Cyanide Detoxifying Enzyme: Rhodanese

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Cyanide Detoxifying Enzyme: Rhodanese Mayank Chaudhary and Reena Gupta* Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla-171 005, India Abstract: Rhodanese is an ubiquitous enzyme active in all living organisms from bacteria to man. It is multifunctional enzyme but plays central role in cyanide detoxification. This enzyme is also widely distributed in plants. It functions through double displacement (ping pong) mechanism. The activity of rhodanese in a particular tissue reflect the ability of that tissue to detoxify cyanide. The level of rhodanese in different tissues of animals is correlated with the level of exposure to cyanide. Mitochondrial bovine rhodanese is the best characterized rhodanese which is 293 amino acids long consisting of inactive N-terminal and catalytic C-terminal domain. It comprises of single polypeptide chain of 32,900 Da folded into two domains of about equal size. Active site of rhodanese has essential sulfhydryl and aromatic groups in close proximity. In addition to this, competitive inhibition of rhodanese by aromatic ions suggests the presence of tryptophanyl residue at the active center. Sequence analysis of rhodanese-like proteins highlights their heterogeneity to form rhodanese superfamily presenting variably arranged rhodanese domains as single or tandem domains, or combined with other protein domains. Many methods for rhodanese assay have been reported but the most common one is colorimetric estimation of thiocyanate formed from the reaction of cyanide with thiosulphate catalyzed by rhodanese.

Keywords: Rhodanese, ubiquitous, cyanide, detoxification, domain, thiosulphate, thiocyanate, mitochondrial enzyme, double displacement mechanism, toluene, ageing, colonic epithelium. INTRODUCTION Cyanogenic glycosides are phytotoxins that occur in at least 2000 plant species, many of which are used as food in tropical countries [1]. Cassava and sorghum are important staple foods containing cyanogenic glycosides [2, 3]. There are approximately 25 cyanogenic glycosides known. The potential toxicity of a cyanogenic plant depends primarily on the potential that its consumption will produce HCN that is highly toxic. Mortality in livestock on consumption of cyanogenic glycosides has been reported [4,5]. Cyanide poisoning in humans has also been reported from eating plant materials such as bitter almond, cassava beans, apricot pits and lima beans [6]. Many naturally occurring substances as well as industrial products contain cyanide [7]. Many studies report death of birds from cyanide poisoning through several routes including exposure to cyanide salts or ingestion of cyanogenic plants [8]. Cyanide is also present in some insecticides, rodenticides, metal polishes, electroplating solutions, gold and silver extraction and fumigants and also in variety of metallurgical processes. The waste discharge from these industries can contain large amounts of cyanide and act as a source of poisoning [9]. Numerous accidental spills of sodium cyanide or potassium cyanide into rivers and streams have resulted in massive killing of fishes, amphibians and aquatic vegetation [10]. Although several chemical methods for treatment of cyanide polluted wastewater are currently available, they are relatively expensive and challenge the

environment for the release of chemical agents potentially causing secondary pollution [11]. The extreme toxicity of cyanide and environmental concerns from its continued industrial use continue to generate interest in facile and sensitive methods for cyanide detection [12]. Cyanide is a potent toxic agent that causes death by inhibition of cellular oxidising enzyme such as Cytochrome oxidase. Inhibition of cytochrome oxidase causes intracellular respiratory cessation and tissue anoxia, mainly leading to the dysfunction of neural cells and symptoms of CNS failure [6,7,13]. Thus cyanide is acutely toxic to mammals by all routes of administration with a very steep and dose dependant curve involving cytochrome oxidase inactivation [12]. The enzyme which plays central role in cyanide detoxification is mitochondrial enzyme named rhodanese. The enzyme was named rhodanese from the german name for thiocyanate (rhodanid) with the ending ‘ese’ indicating that this compound was formed through the enzymatic reaction [14]. The enzyme rhodanese (E.C. 2.8.1.1., thiosulphate: cyanide sulphurtransferase) is an ubiquitous enzyme present in all living organisms from bacteria to man [15-20]. Highest activity of rhodanese was found in kidney, followed by liver, brain, lungs, muscle and stomach in case of humans [21] (Table 1). Jarbak and Westley (1974) studied human liver rhodanese and showed that it differed from bovine liver rhodanese in kinetic behaviour and UV absorption properties [22]. Genetic polymorphism of rhodanese in human erythrocytes has been reported [23]. PHYSIOLOGICAL FUNCTION

*Address correspondence to this author at the Department of Biotechnology, Himachal Pradesh University, Summer Hill, Shimla-171 005, India; Tel: 91177-2831948; Fax: 91-177-2831948; E-mail: [email protected]

2211-55;/12 $58.00+.00

The physiological role of rhodanese (E.C 2.8.1.1., thiosulphate: cyanide sulphurtransferase ) in different species is controversial. The main role attributed to this enzyme is © 2012 Bentham Science Publishers

328 Current Biotechnology, 2012, Volume 1, No. 4

Table 1.

