Utilization of cyanide as nitrogenous substrate by Pseudomonas fluorescens NCIMB 11764: evidence for multiple pathways of metabolic conversion.

The growth of Pseudomonas fluorescens NCIMB 11764 on cyanide as the sole nitrogen source was accomplished by use of a modified fed-batch cultivation procedure. Previous studies showing that cyanide metabolism in this organism is both an oxygen-dependent and an inducible process, with CO2 and ammonia representing conversion products, were confirmed. However, washed cells (40 mg ml-1 [dry weight]) metabolized cyanide at concentrations far exceeding those previously described; 85% of 50 mM KCN was degraded in 6 h. In addition, two other C1 metabolites were detected in incubation mixtures; their identities were confirmed as formamide and formate by 13C nuclear magnetic resonance spectrocopy, high-pressure liquid chromatography, radioisotopic trapping experiments, and other analytical means. The relative yields of all four metabolites (CO2, formamide, formate, and ammonia) were shown to be dependent on the KCN concentration and availability of oxygen; at 0.5 to 10 mM substrate, CO2 was the major C1 product, whereas at 20 and 50 mM substrate, formamide and formate were principally formed. The latter two metabolites also accumulated during prolonged anaerobic incubation, suggesting that P. fluorescens NCIMB 11764 can elaborate several pathways of cyanide conversion. One is formally similar to that proposed previously (R. E. Harris and C. J. Knowles, FEMS Microbiol. Lett. 20:337-341, 1983), involving the oxygen-dependent conversion of cyanide to CO2 and ammonia. The other two, occurring in the presence or absence of oxygen, involve separate reactions to yield, respectively, formate plus ammonia or formamide.(ABSTRACT TRUNCATED AT 250 WORDS)     

Enzymatic assimilation of cyanide via pterin-dependent oxygenolytic cleavage to ammonia and formate in Pseudomonas fluorescens NCIMB 11764

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 micro 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 micro 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.     

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

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 μM) 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 μM). 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, O₂, 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.     

Incorporation of Molecular Oxygen and Water during Enzymatic Oxidation of Cyanide by Pseudomonas fluorescens NCIMB 11764

Cell extracts (high-speed [150,000 x g] supernatants) from Pseudomonas fluorescens NCIMB 11764 catalyzed the oxidation of cyanide to CO(inf2) (and NH(inf3)). Conversion was both oxygen and NADH dependent, with 1 mol of each being consumed per mol of cyanide degraded. Analysis of (sup13)CO(inf2) by mass spectrometry indicated that one atom each of isotopically labelled oxygen 18 from molecular oxygen and water were incorporated during enzymatic conversion. The results confirm earlier reports of oxygenase-mediated cyanide conversion in this organism. A reaction pathway for cyanide oxidation involving initial monooxygenation followed by hydrolysis of a hypothetical oxygenated intermediate to CO(inf2) (and NH(inf3)) is proposed.     

Evidence that bacterial cyanide oxygenase is a pterin-dependent hydroxylase

The soluble cell-free fraction (150,000g high-speed supernatants [HSS]) of Pseudomonas fluorescens NCIMB 11764 contains putative cyanide oxygenase (CNO) responsible for initiating cyanide oxidation and assimilation as a nitrogenous growth substrate. CNO activity, assayed either by cyanide-dependent O(2) or NADH uptake, or by conversion of radioactive K(14)CN to (14)CO(2), was detected at micromolar concentrations (apparent half-saturation constant, 4 microM). Results demonstrating that CNO requires a protein-enriched cell fraction and a low MW redox factor (<500 Da) for which reduced biopterin could substitute are presented. The properties of CNO are consistent with those of a pterin hydroxylase.     

Cyanide oxygenase and cyanase activities of Pseudomonas fluorescens NCIMB 11764

Abstract Pseudomonas fluorescens NCIMB 11764 is able to utilise cyanide (both KCN and Ni(CN)₄²⁻) as a nitrogen source for growth. Under such conditions cyanide oxygenase activity is induced. When potassium cyanate (KOCN) is supplied as the sole nitrogen source for growth, cyanase activity is induced. It has been demonstrated that these two enzymic activities are physiologically distinct, and are not co-induced under any of the growth conditions tested.     

