Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalyzed the degradation of cyanide into products that included C02, formic acid, formamide and ammonia. Cyanide-degrading activity was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM. Two cyanide degrading activities were identified by: (i) the determination of reaction products stoichiometries, (ii) requirements for NADH and oxygen, and (iii) kinetic analysis. The first activity produced CO2 and NH3 as reaction products, was dependent on oxygen and NADH for activity, and displayed an apparent Km for cyanide of 1.2 mM. The second activity generated formic acid (and NH3) pfus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymatic activity was identified as cyanide oxygenase whereas the second activity consists of two enzymes, a cyanide nitrilase (dihydratase) and putative cyanide hydratase. In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (FDH), providing a means of recycling NADH utilized by cyanide oxygenase.

Pseudomonas fluorescens NCIMB 11764 (Pf11764) is uniquely able to grow on the poison cyanide as its sole nitrogen source. It does so by converting cyanide oxidatively to carbon dioxide and ammonia, the latter being assimilated into cellular molecules. This requires a complex enzymatic machinery that includes nitrilase and oxygenase enzymes the nature of which are not well understood. In the course of a proteomics analysis aimed at achieving a better understanding of the proteins that may be required for cyanide degradation by Pf11764, an unknown protein of 17.8 kDa was detected in cells exposed to cyanide. Analysis of this protein by ESI-coupled mass spectrometry and bioinformatics searches gave evidence of strong homology with a protein (Hyp1) of unknown function (hypothetical) present in the bacterium Photorhabdus luminescens subsp. laumondii TTO1 (locus plu_1232). A search of available microbial genomes revealed a number of Hyp1 orthologs the genes of which are found in a conserved gene cluster known as Nit1C. Independent studies revealed that in addition to Hyp1, Pf11764 possesses a gene (nit) specifying a nitrilase enzyme whose closest homologue is a nitrilase found in Nit1C gene clusters (77% amino acid identity). DNA sequence analysis has further revealed that indeed, hyp1Pf11764 and nitPf11764 are contained in a cluster that includes also a gene specifying an oxygenase. Given the possible connection of Nit1C-endoded nitrilase and oxygenase enzymes to enzymatic cyanide degradation, there is strong reason for thinking that the genes specifying these enzymes contribute to bacterial growth on cyanide in those bacteria containing the Nit1C cluster. Because the biological function of the Hyp1 protein is currently unknown, it was cloned and the protein expressed in E. coli so that its properties could further be explored. Unfortunately, the expression of the protein in an insoluble form complicated these analyses. However, at least two lines of evidence suggest a possible role as a regulator of gene expression. First, over-expression of the protein was accompanied by the parallel elevation of the putative vector-encoded b-lactamase, implying that Hyp1Pf11764 can affect the expression of other genes. Second, a comparison of the amino acid sequence of select peptide fragments of Hyp1Pf11764, by conducting searches for homology with proteins in the existing nonredundant protein database, consistently revealed motifs in common with those present in bacterial response regulators that are part of two-component signal transduction systems widely distributed in bacteria.

The involvement of cyanide oxygenase in the metabolism of pyruvate and a-ketoglutarate-cyanohydrin was investigated and shown to occur indirectly by the consumption of free cyanide arising from the cyanohydrins via chemical dissociation. Thus, free cyanide remains the substrate, for which the enzyme displays a remarkably high affinity (Kmapp,4 mM). A model for cyanide utilization is therefore envisioned in which the substrate is initially detoxified by complexation to an appropriate ligand followed by enzymatic oxidation of cyanide arising at sublethal levels via chemical dissociation. Putative cyanide oxygenase in cell extracts consumed both oxygen and NADH in equimolar proportions during cyanide conversion to CO2 and NH3 and existed separately from an unknown heat-stable species responsible for the nonenzymatic cyanide-catalyzed consumption of oxygen. Evidence of cyanide inhibition and nonlinear kinetics between enzyme activity and protein concentration point to a complex mechanism of enzymatic substrate conversion.