Chaudhary and Gupta

Specific Activity of Rhodanese (U/mg Protein) in Selected Tissues of Human and Some Domestic Animals [21] Species

Tissues Sheep

Cattle

Goat

Camel

Horse

Pig

Dog

Chicken

Human

Liver

5.21

4.95

2.93

1.7

1.7

0.56

0.18

0.31

0.15

Rumen

16.3

10.3

3.31

0.28






















Muscular layer

0.09

0.05

0.01

Cortex

1.7

0.82

0.94

0.08

1.4

0.4

0.06

0.08

0.96

Medulla

0.24

0.05

0.25

0.1

Brain

0.48

0.36

0.47

0.13

0.15

Nd

0.32

Nd

0.03

Lung

0.24

0.13

0.36

0.24

0.03

0.04

0.02

0.01

0.02

Epithelial layer

Kidney

Stomach

0.1

0.07

Abomasal fundus

0.09

0.04

0.35

0.07

0.13

Abomasal pylorus

0.1

0.04

0.31

0.02

0.006

0.01

Muscular layer of proventriculus

0.09

Epithelial layer of proventriculus Muscle

0.59 0.04

0.02

0.08

0.05

0.15

0.07

0.04

Nd

0.01

cyanide detoxification [15,16,24,25]. It is responsible for biotransformation of cyanide to thiocyanate using thiosulphate as donor substrate.

-

S2O32 - + CN-

Rhodanese

SCN- + SO32

Liver has always been considered to be the major source of rhodanese and is believed to be the major site of cyanide detoxification [26]. The activity of enzyme in a particular tissue/organ reflect the ability of that tissue/organ to detoxify cyanide [27]. It has been suggested that the level of rhodanese in different tissues of animals is correlated with the level of exposure to cyanide [15,28,29]. Activity of rhodanese is higher in those tissues that are more exposed to cyanide due to high blood supply [21]. The pattern of distribution of rhodanese in different animals appears to be highly species and tissue specific. No significant difference was seen between liver and kidney rhodanese activity in pigeon whereas in Japanese quail, rhodanese activity in kidney was significantly higher than that of liver [30]. Rhodanese activity in digestive tract of chicken was different at different stages of development [21]. All of the tissues in case of cat contain rhodanese activity. In terms of specific activity, colon and rectum mucosal layer and testis were found to be the richest sources of this enzyme followed by ovary, mucosal layer of jejunum and liver [31]. Role of rhodanese in cyanide detoxification and aging can also be attributed due to the fact that injury of mitochondrial function and decline of antioxidant capability are important reasons for aging of human colonic epithelium [32]. Downregulation of rhodanese in aged human colonic epithelium indicated that aging colonic epithelium decreases in the ability of cyanide detoxification (Fig. 1, Table 2).

Fig. (1). The expression of Rack1, EF-Tu and Rhodanese proteins in senescent NIH/3T3 cells. (A)
- Gal activity measured by X-Gal cytochemical staining in the D-Gal-exposed (senescent) and unexposed (control) NIH/3T3 cells; (B) a representative result of Western blot shows the downregulation of Rack1, EF-Tu and Rhodanese expression in the D-Gal-exposed (senescent) NIH/3T3 cells compared with unexposed (control) NIH/3T3 cells.
Actin was used as an internal control for loading [32].

Cyanide Detoxifying Enzyme: Rhodanese

Table 2.

Current Biotechnology, 2012, Volume 1, No. 4

Expression Levels of Three Differential Proteins in the Colonic Epithelium of Young and Old Peoplea [32] Staining Score

Protein Rack 1

Group

0-2

3-4

5-6

Total

Young

6

9

15

30

Old

14

13

3

30

Young

5

15

10

30

Old

17

12

1

30

Young

10

13

7

30

Old

21

6

3

30

EF-Tu

P 0.001

0.000

Rhodanese

0.007

a

The expression levels of three differential proteins were significantly higher in the colonic epithelia in young group than in old group by Mann-Whitney U test.