Isolation and growth of a Pseudomonas species that utilizes cyanide as a source of nitrogen

A simple method of isolating bacteria that utilize cyanide as a source of nitrogen for growth has been developed. This involved supplying hydrogen cyanide as a vapour to glucose-containing minimal-salts agar plates. The bacteria isolated were Gram-negative, oxidase-positive rods producing a fluorescent green pigment and were tentatively identified as strains of Pseudomonas fluorescens. Three organisms were studied further and shown to be P. fluorescens biotype II. One of these (NCIB 11764) was grown in a glucose-containing fed-batch culture with either NH4Cl or KCN as the limiting nutrient. Cyanide-grown bacteria produced stoichiometric amounts of ammonia from cyanide when pulsed with cyanide under aerobic conditions. Stimulation of oxygen uptake was seen on addition of cyanide to suspensions of cyanide-grown but not ammonia-grown bacteria.     

Characterization of the Nit6803 nitrilase homolog from the cyanotroph Pseudomonas fluorescens NCIMB 11764

We report the purification and characterization of a nitrilase (E.C. 3.5.5.1) (Nit11764) essential for the assimilation of cyanide as the sole nitrogen source by the cyanotroph, Pseudomonas fluorescens NCIMB 11764. Nit11764, is a member of a family of homologous proteins (nitrile_sll0784) for which the genes typically reside in a conserved seven-gene cluster known as Nit1C. The physical properties and substrate specificity of Nit11764 resemble those of Nit6803, the current reference protein for the family, and the only true nitrilase that has been crystallized. The substrate binding pocket of the two enzymes places the substrate in direct proximity to the active site nucleophile (C160) and conserved catalytic triad (Glu44, Lys126). The two enzymes exhibit a similar substrate profile, however, for Nit11764, cinnamonitrile, was found to be an even better substrate than fumaronitrile the best substrate previously identified for Nit6803. A higher affinity for cinnamonitrile (Km 1.27 mM) compared to fumaronitrile (Km 8.57 mM) is consistent with docking studies predicting a more favorable interaction with hydrophobic residues lining the binding pocket. By comparison, 3,4-dimethoxycinnamonitrile was a poorer substrate the substituted methoxyl groups apparently hindering entry into the binding pocket. in situ ¹H NMR studies revealed that only one of the two nitrile substituents in the dinitrile, fumaronitrile, was attacked yielding trans-3-cyanoacrylate (plus ammonia) as a product. The essentiality of Nit11764 for cyanotrophy remains uncertain given that cyanide itself is a poor substrate and the catalytic efficiencies for even the best of nitrile substrates (~5 × 10³ M^?1 s^?1) is less than stellar.     

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min⁻¹ ml of culture fluid⁻¹) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by ¹³C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO₂ (and NH₃) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.     

Reduced ferredoxin: CO2 oxidoreductase from Clostridium pasteurianum. Effect of ligands to transition metals on the activity and the stability of the enzyme.

Reduced ferredoxin: CO2 oxidoreductase (CO2-reductase) from Clostridium pasteurianum catalyzes the reduction of CO2 to formate at the expense of reduced ferredoxin, an isotopic exchange between CO2 and formate in the absence of ferredoxin, and the oxidation of formate to CO2 with oxidized ferredoxin. The three activities were found to be equally affected by monovalent anions known to be ligands to transition metals: The enzyme was reversibly inhibited by azide (Ki = 0.004mM), cyanate (Ki = 0.3 mM), thiocyanate (Ki = 1mM), nitrite (Ki = 0.4mM), nitrate (Ki = 6mM), chlorate (Ki = 3mM), fluoride (Ki = 5mM), and by chloride, bromide, iodide (Ki greater than 5mM). There was no observable effect of pH on the inhibition constants. The enzyme was not inhibited by carbon monoxide. The enzyme was irreversibly inactivated by low concentrations (10muM) of cyanide. The rate of inactivation increased with increasing pH with an inflection point near pH 9.5. Reduced ferredoxin and formate rather than oxidized ferredoxin or CO2 protected the enzyme from inactivation by cyanide. The enzyme was protected by azide and cyanate from inactivation. In the presence of high concentrations of the monovalent anions the rate of inactivation by heat (55 degrees C), by molecular oxygen, and by cyanide was decreased by a factor of more than 100. Half maximal protection was observed at the Ki concentrations of the two reversible inhibitors. The data are interpreted to indicate that a transition metal of weak "a class" character and a disulfide are catalytically significant groups of CO2-reductase from C. pasteurianum.     

Intensity of respiration and the metabolism of phospholipids in isolated brain tissues of rats at various temperatures in the presence of KCN]

The intensity of oxygen consumption, as well as phospholipid metabolism of minced rat brain tissue were studied at different temperature of the incubation media (37 degrees, 32 degrees and 27 degrees C) without cyanide and in the media containing KCN (0.5 and 1.0 mM). As shown, both parameters depended directly upon the incubation temperature within the range of 27 degrees-37 degrees. KCN inhibited both processes, but depression of phospholipid metabolism was more expressed. These data suggest that under conditions of cyanide poisoning phospholipid metabolism depends both on the toxic effect of KCN directly and on the temperature, whose reduction reinforces this effect.     

