Metabolism Of Nitriles Research Articles

Metabolism of Aldoximes and Nitriles in Plant-Associated Bacteria and Its Potential in Plant-Bacteria Interactions.

In plants, aldoximes per se act as defense compounds and are precursors of complex defense compounds such as cyanogenic glucosides and glucosinolates. Bacteria rarely produce aldoximes, but some are able to transform them by aldoxime dehydratase (Oxd), followed by nitrilase (NLase) or nitrile hydratase (NHase) catalyzed transformations. Oxds are often encoded together with NLases or NHases in a single operon, forming the aldoxime–nitrile pathway. Previous reviews have largely focused on the use of Oxds and NLases or NHases in organic synthesis. In contrast, the focus of this review is on the contribution of these enzymes to plant-bacteria interactions. Therefore, we summarize the substrate specificities of the enzymes for plant compounds. We also analyze the taxonomic and ecological distribution of the enzymes. In addition, we discuss their importance in selected plant symbionts. The data show that Oxds, NLases, and NHases are abundant in Actinobacteria and Proteobacteria. The enzymes seem to be important for breaking through plant defenses and utilizing oximes or nitriles as nutrients. They may also contribute, e.g., to the synthesis of the phytohormone indole-3-acetic acid. We conclude that the bacterial and plant metabolism of aldoximes and nitriles may interfere in several ways. However, further in vitro and in vivo studies are needed to better understand this underexplored aspect of plant-bacteria interactions.

Substrate specificity of plant nitrilase complexes is affected by their helical twist

Nitrilases are oligomeric, helix-forming enzymes from plants, fungi and bacteria that are involved in the metabolism of various natural and artificial nitriles. These biotechnologically important enzymes are often specific for certain substrates, but directed attempts at modifying their substrate specificities by exchanging binding pocket residues have been largely unsuccessful. Thus, the basis for their selectivity is still unknown. Here we show, based on work with two highly similar nitrilases from the plant Capsella rubella, that modifying nitrilase helical twist, either by exchanging an interface residue or by imposing a different twist, without altering any binding pocket residues, changes substrate preference. We reveal that helical twist and substrate size correlate and when binding pocket residues are exchanged between two nitrilases that show the same twist but different specificities, their specificities change. Based on these findings we propose that helical twist influences the overall size of the binding pocket.

The industrial potential of microbial nitrile biochemistry.

Nitrile compounds are manufactured widely by man as well as in Nature. The industrial potential of using the microbial metabolism of nitriles and related compounds for the manufacture of a variety of chemicals has started to be realised. The microbial metabolism of nitrile-using microorganisms can also be harnessed to biodegrade toxic waste effluent streams that contain nitriles and are formed during the chemical synthesis of nitrile compounds.

A gene cluster responsible for alkylaldoxime metabolism coexisting with nitrile hydratase and amidase in Rhodococcus globerulus A-4.

An enzyme "alkylaldoxime dehydratase (OxdRG)" was purified and characterized from Rhodococcus globerulus A-4, in which nitrile hydratase (NHase) and amidase coexisted with the enzyme. The enzyme contains heme b as a prosthetic group, requires reducing reagents for the reaction, and is most active at a neutral pH and at around 30 degrees C, similar to the phenylacetaldoxime dehydratase from Bacillus sp. OxB-1 (OxdB). However, some differences were seen in subunit structure, substrate specificity, and effects of activators and inhibitors. The corresponding gene, oxd, encoding a 1059-base pair ORF consisting of 353 codons, was cloned, sequenced, and overexpressed in Escherichia coli. The predicted polypeptide showed 30.3% identity to OxdB. The gene is mapped just upstream of the gene cluster encoding the enzymes involved in the metabolism of aliphatic nitriles, i.e., NHase and amidase, and their regulatory and activator proteins. We report here the existence of an aldoxime dehydratase genetically linked with NHase and amidase, and responsible for the metabolism of alkylaldoxime in R. globerulus.

