Daniel A. Kunz

Alternative routes of enzymic cyanide metabolism in Pseudomonas fluorescens NCIMB 11764.

Cell-free extracts from Pseudomonas fluorescens NCIMB 11764 catalysed the degradation of cyanide into products that included CO2, formic acid, formamide and ammonia. Cyanide-degrading activity (CDA) was localized to cytosolic cell fractions and was observed at substrate concentrations as high as 100 mM (2600 mg CN-I-1). At least two different CDAs could be distinguished by: (i) the determination of reaction product 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) plus formamide as reaction products, was oxygen independent, and had an apparent Km of 12 mM for cyanide. The first enzymic activity was identified as cyanide oxygenase as previously described [Harris, R. E. & Knowles, C. J. (1983) FEMS Microbiol Lett 20, 337-341] whereas the second activity is believed to consist of two enzymes, a cyanide nitrilase (dihydratase) and hydratase (EC 4.2.1.66). In addition to these enzymes, cyanide-grown cells were also induced for formate dehydrogenase (EC 1.2.1.2) thereby providing a means of recycling NADH utilized by cyanide oxygenase. A mutant strain having lost the ability to grow on cyanide as a nitrogen source was isolated and shown to be defective in cyanide oxygenase, but not the cyanide nitrilase/hydratase enzymes. This finding together with results showing that the substrate affinity of cyanide oxygenase was tenfold greater than for the nitrilase/hydratase enzymes, indicates that it is this enzyme that is most important in cyanide assimilation.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

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

ABSTRACT 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.

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 organisms unique cyanotrophic ability. This is the first sequenced genome of a cyanide-assimilating bacterium.

Complete Genome Sequence of a Cyanotroph, Pseudomonas fluorescens NCIMB 11764, Employing Single-Molecule Real-Time Technology.

ABSTRACT We report here the application of single-molecule real-time sequencing for determining the entire genome structure of the cyanotroph Pseudomonas fluorescens NCIMB 11764.

Linkage of the Nit1C gene cluster to bacterial cyanide assimilation as a nitrogen source

A genetic linkage between a conserved gene cluster (Nit1C) and the ability of bacteria to utilize cyanide as the sole nitrogen source was demonstrated for nine different bacterial species. These included three strains whose cyanide nutritional ability has formerly been documented (Pseudomonas fluorescens Pf11764, Pseudomonas putida BCN3 and Klebsiella pneumoniae BCN33), and six not previously known to have this ability [Burkholderia (Paraburkholderia) xenovorans LB400, Paraburkholderia phymatum STM815, Paraburkholderia phytofirmans PsJN, Cupriavidus (Ralstonia) eutropha H16, Gluconoacetobacter diazotrophicus PA1 5 and Methylobacterium extorquens AM1]. For all bacteria, growth on or exposure to cyanide led to the induction of the canonical nitrilase (NitC) linked to the gene cluster, and in the case of Pf11764 in particular, transcript levels of cluster genes (nitBCDEFGH) were raised, and a nitC knock-out mutant failed to grow. Further studies demonstrated that the highly conserved nitB gene product was also significantly elevated. Collectively, these findings provide strong evidence for a genetic linkage between Nit1C and bacterial growth on cyanide, supporting use of the term cyanotrophy in describing what may represent a new nutritional paradigm in microbiology. A broader search of Nit1C genes in presently available genomes revealed its presence in 270 different bacteria, all contained within the domain Bacteria, including Gram-positive Firmicutes and Actinobacteria, and Gram-negative Proteobacteria and Cyanobacteria. Absence of the cluster in the Archaea is congruent with events that may have led to the inception of Nit1C occurring coincidentally with the first appearance of cyanogenic species on Earth, dating back 400-500 million years.

POSTER: “Pseudomonas fluorescens Strain NCIMB 11764, a unique bacterium with cyanide adaptation features”

Cyanide is a potent poison that can be found naturally in some environments, and released as a waste in human activities. Cyanide waste is indeed a public health and an environmental concern. Its removal is an important issue, which has been addressed by chemical-physical methods or by alternatives such as bioremediation, which utilize organisms that can degrade cyanide. Pseudomonas fluorescens NCIMB11764 (Pf11764) bacterium is capable of utilize cyanide as its sole nitrogen source. However, the complete pathways involved in its metabolism are unclear. Therefore, to learn more about the unique genetic features of Pf11764, we characterized the bacteriums genome using next-generation sequencing technology. Our main objectives were to characterize and identify the genes involved in cyanide degradation and to study potential resistance pathways present in this genome.