Cyanase-mediated utilization of cyanate in Pseudomonas fluorescens NCIB 11764.

Pseudomonas fluorescens NCIB 11764 was capable of utilizing cyanate (OCN-) as a sole nitrogen source for growth. Crude cell extracts from cells grown on cyanate, but not on ammonium sulfate, were induced for an enzyme catalyzing cyanate conversion to ammonia. Enzymatic activity was shown to be bicarbonate dependent and specific for cyanate as a substrate, suggesting that cyanate utilization in this organism is facilitated by an enzyme resembling cyanase (cyanate amidohydrolase; EC 3.5.5.3), as described previously in Escherichia coli and Flavobacterium sp.     

Accumulation of α-Keto Acids as Essential Components in Cyanide Assimilation by Pseudomonas fluorescens NCIMB 11764

Pyruvate (Pyr) and α-ketoglutarate (αKg) accumulated when cells of Pseudomonas fluorescens NCIMB 11764 were cultivated on growth-limiting amounts of ammonia or cyanide and were shown to be responsible for the nonenzymatic removal of cyanide from culture fluids as previously reported (J.-L. Chen and D. A. Kunz, FEMS Microbiol. Lett. 156:61–67, 1997). The accumulation of keto acids in the medium paralleled the increase in cyanide-removing activity, with maximal activity (760 μmol of cyanide removed min⁻¹ ml of culture fluid⁻¹) being recovered after 72 h of cultivation, at which time the keto acid concentration was 23 mM. The reaction products that formed between the biologically formed keto acids and cyanide were unambiguously identified as the corresponding cyanohydrins by ¹³C nuclear magnetic resonance spectroscopy. Both the Pyr and α-Kg cyanohydrins were further metabolized by cell extracts and served also as nitrogenous growth substrates. Radiotracer experiments showed that CO₂ (and NH₃) were formed as enzymatic conversion products, with the keto acid being regenerated as a coproduct. Evidence that the enzyme responsible for cyanohydrin conversion is cyanide oxygenase, which was shown previously to be required for cyanide utilization, is based on results showing that (i) conversion occurred only when extracts were induced for the enzyme, (ii) conversion was oxygen and reduced-pyridine nucleotide dependent, and (iii) a mutant strain defective in the enzyme was unable to grow when it was provided with the cyanohydrins as a growth substrate. Pyr and αKg were further shown to protect cells from cyanide poisoning, and excretion of the two was directly linked to utilization of cyanide as a growth substrate. The results provide the basis for a new mechanism of cyanide detoxification and assimilation in which keto acids play an essential role.Cyanide is a notorious poison. Its inhibitory effect on respiration has been known since the 1920s, when Warburg and Keilin first demonstrated that it combines with trivalent iron in cytochrome oxidase (38, 40, 44). Although highly toxic, it is a normal part of our environment for which mechanisms of biological formation (cyanogenesis) and detoxification exist (8, 22, 42). Cyanide also arises from various industrial practices such as steel coking, electroplating, and mining, but significant accumulations in the environment probably do not occur because of its highly reactive nature (13, 18, 41, 46). The interactions between microorganisms and cyanide, however, remain of interest, since the mechanisms of tolerance and assimilation are poorly understood. A number of reports documenting the ability of microorganisms to grow on cyanide have appeared, but the biochemical basis of these abilities has remained largely obscure. Most studies have reported its ability to serve as a nitrogen source only, since at the concentrations needed for it to serve as a carbon source, it is too toxic (15, 24). As far as is known, growth on cyanide requires that it be enzymatically converted to ammonia. Once formed it can then be readily incorporated into cellular macromolecules by established mechanisms (31). Two separate conversions have been described. They are hydrolytic and oxidative conversion, and they yield formic acid and carbon dioxide as reaction by-products, respectively. The enzyme responsible for hydrolytic conversion has variously been described as cyanidase, cyanide dihydratase, or cyanide nitrilase (CNN), and it catalyzes the reaction shown in equation 1. 1 Mechanistically, CNNs resemble other nitrilases (e.g., EC 3.5.5.1) that catalyze the direct conversion of organic nitriles into an organic acid and ammonia but for which the substrate range appears to be limited to cyanide. The involvement of CNNs in cyanide metabolism has been reported for Alcaligenes xylosooxidans subsp. denitrificans (19, 20), Bacillus pumilus (30), and Pseudomonas sp. (45). Oxidative conversion is mediated by an enzyme described as cyanide oxygenase (CNO). This enzyme has been described for Pseudomonas fluorescens NCIMB 11764 only (15, 23–26). Recent work in our laboratory has shown that CNO functions as a monooxygenase, since a single atom of molecular oxygen was shown to be incorporated during conversion (43). Since the other atom of oxygen in CO₂ was shown to be derived from water, a reaction mechanism in which cyanide undergoes initial monooxygenative attack to give an unknown intermediate (X-OH) as shown in equation 2 was proposed (43). 2 Further hydrolysis of X-OH is then expected to give CO₂ and NH₃ as shown in equation 3. 3 The nature of X-OH and whether an additional enzyme is required for its conversion are unknown. Interestingly, NCIMB 11764 also elaborates a CNN, but only CNO has been shown to be physiologically required for cyanide utilization (26). This conclusion was reached after it was found that mutants unable to grow on cyanide did not make CNO but could still elaborate CNN.CNO is induced when cyanide (KCN) is added to nitrogen-limited cells (4, 26). This approach for obtaining cells induced for the enzyme is more convenient than growing cells on cyanide, which requires several days of fed-batch cultivation. During the course of experiments aimed at optimizing CNO induction, we discovered that the consumption of cyanide and the appearance of CNO activity in cell extracts were not concomitant (3, 4). Further experiments showed that cyanide consumption independent of that catalyzed by CNO occurred nonenzymatically and that a reaction between cyanide and a metabolite excreted into the medium was responsible for cyanide’s removal (4). Since cyanide-removing activity in culture fluids consistently copurified with iron-chelating activity, it was concluded that the responsible metabolite was a siderophore, but further identification of this siderophore was not achieved. Here we report that the compounds responsible for nonenzymatic cyanide removal are α-keto acids, namely, pyruvate (Pyr) and α-ketoglutarate (αKg). These findings help explain the earlier reported involvement of a putative siderophore, since these compounds can act as iron chelators (10, 35). However, the additional ability to serve also as effective cyanide-scavenging agents has not generally been recognized. Both Pyr and αKg were excreted into the medium when P. fluorescens NCIMB 11764 was grown on nitrogen-limiting amounts of ammonia or cyanide as a nitrogen source, and we now demonstrate that these metabolites play an essential role in the utilization of cyanide as a growth substrate.     