Comparative Developmental Toxicities of Aliphatic Nitriles: In Vivo and in Vitro Observations

The effects on embryonic development of a series of eight saturated (acetonitrile, propionitrile, and n-butyronitrile) and unsaturated (acrylonitrile, methacrylonitrile, allylnitrile, cis-2-pentenenitrile, and 2-chloroacrylonitrile) nitriles were compared in vitro using the whole embryo culture system. Day 10 rat embryos were cultured for 46 h in rat serum in the presence of either of these chemicals. All the tested chemicals produced concentration-dependent decreases in growth and differentiation and increases in the incidences of morphologically abnormal embryos. A wide range of embryotoxic potency was observed, with 2-chloroacrylonitrile and acetonitrile at the extremes (lowest effect levels of 50 μM and 40 mM, respectively). No common pattern could be drawn for all the eight nitriles tested in vitro, although there were some similarities between the malformations elicited by propionitrile and n-butyronitrile or between those elicited by the five unsaturated nitriles. Presence of a rat hepatic microsomal fraction and NADPH in the culture medium enhanced the embryotoxic effects of the five unsaturated nitriles tested but had no effects on saturated nitriles embryotoxicity. In addition to these in vitro experiments, pregnant rats were given a single oral dose of each compound on Day 10 of gestation and the embryos were evaluated on Day 12 of gestation, i.e., at a time of development corresponding to the developmental stage at the end of the whole embryo culture. All the nitriles investigated produced the characteristic defects developed by embryos exposed to sodium cyanide in utero or in culture. Our results provide further evidence that maternal production of cyanide may contribute to the developmental toxicity of saturated and unsaturated nitriles and suggest that distinct metabolites derived from microsomal metabolism of unsaturated nitriles may also play a role.

In Vitro Metabolism of Aromatic Nitriles

Studies on the metabolic fate of aromatic nitriles, in contrast to their aliphatic counterparts, have been minimal and the subject of controversy. Thein vitrometabolic fate of several aromatic nitriles with varying substituents was investigated by using rat liver subcellular fractions, with a particular emphasis on the nitrile moiety. Benzonitriles and 4‐cyanophenols underwent oxidative metabolism to produce ring‐hydroxy‐lated metabolites. On the other hand, 2‐cyanophenol was resistant to metabolism.o‐Tolunitrile was metabolized and producedo‐cyanobenzyl alcohol and phthalide. Phthalide, however, was chemically derived fromo‐cyanobenzyl alcohol, the initial metabolite. 4‐Nitrobenzonitrile was resistant to oxidation on the ring, but was readily reduced to the corresponding amine metabolite under both aerobic and anaerobic conditions. Nitroxynil (3‐iodo‐4‐hydroxy‐5‐nitrobenzonitrile) was metabolized to produce 3‐iodo‐4‐hydroxy‐5‐nitrobenzamide and 3‐iodo‐4‐hydroxy‐5‐nitrobenzoic acid. The enzyme(s) responsible for this hydrolytic metabolism was primarily localized in the cytosol. Among the nitriles tested,o‐tolunitrile and nitroxynil produced metabolites in which the nitrile moiety was modified. Nitroxynil, however, was the only compound that was directly metabolized on the nitrile moiety by the rat liver enzyme(s).

Microbial metabolism of nitriles and cyanides

Abstract Pseudomonas putida isolated from contaminated industrial wastewaters and soil sites was able to utilize acetonitrile and sodium cyanide as a sole source of carbon and nitrogen. The P. putida cells were immobilized in calcium alginate. The rate of degradation of acetonitrile (160 mM) and sodium cyanide (40 mM) by the immobilized cells of P. putida was studied by determining the products: NH3 and CO2. The product‐dependent kinetics of sodium cyanide and acetonitrile indicate that the rate of degradation of these compounds is in the first order kinetics. The present study concludes that remediation of organic nitriles and inorganic cyanides can be achieved with immobilized cells of P. putida.