Down-regulation or suppression of colonic rhodanese that prevents detoxification of H2S is involved in inflammatory bowel disease (IBD). The delayed enhancement of rhodanese activity in RBCs, a possible compensative event, might be available as a disease marker for IBD [33]. Impaired rhodanese expression is associated with increased whole cell reactive oxygen species as well as higher mitochondrial superoxide production and predicts mortality in hemodialysis patients [34]. Rhodanese is responsible for the import of 5S rRNA into mitochondria as silencing of the rhodanese gene caused not only a proportional decrease of 5S rRNA import but also a general inhibition of mitochondrial translation, indicating the functional importance of the imported 5 S rRNA inside the organelle [35]. The capacity of Bacillus stearothermophilus to detoxify cyanide was greatly increased in mutants showing 5 to 6 folds of rhodanese activity than wild type [36]. Moreover, rhodanese from cyanogenic bacterium Pseudomonas aeruginosa has been reported to contribute cyanide detoxification [14,37]. Mycobacterium tuberculosis also encodes a probable rhodanese-like thiosulfate sulfurtransferase [38]. Coexistence of several rhodanese like proteins in same organism suggests its different physiological roles [14]. Distribution of rhodanese in both larvae and adults of insects that are not exposed to cyanides through feeding on cyanogenic plants indicated other possible roles of this enzyme. This enzyme is also involved in formation of iron sulphur centres [39], participation in energy metabolism [40,41], biosynthesis of molybdenum cofactor in humans [42]. It also functions as thioredoxin oxidase [43]. It has been suggested that the reversible sulphur transfer to and from rhodanese is an important feature of the role played by this enzyme in energy metabolism [39,40,44]. Rhodanese controls the rate of respiratory chain and ATP production by reversible sulphuration of key iron- sulphur centres and affects activity of enzymes like succinate dehydrogenase, NADH dehydrogenase. Rhodanese is also involved in biosynthesis of iron-sulfur clusters [45]. The iron-sulfur clusters of ferroxidans, succinate dehydrogenase, mitochondrial NADH dehydrogenase can be reconstituted by incubation with rhodanese, a sulfur donor, a sulfur acceptor and an iron source [40,41,46]. It also prevents against oxidative stress in Azotobacter vinelandii [47]. In addition to this it plays role

329

in sulphur and selenium metabolism. The expression of rhodanese like protein from Acidithiobacillus ferrooxidansis induced during growth on sulphur compounds to generate thiosulphate which is used as electron donor by this chemolithotrophic bacterium [48]. Rhodanese also generate reactive form of selenium for synthesis of selenophosphate (SePO32-) which is active selenium donor compound required by bacteria and eukaryotes for synthesis of Se Cys-tRNA [49]. These enzymes are involved in selenium metabolism as incubation of bovine rhodanese with selenite in the presence of glutathione resulted in formation of an equimolar rhodanese-selenium adduct that was capable of serving as a selenium donor for selenophosphate synthetase [50]. In rhodopseudomonas spheroids rhodanese catalyses the formation of cysteine from cysteine trisulphide [51]. A possible role of the enzyme in modulating S-amino levulinate synthetase activity has also been reported [52]. STRUCTURE Rhodanese enzyme of bovine liver mitochondria is a single polypeptide chain of 32,900 Da which is folded into two domains of about equal size [53,54]. Another rhodanese from bovine liver is a dimer of 37,000 Da with two catalytic sites which is dissociable into identical subunits [55]. Rhodanese enzyme of 14,000 Da molecular weight has been purified from E. coli [56]. Bovine liver rhodanese contains 293 amino acids. The protein consists of two equally sized globular domains, the inactive N-terminal and catalytic Cterminal showing identical
/ topology. Each domain is 120 amino acids long with low sequence similarity. Only Cterminal carries 6 amino acid active site loop having cysteine at first position involved in the catalytic process. The counterpart of cys residue in N-terminal domain is an aspartate residue which has no involvement in catalysis [57]. The pecularity of rhodanese resides in pattern of amino acid sequences at both N- and C- terminal. These sequences called ‘rhodanese signatures’ have remarkable degree of conservation among rhodanese and therefore are used to recognize these proteins encoded by different genomes (Fig. 2). Pseudomonas aeruginosa rhodanese shows 79% and 22% sequence identity with Azotobacter vinelandii rhodanese and bovine liver rhodanese respectively, but no striking differences emerge by comparing their 3 Dstructures as shown in Fig. (3). RHODANESE SUPERFAMILY Rhodanese is widely distributed in eubacteria, archae and eukarya. Analysis of sequences highlights that they are highly heterogenous despite conservation of rhodanese signatures [58]. Genome analysis of Pseudomonas aeruginosa reveals the presence of 10 rhodanese-like proteins belonging to each of the rhodanese groups proposed below [19]. Group I - Single Domain Proteins This family includes those proteins that contain either a catalytic or an inactive rhodanese domain. e.g. E. coli GlpE, contains a single 108 amino acid rhodanese domain