Utilization of cyanide as the sole nitrogen source by Pseudomonas fluorescens NCIMB 11764 (Pf11764) occurs via oxidative conversion to carbon dioxide and ammonia, the latter satisfying the nitrogen requirement. Substrate attack is initiated oxygenolytically by an enzyme referred to as cyanide oxygenase (CNO), which exhibits properties of a pterin-dependent hydroxylase. The pterin requirement for Pf11764 CNO was 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. A related CNO-mediated mechanism of cyanide utilization was identified in cyanide-degrading P. putida BCN3. This conclusion was based on (i) the recovery of CO2 and NH3 as enzymatic reaction products, (ii) the dependency of substrate conversion on both O2 and NADH, and (iiii) utilization of cyanide, O2 and NADH in a 1:1:1 reaction stoichiometry. In contrast to findings reported for Pf11764, it was not possible to demonstrate a need for exogenously added pterin as a cofactor for the PpBCN3 enzyme system. However, results which showed that cells of PpBCN3 contained approximately seven times the amount of pterin as Pf11764 (of which a significant portion was protein-bound) were interpreted as indicating that sufficient bound CNO-cofactor exists, thus eliminating any need for a supplemental source.

Ten bacteria capable of growth on the metal-cyano complex, tetracyanonickelate (II) {K2 [Ni(CN)J } (TCN), supplied as the sole nitrogen source, were isolated. Seven isolates were identified as pseudomonads while the remaining three were classified as Klebsiella species. In addition to TCN, all isolates were able to utilize KCN although it was significantly more toxic. The degradation of TCN was most complete when supplied at growth-limiting concentrations, did not occur when ammonia was present, and resulted in the formation of nickel cyanide [Ni(CN)2] as a degradation product.

Pseudomonas fluorescens NCIMB 11764 was capable of utilizing cyanide as a sole nitrogen source for growth. Cyanate (OCN") and S-cyanoalanine could also serve as nitrogenous substrates, but do not appear to play a role as intermediates in cyanide metabolism. Growth of this strain on cyanate as the sole nitrogen source led to the induction of an enzyme characterized as a cyanase (EC 3.5.5.3) based on its stoichiometric conversion of cyanate to ammonia, and dependence on bicarbonate for maximal activity. However, since cyanase activity was not elevated in cyanide-grown cells it was concluded that it serves no role in cyanide metabolism. Related studies aimed at examining a possible role for S-cyanoalanine as a cyanide-assimilation intermediate showed that while this compound also serves as a nitrogen source, it also is not important in cyanide metabolism. Studies focused on the utilization of free cyanide as a growth substrate led to the development of a fed-batch cultivation procedure greatly facilitating further experimentation aimed at the identification of cyanide metabolites. In addition to CO_2 and NH_3 as described earlier, two additional metabolites including formamide and formate were detected by using nC-NMR, HPLC, radioisotrapping methods and other analytical means. The formation of metabolites was shown to be induced after growth on cyanide with the relative product yields dependent on the availability of oxygen. These findings support earlier work in which an oxygen-dependent mechanism was proposed for the formation of C02 and NH3. However, at least two additional oxygen-independent pathways of cyanide conversion can be elaborated by this organism. One of these involves conversion to formate and ammonia while the other leads to the formation of formamide, which is not further degraded. Thus, growth on cyanide appears to occur by several mechanisms of chemical transformation presumably serving both detoxification and nutritional roles. Since two of these mechanisms generate ammonia, which is readily assimilated, growth is presumed to proceed via ammonia as a provisionary nitrogenous substrate.

Utilization of cyanide as a nutritional nitrogen source in P. fluorescens NCIMB 11764 was shown to involve a novel metabolic mechanism involving nonenzymatic neutralization outside of cells prior to further enzymatic oxidation within. Several cyanide degrading enzymes were produced by NCIMB 11764 in response to growth or exposure to cyanide, but only one of these cyanide, oxygenase (CNO), was shown to be physiologically required for assimilation of cyanide as a growth substrate.