The metabolism of acetonitrile to cyanide by isolated rat hepatocytes

The metabolism of saturated nitriles, including acetonitrile, has been assumed to occur by a cytochrome P-450-dependent oxidation at the α-carbon, yielding a cyanohydrin intermediate which may spontaneously degrade to hydrogen cyanide and an aldehyde. However, results of studies in our laboratory suggest that formaldehyde is not a metabolite of acetonitrile. Since acetonitrile is structurally similar to iodomethane, a substrate for glutathione (GSH) S-transferases, we hypothesized that the metabolism of acetonitrile to cyanide might also occur by a nucleophilic substitution reaction involving GSH. The present studies were conducted to investigate these hypotheses and to further our study of the effects of acetone on acetonitrile metabolism. Female Sprague-Dawley rats were pretreated with buthionine sulfoximine BSO (4 mmol/kg ip, at −4 and −2 hr), cobalt heme (90 μmol/kg sc, at −48 hr), acetone (1960 mg/kg po, at −24 hr), or vehicle, and hepatocytes were isolated after collagenase perfusion of the liver. BSO reduced the cellular GSH content by > 80%, but did not appear to affect the metabolism of acetonitrile: the liberation of cyanide correlated with cytochrome P-450, and not GSH, concentrations. Cobalt heme depleted hepatocellular cytochrome P-450 (−45%) content, decreased cell yield and viability, and resulted in a marked reduction in the metabolism of acetonitrile to cyanide. Cobalt heme did not affect the recovery of sodium cyanide from hepatocyte suspensions. Pretreatment of rats with acetone resulted in a twofold increase in the metabolism of acetonitrile to cyanide. Addition of acetone in vitro inhibited acetonitrile metabolism, with an IC50 of 319 μ m. These results suggest that the metabolism of acetonitrile to cyanide occurs by a cytochrome P-40-dependent pathway, and not by a nucleophilic substitution reaction with GSH.

Influence of ethanol on the in vivo and in vitro metabolism of nitriles in mice.

The effect of ethanol on metabolism of 20 nitriles was studied in vivo and in vitro in mice. The hepatic microsomal metabolizing activity for nitriles was at a maximum 13 h after ethanol dosing (4.0 g/kg). Using microsomes from mice pretreated with ethanol under the above conditions, enhancement of the in vitro metabolism of nitriles was 1.00-1.83 compared with the glucose-treated control. When mice were orally given nitriles 13 h after dosing with either ethanol (4.0 g/kg) or glucose (7.0 g/kg), the hepatic metabolizing activity of nitriles for the ethanol-treated group was always higher than that for the glucose group, although no change in the content of hepatic microsomal P-450 was observed between the two groups. However, ethanol added to the incubation mixture inhibited the in vitro metabolism of most nitriles. The results in the present study suggest that ethanol can enhance the acute toxicity of nitriles.

Structure-acute toxicity relationship of dinitriles in mice.

Acute toxicity and metabolism of seven dinitriles in mice was studied in relation to the chemical structures. The oral LD50 for each nitrile was determined under different conditions for mice pretreated with either carbon tetrachloride (CCl4) or olive oil. All test nitriles were metabolized into cyanide in vivo and in vitro. The cyanide level was variable among the compounds (0.35-0.74 micrograms CN/g brain) at death in the brains of mice, the level for malono- and adiponitrile being comparable to that found in mice killed by dosing with potassium cyanide. After receiving each nitrile, the mean survival time of mice pretreated with CCl4 was prolonged and their brain cyanide level decreased when compared with the corresponding control. With malononitrile, the former did not significantly change and the latter decreased slightly. Brain cyanide levels of control mice at death showed a peak at the lower log P region, while those of CCl4-pretreated animals remained lower independently of log P, with the exception of malononitrile. Microsomal metabolism of nitriles to cyanide was greatly inhibited when microsomes were prepared from livers of mice pretreated with CCl4. The relationship between log (1/LD50-CCl4), LD50 in mice pretreated with CCl4, and log P fits a parabolic plot.

Structure-toxicity relationship of aliphatic nitriles

Acute toxicity, in vitro metabolism and structure-toxicity relationship of aliphatic mononitriles were examined in mice pretreated with carbon tetrachloride (CCl 4). The LD 50 in mice pretreated with CCl 4 (LD 50-CCl 4) was increased in most nitriles compared to that in untreated animals (LD 50-cont.) with different degrees among compounds. Microsomal metabolism of nitriles to CN − was completely inhibited when microsomes were prepared from livers of mice pretreated with CCl 4. Log (1/LD 50-CCl 4) was a linear function of partition coefficient, log P, i.e., log (1/LD 50-CCl 4) = −0.371 log P-0.152, and the equation was statistically significant ( P < 0.01), provided aceto- and 3-hydroxypropionitrile were omitted from the analysis. These two compounds seem to be different from the others in exerting the biological effect.