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Chaudhary and Gupta

Fig. (2). Partial sequence alignment of the rhodanese domains from representative members of the rhodanese superfamily: Rhobov (P00586), E. coli SseA (P31142), A. vinelandii RhdA (P52197), P. aeruginosa RhdA (Q9HUK9), human Cdc25A (P30304), E. coli GlpE (P0A6V5), W. succinogenes Sud (Q56748), and E. coli ThiI (P77718). Rhodanese signatures are highlighted in grey. Residues forming the six-amino acid active-site loop of the catalytic domain are underlined. Amino acid sequences were retrieved from Swiss-Prot/TrEMBL database (www.expasy.org/sprot/) and aligned with the program CLUSTAL_W with default parameters. Identical residues, conserved and semiconserved substitutions are indicated by asterisks, colons, and periods, respectively. Note that translation of Rhobov reading frame differs from chemically determined amino acid sequence by an N-terminal Methionine and the three residue C-terminal extension Gly-Lys-Ala (not shown in the figure) [14].

demonstrating that a single domain can be self sufficient for thiocyanate formation from thiosulphate in presence of cyanide [59]. Single catalytic rhodanese domain proteins are encoded by both eukaryotic and prokaryotic genomes. These proteins have been associated with stress conditions as HSP 67-B2 in Drosophila melanogaster [60], shock protein Q9KN65 in Vibrio cholera [61], leaf senescence in plants as

Sen1 in Arabidopsis thaliana [62], Ntdin in Nicotianatabacum, sulphur oxidation as P15 from bacterium Acidithiobacillus ferrooxidans [63], Ygr203w protein in Saccharomyces cerevisiae [64]. A hyperthermophile bacterium, Aquifex aeolicus is also characterized by the presence of single domain rhodanese [65].

Cyanide Detoxifying Enzyme: Rhodanese

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331

vinelandii, Pseudomonas aeruginosa, Arabidopsis thaliana, Leishmania major and E. coli [37]. Group III – Multidomain Proteins In these the catalytic rhodanese domain is often combined with other characterized protein domains. This family includes ThiI and ThiF/MoeB-related proteins which are involved in metabolism of sulphur containing biomolecules thiamine and molybdoprotein, respectively [70], Human cytosolic MOCS3 protein which contains an Nterminal MoeB-like domain and a C-terminal module displaying similarities with rhodanese [71], E. coli YbbB protein, which consists of rhodanese catalytic domain and second domain containing a P-loop (Walker A) motif [50]. In the proposed ThiI reaction mechanism, a partner desulfurase (IscS) first catalyzes the transfer of sulfur from free cys to the cys catalytic residue of the rhodanese homology domain of ThiI which in turn becomes the sulfur source for thiamine and 4-thiouridine biosynthesis [72]. The rhodanese domain of ThiI is also necessary for synthesis of Thiazole moiety of thiamine in Salmonella enterica [73]. Group IV – Elongated Active-Site Loop Proteins

Fig. (3). Three-dimensional structure of Rhobov (1RHD), A. vinelandii RhdA (1E0C) and P. aeruginosa RhdA. The three dimensional structure of Rhobov and A. vinelandii RhdA were recovered from the Protein Data Bank (www.rcsb.org). Homology modelling of P. aeruginosa RhdA has been obtained using A. vinelandii RhdA as the template and the SWISS-MODEL facility (http://swissmodel.expasy.org/ SWISS-MODEL.html) [14].

