and was strongly stimulated by ascorbate. Unlike most other characterized ␣-ketoglutaratedependent dioxygenases, AtsK accepted a range of ␣-keto acids as co-substrates, including ␣-ketoglutarate (K m 140 M), ␣-ketoadipate, ␣-ketovalerate, and ␣-ketooctanoate. The measured K m values for hexyl sulfate and SDS were 40 and 34 M, respectively. The apparent M r of the purified enzyme of 121,000 was consistent with a homotetrameric structure, which is unusual for this enzyme superfamily, members of which are usually monomeric or dimeric. The properties and amino acid sequence of the AtsK enzyme thus define it as an unusual oxygenolytic alkylsulfatase and a novel member of the ␣-ketoglutarate-dependent dioxygenase family.Bacterial enzymes that cleave aliphatic sulfate esters to release the sulfate moiety have been the subject of considerable study, motivated originally by an awareness of the large scale release of synthetic alkyl sulfate esters into the environment. Due to their amphiphilic properties, long chain aliphatic sulfate esters such as sodium dodecyl sulfate (SDS) are in common use as components of surfactant formulations and are consequently discharged into wastewater. A range of bacterial strains able to degrade aliphatic sulfate esters has been isolated from contaminated sources such as sewage sludge on the basis of their ability to utilize aliphatic sulfate esters as carbon sources for growth (for a review, see Ref. 1). In most cases, degradation of alkyl sulfate esters was found to be initiated by alkylsulfatase enzymes that catalyze the hydrolytic cleavage of the ester bond to liberate inorganic sulfate. The resulting parent alcohol is further degraded (1) or incorporated into cellular lipids (2). Cleavage of the sulfate moiety has been studied in some detail, and several alkylsulfatase enzymes have been purified from cell extracts (3-7).The finding that many isolates from environmental sites that had not been contaminated by detergents also exhibit alkylsulfatase activity (8) suggests that such enzymes may play a role in natural environments as well. Naturally occurring alkyl sulfates include methyl, ethyl, and propyl sulfate in avian eggs (9) and the long chain alkyl sulfates that have been found in membrane structures from unicellular algae (10) and seaweed (11). In aerobic soils, 40 -50% of the total sulfur is present as sulfate esters bound to the soil organic matter (12, 13), although the molecular s...

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“…Thus, sulphate esters were generally the most readily degradable and had the highest fraction of Biolog-positive isolates, while the sulphonates were more recalcitrant and had the lowest fraction of Biolog-positive results. The SDS-degrading isolate, Pseudomonas C12B, gave positive results for all the classes of sulphated surfactants, a versatility which matches the known battery of sulphatase activity in this organism (Bartholomew et al, 1978;Cloves et al, 1980;Shaw etal., 1980;Bateman etal., 1986;Hales etal., 1986). The LABS-degrading consortium appears to be metabolically diverse and is capable of biodegrading all the sulphated and sulphonated surfactants used during this study.…”

Section: Discussionmentioning

confidence: 91%

Rapid screening for bacterial phenotypes capable of biodegrading anionic surfactants: development and validation of a microtitre plate method

Lee¹,

Russell

White

1995

Microbiology

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The Biolog microtitre plate assay, which is based on tetrazolium dye reduction as an indicator of sole-carbon-source utilization, has been evaluated as a rapid method to investigate the biodegradation of five classes of anionic surfactant by pure and mixed cultures of bacteria. The assay gave reproducible results over a fourfold range of inoculum optical density, and the surfactant concentration was selected t o provide a compromise between the length of the lag period prior to colour production and the maximum colour produced. A kinetic model was developed and used t o analyse the appearance of colour in the assay and was found t o give rise to three biologically significant parameters describing the processes underlying the assay. No false-positives were obtained with environmental isolates. The small number of falsenegatives obtained (< 8% of the total) could be explained by the methodology used to prepare the bacterial inoculum. All isolates which were positive in the Biolog assay were shown t o be both primary and ultimate degraders of the test surfactant. These results show that the method provides a useful means of studying the biodegradation of anionic surfactants by both pure and mixed cultures of bacteria and will find use in the rapid analysis of biodegradation kinetics and specificities of larger numbers of individual isolates than hitherto possible. In addition, an important benefit of the methodology is that it can be used for direct analysis of the biodegradation potential of whole bacterial communities without having t o make an artificial selection during laboratory growth.UK

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Purification and properties of the P2 primary alkylsulphohydrolase of the detergent-degrading bacterium pseudomonas C12B

Cited by 36 publications

References 36 publications

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

Purification, properties, and sequence of glycerol trinitrate reductase from Agrobacterium radiobacter

Characterization of a Sulfur-regulated Oxygenative Alkylsulfatase from Pseudomonas putida S-313

Rapid screening for bacterial phenotypes capable of biodegrading anionic surfactants: development and validation of a microtitre plate method

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