Metabolism of nitriles in microorganisms. Part V. Purification, crystallization and some properties of .BETA.-cyano-L-alanine-degrading enzyme in Pseudomonas sp. 13.

A β-cyano-L-alanine (β-CNAla)-degrading enzyme in Pseudomonas sp. 13 was purified and isolated as a crystalline preparation. The purification procedure involved ethanol fractionation, ammonium sulfate fractionation, column chromatography on DEAE-cellulose and crystallization in the presence of ammonium sulfate, resulting in 660-fold purification with a yield of 21%.The crystalline enzyme was homogeneous as judged by ultracentrifugation and SDS-polyacrylamide gel electrophoresis. The enzyme has a molecular weight of approximately 1,000,000, and consists of about 30 apparently identical subunits, each of a molecular weight of approximately 35,000.The enzyme catalyzed the hydrolysis of β-CNAla to form asparagine and aspartic acid. The enzyme reaction was inhibited strongly by thiol reagents.

Metabolism of nitriles in microorganisms. Formation of .BETA.-cyanoalanine by cyanide-resistant strain Enterobacter sp. 10-1.

A cyanide-tolerant strain was isolated from a soil sample, and identified as belonging to the genus Enterobacter. The microorganism accumulated a compound which produced a green color with ninhydrin, in culture fluid containing sodium cyanide and sodium fumarate. This compound was isolated from the culture fluid in crystalline form by using ion exchange resin column chromatography, and was identified as β-cyano-l-alanine (β-CNAla). The compound accumulated in the culture fluid with the growth of the microorganism, and the amount of β-CNAla accumulated depended on the amount of sodium cyanide added. Both intact cells and a cell-free extract of the microorganism catalyzed the formation of β-CNAla from O-acetyl-l-serine with sodium cyanide as substrates. The β-CNAla-forming enzyme was found to be localized in both the cytoplasm and cytoplasmic membrane of this icroorganism.

Studies on the Metabolism of Nitriles

Studies on the Metabolism of Nitriles

The metabolism of certain acids, amides and nitriles within plant tissues.

The plant growth-regulating activities of the first six members of the homologous series ω -(2:4-dichlorophenoxy)alkanecarboxylic acids and the corresponding amides and nitriles have been assessed in the wheat cylinder, pea curvature, pea segment and tomato-leaf epinasty tests. The breakdown of these compounds when metabolized under standard conditions in wheat coleoptile and pea-stem tissues has also been investigated using paper chromatography together with chromogenic and bio-assay procedures. In addition, colorimetric analysis was used. The alternation in activity shown by the homologous series of acids and the production of 2:4-dichlorophenoxyacetic acid and of 2:4-dichlorophenol from alternate members indicates that β -oxidation of the side-chain of these acids occurs within wheat and pea tissues. The behaviour of the amides was similar to that of the acids and explicable in terms of hydrolysis to corresponding acid within the tissue followed by β -oxidation. Only the first member of the nitrile series was active in the pea and tomato tests, though in the wheat cylinder test all showed some activity, that of the propionic derivative, however, being only slight. With the exception of this compound, the nitriles yielded 2:4-dichlorophenoxyacetic acid and all gave some 2:4-dichlorophenol when metabolized in wheat tissue. These results can be interpreted in terms of modification of the —CH 2 CN grouping by two mechanisms, viz. hydrolysis to corresponding acid, and a type of breakdown which we refer to as α -oxidation which leads to the production of the lower carboxylic acid. The acids produced by either route are then subject to β -oxidation. A study of the metabolism of 3-indolylacetonitrile provided further evidence that α -oxidation of substituted nitriles can occur in plant tissue. In this instance, the end-product of the reaction was proved to be 3-indolecarboxylic acid and the occurrence of 3-indolealdehyde as an intermediate compound was established. The mechanism of α -oxidation of nitriles is discussed and it is suggested that this may be a general reaction occurring in both plant and animal biochemistry.