The cyanide hydratase enzyme of Fusarium lateritium also has nitrilase activity

The filamentous fungus Fusarium lateritium produces cyanide hydratase when grown in the presence of cyanide. The cyanide hydratase protein produced at a high level in Escherichia coli shows a low but significant nitrilase activity with acetonitrile, propionitrile and benzonitrile. The nitrilase activity is sufficient for growth of the recombinant strain on acetonitrile, propionitrile or benzonitrile as the sole source of nitrogen. The recombinant enzyme shows highest nitrilase activity with benzonitrile. Site-directed mutagenesis of the F. lateritium cyanide hydratase gene indicates that mutations leading to a loss of cyanide hydratase activity also lead to a loss of nitrilase activity. This suggests that the active site for cyanide hydratase and nitrilase activity in the protein is the same. This is the first evidence of cyanide hydratase having nitrilase activity.     

Binding of metal cyanide complexes to bovine liver rhodanese in the crystalline state

Bovine liver rhodanese, which catalyzes the transfer of sulfur atoms between a variety of sulfur donor and sulfur acceptor substrates, is inhibited by metal cyanide complexes [Volini, M., Van Sweringen, B., & Chen, F.-Sh. (1978) Arch. Biochem. Biophys. 191, 205-215]. Crystallographic studies are described which reveal the binding mode of four different metal cyanides to bovine liver rhodanese: Na[Au(CN2], K2[Pt(CN)4], K2[Ni(CN)4], and K2[Zn(CN)4]. It appears that these complexes bind at one common site at the entrance of the active site pocket, interacting with the positively charged side chains of Arg-186 and Lys-249. This observation explains the inhibition of rhodanese by this class of compounds. For the platinum and nickel cyanide complexes virtually no other binding sites are observed. The gold complex binds, however, to three additional cysteine residues, thereby also displacing the extra sulfur atom which was bound to the essential Cys-247 in the sulfur-rhodanese complex. The zinc complex binds to completely different additional sites and forms complexes with the side chains of Asp-101 and His-203. Possible reasons for these different binding modes are discussed in terms of the preference for "hard" and "soft" ligands of these four metal ions.     

Cyanide Degradation under Alkaline Conditions by a Strain of Fusarium solani Isolated from Contaminated Soils

Several cyanide-tolerant microorganisms have been selected from alkaline wastes and soils contaminated with cyanide. Among them, a fungus identified as Fusarium solani IHEM 8026 shows a good potential for cyanide biodegradation under alkaline conditions (pH 9.2 to 10.7). Results of K(sup14)CN biodegradation studies show that fungal metabolism seems to proceed by a two-step hydrolytic mechanism: (i) the first reaction involves the conversion of cyanide to formamide by a cyanide-hydrolyzing enzyme, cyanide hydratase (EC 4.2.1.66); and (ii) the second reaction consists of the conversion of formamide to formate, which is associated with fungal growth. No growth occurred during the first step of cyanide degradation, suggesting that cyanide is toxic to some degree even in cyanide-degrading microorganisms, such as F. solani. The presence of organic nutrients in the medium has a major influence on the occurrence of the second step. Addition of small amounts of yeast extract led to fungal growth, whereas no growth was observed in media containing cyanide as the sole source of carbon and nitrogen. The simple hydrolytic detoxification pathway identified in the present study could be used for the treatment of many industrial alkaline effluents and wastes containing free cyanide without a prior acidification step, thus limiting the risk of cyanhydric acid volatilization; this should be of great interest from an environmental and health point of view.     

sti35, a stress-responsive gene in Fusarium spp.

A stress-induced mRNA was identified in the phytopathogenic fungus Fusarium oxysporum f. sp. cucumerinum. Treatment of the fungus with ethanol resulted in the induction of a major mRNA species encoding a protein of approximate Mr 37,000. A full-length cDNA clone of the induced message was obtained. RNA blot analysis indicated that the mRNA was induced by various other stresses, including treatment with copper(II) chloride and heat (37 degrees C). However, it was not greatly induced by treatment with phaseollinisoflavan, an antifungal isoflavonoid produced by Phaseolus vulgaris (French bean). In contrast, phaseollinisoflavan induced the homologous mRNA in the related bean pathogen Fusarium solani f. sp. phaseoli. A genomic clone of the F. solani f. sp. phaseoli gene was obtained, and both this and the cDNA clone from F. oxysporum f. sp. cucumerinum were sequenced. The latter indicated an open reading frame of 320 codons encoding a 34,556-dalton polypeptide. The corresponding reading frame in F. solani f. sp. phaseoli was 324 codons, 89% identical to the F. oxysporum f. sp. cucumerium sequence, and was interrupted by a short intron. The gene was designated sti35 (stress-inducible mRNA). Although computer homology searches were negative, the cloned gene was observed to cross-hybridize to DNAs of other filamentous fungi, Saccharomyces cerevisiae, and soybean. Thus, sti35 appears to be a common gene among a variety of eucaryotes.     

Heterologous expression of mitochondria‐targeted microbial nitrilase enzymes increases cyanide tolerance in Arabidopsis

Anthropogenic activities have resulted in cyanide (CN) contamination of both soil and water in many areas of the globe. While plants possess a detoxification pathway that serves to degrade endogenously generated CN, this system is readily overwhelmed, limiting the use of plants in bioremediation. Genetic engineering of additional CN degradation pathways in plants is one potential strategy to increase their tolerance to CN. Here we show that heterologous expression of microbial nitrilase enzymes targeted to the mitochondria increases CN tolerance in Arabidopsis. Root length in seedlings expressing either a CN dihydratase from Bacillus pumilis or a CN hydratase from Neurospora crassa was increased by 45% relative in wild‐type plants in the presence of 50 μm KCN. We also demonstrate that in contrast to its strong inhibitory effects on seedling establishment, seed germination of the Col‐0 ecotype of Arabidopsis is unaffected by CN.     

Natural attenuation potential of cyanide via microbial activity in mine tailings

Biological removal by indigenous microflora of cyanide, contained in old (6-9 years) and fresh tailings (3 months), was studied in order to assess its natural attenuation potential via biodegradation. To investigate the presence of indigenous microflora in tailings, total heterotrophic and cyanide resistant bacteria were counted using the spread-plate method. The free cyanide mineralization potential was estimated using K14CN in the presence of various unlabeled cyanide concentrations (0, 5, and 10 mg CN/kg). The biodegradation of cyanide contained initially in the samples was also investigated by monitoring formate, formamide, ammonia and total cyanide (CNT) concentrations over 111 days. The enumeration of total heterotrophic and cyanide-resistant bacteria in old tailings showed an average population of 105 cfu/g. However, no growth was detected in fresh tailings. Nevertheless, cyanide mineralization tests indicated the presence, in both old and fresh tailings, of a cyanide-degrading microflora. In old tailings, maximum mineralization percentages of free cyanide ranging from 85% to 100% were obtained after 65 days at all concentrations tested. A mineralization percentage of 83% after 170 days was also observed in fresh tailings. No decrease of total cyanide concentration in old tailings was observed when the biodegradation of endogenous cyanide was tested whereas a significant decrease was recorded in fresh tailings after 96 days. The presence of strong metal-cyanide complexes resistant to biodegradation could explain the absence of biodegradation in old tailings. This study demonstrated the presence of an indigenous free cyanide-degrading microflora in both old and fresh tailings, and suggests that natural attenuation of cyanide in gold mine tailings is likely to occur via microbial activity.