Evidence That Bacterial Cyanide Oxygenase Is a Pterin-Dependent Hydroxylase

Kunz, Daniel A.; Fernandez, Ruby F.; Parab, Preeti

doi:10.1006/bbrc.2001.5611

Cited by 35 publications

(25 citation statements)

References 18 publications

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“…A number of methods are currently available for cyanide remediation with varying degrees of effectiveness, namely biological treatment [3,4], chemical oxidation [5,6], electrochemical decomposition [7][8][9][10], photocatalytic [11][12][13][14][15] and catalytic oxidation [16][17]. The most common method for treating cyanides is alkaline chlorination which results in the formation of a highly toxic cyanogen chloride intermediate and, if organic material is present, chlorinated VOC's.…”

Section: Introductionmentioning

confidence: 99%

Novel Ni−Fe-oxide systems for catalytic oxidation of cyanide in an aqueous phase

Stoyanova

Christoskova

2005

Open Chemistry

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Catalytic activity of mixed Ni-Fe oxide systems with respect to air oxidation of aqueous cyanide solution at 308 K was investigated. The catalysts employed were prepared by an oxidation-precipitation method at room temperature and were characterized by powder Xray diffraction (XRD), Mössbauer spectroscopy, and chemical analysis. The cyanide oxidation rate was found to be dependent on the catalyst's calcination temperature, pH of the medium, and catalyst loading. Results revealed that the catalyst calcined at 120 • C is the most active where up to 90 % of free cyanide (4 mM) was removed after oxidation for 30 minutes in the presence of 2.5 g/L catalyst at pH 9.5. The cyanide conversion becomes less favorable as the pH of the solution increases (with other constant parameters). The selectivity data showed that carbon dioxide is the main oxidation product, regardless of pH of the solution.

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Section: Introductionmentioning

confidence: 99%

Novel Ni−Fe-oxide systems for catalytic oxidation of cyanide in an aqueous phase

Stoyanova

Christoskova

2005

Open Chemistry

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“…CNO has thus far been found only in Pseudomonas fluorescens strain NCIMB 11764. In contrast to CNN, CNO is able to attack cyanide at much lower concentrations (apparent K m , 3.5 M) (24). This helps explain the ability of P. fluorescens NCIMB 11764 to grow on cyanide at low nontoxic concentrations, such as those provided under fed-batch conditions (15,25).…”

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“…Cell extracts were prepared as described previously (24,26), and enzyme activities were recovered in 150,000-ϫ-g high-speed supernatants (HSS). Each HSS was concentrated by passage through 15-ml (Centriprep-10) or 2-ml (Centricon-10) ultrafiltration concentrators (nominal molecular mass cutoff, 10 kDa; Amicon).…”

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“…Fixed-time measurements for CNO and FDH were determined by measuring the extents of K 14 CN and [ 14 C]formate conversion to 14 CO 2 , respectively. Reactions were carried out in crimp-sealed vial or bottles as described previously (24,44). For CNO assays, reaction mixtures (0.25 ml) contained enzyme (0.125 to 0.5 mg protein), NADH (1 mM), H 2 biopterin (0.5 ⌴), and radiolabeled substrate (1 Ci [48 KBq]; approximately 100 M) in Na-K buffer.…”

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Enzymatic Assimilation of Cyanide via Pterin-Dependent Oxygenolytic Cleavage to Ammonia and Formate in Pseudomonas fluorescens NCIMB 11764

Fernandez

Dolghih

Kunz

2004

Appl Environ Microbiol

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Utilization of cyanide as a nitrogen source by Pseudomonas fluorescens NCIMB 11764 occurs via oxidative conversion to carbon dioxide and ammonia, with the latter compound satisfying the nitrogen requirement. Substrate attack is initiated by cyanide oxygenase (CNO), which has been shown previously to have properties of a pterin-dependent hydroxylase. CNO was purified 71-fold and catalyzed the quantitative conversion of cyanide supplied at micromolar concentrations (10 to 50 M) to formate and ammonia. The specific activity of the partially purified enzyme was approximately 500 mU/mg of protein. The pterin requirement for activity could be satisfied by supplying either the fully (tetrahydro) or partially (dihydro) reduced forms of various pterin compounds at catalytic concentrations (0.5 M). These compounds included, for example, biopterin, monapterin, and neopterin, all of which were also identified in cell extracts. Substrate conversion was accompanied by the consumption of 1 and 2 molar equivalents of molecular oxygen and NADH, respectively. When coupled with formate dehydrogenase, the complete enzymatic system for cyanide oxidation to carbon dioxide and ammonia was reconstituted and displayed an overall reaction stoichiometry of 1:1:1 for cyanide, O 2 , and NADH consumed. Cyanide was also attacked by CNO at a higher concentration (1 mM), but in this case formamide accumulated as the major reaction product (formamide/formate ratio, 0.6:0.3) and was not further degraded. A complex reaction mechanism involving the production of isocyanate as a potential CNO monooxygenation product is proposed. Subsequent reduction of isocyanate to formamide, whose hydrolysis occurs as a CNO-bound intermediate, is further envisioned. To our knowledge, this is the first report of enzymatic conversion of cyanide to formate and ammonia by a pterin-dependent oxygenative mechanism.

