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

Abstract

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.

The cyanotrophic bacterium Pseudomonas pseudoalcaligenes CECT5344 responds to cyanide by defence mechanisms against iron deprivation, oxidative damage and nitrogen stress

“…The phosphoribosylglycinamide formyltransferase activity of P. pseudoalcaligenes CECT5344 was determined, and we found that enzymatic activity was 41% higher in cyanide-grown cells than in ammoniagrown cells (data not shown). Although putative siderophore production in response to cyanide was initially reported by Chen and Kunz (1997) fluorescens, this iron-chelating activity was proposed later to be due to the extracellular accumulation of a-ketoacids which carry out the non-enzymatic scavenging of cyanide in this bacterium (Kunz et al, 1998). Thus, our finding that cyanide induces a putative formyltransferase probably involved in the synthesis of a siderophore points out again on the production of this type of specific iron chelators in response to cyanide.…”

“…Besides, bacteria and fungi have developed metabolic pathways to degrade cyanide by a number of oxidative, reductive and hydrolytic pathways, as well as through substitution/addition reactions involving synthesis and subsequent hydrolysis of nitriles (Ebbs, 2004) and cyanohydrines (Kunz et al, 1998).…”

Early changes in apoplast composition associated with defence and disease in interactions between Phaseolus vulgaris and the halo blight pathogen Pseudomonas syringae Pv. phaseolicola

“…This mutation is consistent with the poor oxidation of 2-oxoglutarate by Pph in Biolog assays and indicates that import rather than metabolism is limiting. Interestingly, extracellular accumulation of pyruvate and 2-oxoglutarate by Pseudomonads has been implicated in both cyanide (CN) and ROS detoxification as these α-keto acids react non-enzymatically with CN to form nitriles and with H 2 O 2 to form decarboxylation products (Andrae et al 1985;Kunz et al 1998;Mailloux et al 2009). As ROS production is a hallmark of the plant defence response, the benefit to P. syringae of excreting keto-acids as additional antioxidants is obvious but remains to be directly tested in planta.…”

Biochemical and molecular characterization of ?-ketoisovalerate decarboxylase, an enzyme involved in the formation of aldehydes from amino acids by

“…Samples were incubated at 37°C for 20 min and the reaction was stopped by adding ethyl acetate (1:1, v/v) prior to chromatographic analysis. To detect isobutyraldehyde production, samples were derivatized with 2,4-dinitrophenylhydrazine (DNPH, Panreac) to form the corresponding dinitrophenylhydrazone derivative as described by Kuntz et al [33]. The chromatographic conditions employed were those described by Schmidt et al [34].…”