The effect of inhibitors on the oxygen kinetics of cytochrome c oxidase.

1. The oxygen kinetics of purified beef heart cytochrome c oxidase were investigated. 2. The effect of addition of various fixed concentrations of the inhibitors CO, HN3, HCOOH, HCN and H2S on the double reciprocal plot of respiration rate against oxygen concentration was studied. 3. CO is strictly competitive, azide and formate are uncompetitive, and cyanide and sulfide are non-competitive inhibitors towards oxygen. 4. Binding constants for the various inhibitors from secondary plots of the oxygen kinetics at pH 7.4 are: CO: Ki = 0.32 micronM, azide: Ki = 33 micronM; formate: Ki = 15 mM; cyanide: Ki = 0.2 micronM and sulfide: Ki = 0.2 micronM. 5. The possible significance of these results in the elucidation of the reaction mechanism is discussed.     

Bacterial cyanide oxygenase is a suite of enzymes catalyzing the scavenging and adventitious utilization of cyanide as a nitrogenous growth substrate

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 (approximately 300 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.     

Cyanide utilization in Pseudomonas fluorescens NCIMB 11764 involves a putative siderophore

Cyanide utilization in P. fluorescens NCIMB 11764 requires the induction of cyanide oxygenase. The enzyme is induced during growth on cyanide as the sole nitrogen source or when cyanide is added to stationary-phase cells grown on limiting ammonia, however, enzyme induction and cyanide degradation were found to occur non-concomitantly. Cyanide removal by chloramphenicol-treated cells and a mutant strain (JL102) defective in cyanide oxygenase were also observed. We now report a non-enzymatic mechanism for cyanide biotransformation by an iron-chelating species identified as a putative siderophore. Partially purified siderophore preparations removed cyanide at initial rates as high as 7.6 mmol min⁻¹ mg⁻¹. The reaction product was further shown to be enzymatically oxidized to NH₃ (and CO₂) and also supported growth. The results indicate that both cyanide oxygenase and a putative siderophore component are involved in cyanide utilization.     