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“…In P. fluorescens NCIMB 11764, this occurs by a novel enzymatic mechanism in which CN is cleaved oxygenatively to give formate and ammonia via reactions involving formamide as an inferred intermediate (equations 1 and 2) (6). The responsible enzyme, variously described as cyanide oxygenase (CNO), is located in the cytosolic fraction of cells induced with CN and requires both reduced pyridine nucleotide (NADH) and a source of reduced pterin as a cofactor (6,15). …”

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Bacterial Cyanide Oxygenase Is a Suite of Enzymes Catalyzing the Scavenging and Adventitious Utilization of Cyanide as a Nitrogenous Growth Substrate

Fernandez

Kunz

2005

J Bacteriol

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Cyanide oxygenase (CNO) from Pseudomonas fluorescens NCIMB 11764 catalyzes the pterin-dependent oxygenolytic cleavage of cyanide (CN) to formic acid and ammonia. CNO was resolved into four protein components (P1 to P4), each of which along with a source of pterin cofactor was obligately required for CNO activity. Component P1 was characterized as a multimeric 230-kDa flavoprotein exhibiting the properties of a peroxide-forming NADH oxidase (oxidoreductase) (Nox). P2 consisted of a 49.7-kDa homodimer that showed 100% amino acid identity at its N terminus to NADH peroxidase (Npx) from Enterococcus faecalis. Enzyme assays further confirmed the identities of both Nox and Npx enzymes (specific activity, 1 U/mg). P3 was characterized as a large oligomeric protein (ϳ300 kDa) that exhibited cyanide dihydratase (CynD) activity (specific activity, 100 U/mg). Two polypeptides of 38 kDa and 43 kDa were each detected in the isolated enzyme, the former believed to confer catalytic activity based on its similar size to other CynD enzymes. The amino acid sequence of an internal peptide of the 43-kDa protein was 100% identical to bacterial elongation factor Tu, suggesting a role as a possible chaperone in the assembly of CynD or a multienzyme CNO complex. The remaining P4 component consisted of a 28.9-kDa homodimer and was identified as carbonic anhydrase (specific activity, 2,000 U/mg). While the function of participating pterin and the roles of Nox, Npx, CynD, and CA in the CNO-catalyzed scavenging of CN remain to be determined, this is the first report describing the collective involvement of these four enzymes in the metabolic detoxification and utilization of CN as a bacterial nitrogenous growth substrate.From its role as an important prebiotic reactant to its notorious reputation as a genocidal and homicidal agent, the significance of cyanide (referred to herein as CN and indicating the free form, which exists as either CN Ϫ or HCN) to biology has a long history. However, gaps in our understanding of how this toxic substance is tolerated and metabolized in nature remain. Effective means of detoxifying cyanide have presumably evolved given the preponderance of cyanogenic organisms in nature. For example, a large number of plant species (ϳ2,000) as well as some fungi and bacteria are known to be cyanogenic (25,28). Bacteria exhibit a range of natural resistance to cyanide related in part to their ability to synthesize cyanide-detoxifying enzymes. The added ability to assimilate metabolites derived therefrom accounts further for the un-

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Evidence That Bacterial Cyanide Oxygenase Is a Pterin-Dependent Hydroxylase

Cited by 35 publications

References 18 publications

Novel Ni−Fe-oxide systems for catalytic oxidation of cyanide in an aqueous phase

Novel Ni−Fe-oxide systems for catalytic oxidation of cyanide in an aqueous phase

Enzymatic Assimilation of Cyanide via Pterin-Dependent Oxygenolytic Cleavage to Ammonia and Formate in Pseudomonas fluorescens NCIMB 11764

Bacterial Cyanide Oxygenase Is a Suite of Enzymes Catalyzing the Scavenging and Adventitious Utilization of Cyanide as a Nitrogenous Growth Substrate

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