Proteins displaying only the inactive domain, i.e., lacking the active cys residue, are less common and restricted to the eukaryotic dual specific MAPK-phosphatases, yeast (Ubp4, Ubp7, Ubp8) and mammalian (Ubp-Y) ubiquitin hydrolases [66,67]. The only reported case of a single domain endowed with different activity from TST is the polysufide: cyanide ST Sud from eubacterium Wolinella succinogenes, a periplasmic protein which uses the same cys residue not only for catalyzing the sulfur transfer but only for additional hydrolase activity [68]. Group II – Tandem Domain Proteins This group contains both domains but utilizes 3mercaptopyruvate as specific sulphur donor to catalyze sulphur transfer reaction. Transfer of sulphur atom seems to occur via a single displacement mechanism without formation of stable persulphide intermediate [69]. The main differences between thiosulphate sulphurtransferase (TST) and mercaptopyruvate sulphurtransferase (MST) occur at the level of active site loop amino acid sequences. The amino acid properties of active site loop of known TST and MST correlates with the distinct ionic charge of their substrates [58]. These proteins have been characterized from different eukaryotic and prokaryotic sources including Azotobacter

This family of proteins contains an elongated stretch of rhodanese active-site loop. It includes Cdc25A phosphatases that are involved in dephosphorylation of cyclin-CDK complexes for the progression of the cell cycle. In this case, the catalytic cys residue directly attacks the phosphate atom of the phosphorylated cyclin-CDK complex forming phosphocysteine intermediate in the rhodanese homology [74]. Human Cdc25A catalytic domain shows structural similarity with each half of tandem domain rhodanese [58,67]. Other proteins present with similar amino acid sequence are CDC25 protein from Arabidopsis thaliana, Acr2 protein from Saccharomyces cerevisiae, YnjE from Escherichia coli [75] and Yg4E protein. ACTIVE SITE The active site of rhodanese consists of sulfhydryl and aromatic groups in close proximity. The strongest evidence supporting this fact is the protection of the essential enzymic sulfhydryl group by aromatic inhibitors competitive with thiosulfate. The inactivation of rhodanese with a small molar excess of hydrogen peroxide that relies on modification limited to the active site, consisting of the oxidation of the essential sulfhydryl to sulfenyl group (-S-OH) also supports this [76]. The catalytic site of rhodanese is located in the bottom of the crevice formed by the two domains of the enzyme [77]. The complete loss of enzymic activity on reaction with alkylating agents, aromatic nitro compounds or aliphatic mercaptans indicate the importance of sulfhydryl groups for catalysis. Three different types of reagents inactivate rhodanese by their effects on one of the two sufhydryl groups of the rhodanese monomer (Fig. 4). The aromatic nitro compounds inhibit by causing intermolecular oxidation between two enzyme monomers; the mercaptans, by forming mixed disulfides with enzyme monomer; the alkylating agents, by direct substitution on the enzyme cysteinyl sulfur [78]. The presence of tryptophanyl residue at active center of rhodanese has also been suggested. The

332 Current Biotechnology, 2012, Volume 1, No. 4

inhibitor action of aromatic sulfonates is correlated with effectiveness as an electron acceptor. This behaviour is consistent with inhibitor action by complex formation with tryptophanyl residue in the active site. Rhodanese catalysed thiocyanate formation is inhibited by aromatic ions but not by aliphatic ions because aromatic thiosulfonates have higher inverse Michaelis constant values than aliphatic thiosulfonates. Rhodanese inactivation by number of thiol reagents provided main evidence for the need of free thiol group in the active site of the enzyme. The active form of rhodanese contains four cysteine residues (63, 247, 254, and 263) that are all reduced. The cysteine 247 is at the active site of rhodanese and it forms a persulfide linkage with the sulphur transferred from thiosulfate [79]. Cationic group at active site is also necessary for maximal activity [80]. The presence of strong apolar environment in the active site has been suggested by the need of intact tryptophan residue [81].

Chaudhary and Gupta

thiocyanate ion, there by regenerating the free enzyme [56, 84]. The enzyme functions through ping pong mechanism (Fig. 5). SSO32-

E

SCN-

(Thiosulphate)

(Rhodanese)

(Thiocyanate)

(E.SCN-) ***

(E.SSO32-) *

SO32-

ES **

(Sulphite)

CN(Cyanide ion)

Fig. (5). Mechanism of action of Rhodanese. * Rhodanese thiosulphate complex, ** Rhodanese sulphate complex, *** Rhodanese thiocyanate complex.