Purification and properties of a cyanide-degrading enzyme from Burkholderia cepacia strain C-3

A cyanide-hydrolysing enzyme from Burkholderia cepacia strain C-3 isolated from soil was purified to electrophoretic homogeneity by ammonium sulphate precipitation and column chromatography on HiTrap Q (DEAE-agarose) and phenyl-Sepharose HP. The enzyme was purified 48-fold with a 0.8% yield and a final specific activity of 26.8 u/mg protein. The purified enzyme was observed as a single polypeptide band of molecular mass 38 kDa during both denaturing and non-denaturing gel electrophoresis. Enzymatic activity was optimal at pH 8.0–8.5 and at 30–35 °C. Activity was stimulated by Mo²⁺, Sn²⁺, and Zn²⁺, and inhibited by Al³⁺, Co²⁺, Cu²⁺ and Hg²⁺. The enzyme was specific for cyanide and thiocyanate with formate and ammonia as the main products from KCN degradation. Its K _m and V _max values were 1.4 mM and 15.2 u/mg protein, respectively. Apparent substrate inhibition occurred at cyanide concentrations greater than 2 mM.     

Biodegradation of cyanide by Rhodococcus UKMP-5M

A new bacterial strain, Rhodococcus UKMP-5M isolated from petroleum-contaminated soils demonstrated promising potential to biodegrade cyanide to non-toxic end-products. Ammonia and formate were found as final products during growth of the isolate with KCN as the sole nitrogen source. Formamide was not detected as one of the end-products suggesting that the biodegradation of cyanide by Rhodococcus UKMP-5M may have proceeded via a hydrolytic pathway involving the bacterial enzyme cyanidase. No growth of the bacterium was observed when KCN was supplied as the sole source of carbon and nitrogen even though marginal reduction in the concentration of cyanide was recorded, indicating the toxic effect of cyanide even in cyanide-degrading microorganisms. The cyanide biodegradation ability of Rhodococcus UKMP-5M was greatly affected by the presence of organic nutrients in the medium. Medium containing glucose and yeast extract promoted the highest growth rate of the bacterium which simultaneously assisted complete biodegradation of 0.1 mM KCN within 24 hours of incubation. It was found that growth and cyanide biodegradation occurred optimally at 30°C and pH 6.3 with glucose as the preferred carbon source. Acetonitrile was used as an inducer to enhance cyanide biodegradation since the enzymes nitrile hydratase and/or nitrilase have similarity at both the amino acid and structural levels to that of cyanidase. The findings from this study should be of great interest from an environmental and health point of views since the optimum conditions discovered in the present study bear a close resemblance to the actual scenario of cyanide wastewater treatment facilities.     

Draft Genome Sequence of the Cyanide-Utilizing Bacterium Pseudomonas fluorescens Strain NCIMB 11764

We report here the 6.97-Mb draft genome sequence of Pseudomonas fluorescens strain NCIMB 11764, which is capable of growth on cyanide as the sole nitrogen source. The draft genome sequence allowed the discovery of several genes implicated in enzymatic cyanide turnover and provided additional information contributing to a better understanding of this organism''s unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium.     

Nickel-specific, slow-binding inhibition of carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide

The inhibition of purified carbon monoxide dehydrogenase from Rhodospirillum rubrum by cyanide was investigated in both the presence and absence of CO and electron acceptor. The inhibition was a time-dependent process exhibiting pseudo-first-order kinetics under both sets of conditions. The true second-order rate constants for inhibition were 72.2 M-1 s-1 with both substrates present and 48.9 and 79.5 M-1 s-1, respectively, for the reduced and oxidized enzymes incubated with cyanide. CO partially protected the enzyme against inhibition after 25-min incubation with 100 microM KCN. Dissociation constants of 8.46 microM (KCN) and 4.70 microM (CO) were calculated for the binding of cyanide and CO to the enzyme. Cyanide inhibition was fully reversible under an atmosphere of CO after removal of unbound cyanide. N2 was unable to reverse the inhibition. The competence of nickel-deficient (apo) CO dehydrogenase to undergo activation by NiCl2 was unaffected by prior incubation with cyanide. Cyanide inhibition of holo-CO dehydrogenase was not reversed by addition of NiCl2. 14CN- remained associated with holoenzyme but not with apoenzyme through gel filtration chromatography. These findings suggest that cyanide is a slow-binding, active-site-directed, nickel-specific, reversible inhibitor of CO dehydrogenase. We propose that cyanide inhibits CO dehydrogenase by being an analogue of CO and by binding through enzyme-bound nickel.