ASSAY METHODS FOR RHODANESE

Fig. (4). Loss of enzymic sulfhydryl groups on inactivation by mdinitrobenzene,
-mercaptoethanol, and iodoacetate. The symbols refer to the following treatments: , dinitrobenzene; , mercaptoethanol; , iodoacetate; , treatment with dinitrobenzene to 6.4% of initial activity followed by reactivation with Na2S2O3; , treatment with mercaptoethanol to 40% of the initial activity and reactivation with Na2S2O3. The curve was drawn on the assumption that one intact -SH per rhodanese monomer was essential for activity. The percentage enzyme activity was calculated based on a specific activity of 260 units per mg of protein for pure native rhodanese (4, 5). The abscissa represents the difference in the number of -SH groups per monomer between the native and the modified enzyme [81].

MECHANISM OF ACTION Rhodanese catalyzes the reaction via double displacement mechanism involving the formation of persulphide containing intermediate (Rhod-S), in which the transferring sulphur is bound to catalytic cys residue [58]. The mechanism involves binding of thiosulphate to a metal ion in the enzyme. This causes weakening of S-S bond making it more susceptible to attack by strong enzymic nucleophile [82]. The enzyme substrate (E-S) complex differing in reactivity depending on the nature of sulphur donor substrate [83] is formed by discharging sulphite ion from enzyme-thiosulphate complex. The acceptor substrate, cyanide ion then combines with E-S intermediate to form

Various methods for rhodanese assay have been reported. The most common of all is colorimetric measurement of formation of thiocyanate from cyanide and thiosulphate, which is reacted with ferric nitrate reagent to form red-brown acidic iron-thiocyanate complex. This complex is then estimated colorimetrically at 460 nm [85-87]. This method is suitable for measuring rhodanese activity of any degree of purity. This method is not applicable for plant homogenate [88] and microorganisms because thiocyanate production occurred by both enzymatic and non-enzymatic method. As a result a new and improved assay for rhodanese in Thiobacillus sp. was given [89]. This method was devised to determine the contributions of each of these reactions to total thiocyanate production. Determination of rhodanese activity in soil samples involves incubation of buffered substrates and toluene [90]. In addition to this, another method for determination of enzyme in polyacrylamide gel after electrophoresis was also developed [91]. Another method of rhodanese determination using Ion-Selective membrane electrodes has been reported [92]. This method uses cyanide-sensitive membrane electrodes to monitor rates of enzyme catalyzed reactions under controlled conditions. Data on electrode parameters and experimental conditions are then critically evaluated for analytical purposes. This method is more rapid and convenient but less sensitive when compared to colorimetric method. Determination of rhodanese activity by NMR has also been reported [93]. This method is suitable for studying enzymic reactions which result in either major chemical change or deuteration of substrate as it detects changes in the chemical environment of all 1H nuclei present in the reaction mixture. Rhodanese from Thiobacillus A2 was shown by NMR spectroscopy to use dihydrolipoate or dihydrolipoamide as acceptor of sulphane moiety from thiosulphate with the production of
-lipoate and lipoamide respectively. A simple method based on the differential colour formation profile was reported [94]. Positive reaction was indicated by brownish-red colour, weakly positive by pale orange colour and negative result by honey yellow or faint yellow. A method for assay of rhodanese activity using

Cyanide Detoxifying Enzyme: Rhodanese

pH-STAT apparatus was also reported [95]. A colorimetric method based on determination of sulphite was also devised [96]. CONCLUSION Rhodanese catalyzes the transfer of sulphanesulphur from a donor molecule such as thiosulphate to a nucleophilic acceptor such as cyanide involved in some cellular processes (e.g., biosynthesis of cofactors and thionucleosides) and in environmental adaptation (e.g., cyanide detoxification). The increasing number of novel rhodanese-like proteins with diverse and highly specified functions supports the notion that rhodaneses are involved in different cellular processes and coexist within the same organism since they accomplish essential cell functions. The availability of large-scale genomic databases holds out the prospect of discovering novel rhodanese classes which can infer an even wider range of functions than those presently known.

Current Biotechnology, 2012, Volume 1, No. 4 [15]

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CONFLICT OF INTEREST The authors declare no conflict of interest.

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ACKNOWLEDGEMENTS The financial grant in the form of a Research Fellowship (DBT) given by Department of Biotechnology, Ministry of Science and Technology, Govt. of India to Mayank Chaudhary and to Department of Biotechnology, H.P. University, Shimla (India) is thankfully acknowledged. REFERENCES [1] [2] [3] [4]

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Revised: August 23, 2012

Accepted: August 23, 2012