Reaction of rhodanese with dithiothreitol

Pecci, Laura; Pensa, Bernardo; Costa, Mara; Cignini, P. L.; Cannella, C.

doi:10.1016/0005-2744(76)90163-7

Cited by 25 publications

(10 citation statements)

References 14 publications

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“…In general, a higher k cat resulted in an increase of K m for thiosulfate and a decrease in K m for dithiothreitol (Table 1). It has been reported before that dithiothreitol can inactivate the bovine liver rhodanese at certain dithiothreitol/enzyme ratios [15,17]. Here, the inactivated rhodanese was completely reactivated in the presence of high amounts of thiosulfate [17].…”

Section: Resultsmentioning

confidence: 98%

“…In vitro , thiosulfate sulfurtransferase activity can be measured as described by Pecci et al . [15], using reduced dithiothreitol for cleavage of the persulfide, releasing sulfite, hydrogen sulfide and oxidized dithiothreitol. In addition, the method described by Sörbo [16] can be used, applying thiosulfate as sulfur donor and cyanide as sulfur acceptor, yielding thiocyanate (rhodanide) and sulfite.…”

Section: Resultsmentioning

confidence: 99%

“…Dithiothreitol:thiosulfate oxidoreductase activities of MOCS3‐RLD and variants were measured by the method of Pecci et al . [15] as rate of dithiothreitol oxidation. Reaction mixtures contained 50 m m Tris (pH 8.0), 50 m m sodium thiosulfate and 50 m m dithiothreitol (reduced) in a volume of 1 mL.…”

Section: Methodsmentioning

confidence: 99%

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Site‐directed mutagenesis of the active site loop of the rhodanese‐like domain of the human molybdopterin synthase sulfurase MOCS3

Krepinsky

Leimkühler

2007

The FEBS Journal

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Sequence alignments of human molybdopterin synthase sulfurase, MOCS3, showed that the N‐terminal domain is homologous to Escherichia coli MoeB, whereas the C‐terminal domain is homologous to rhodanese‐like proteins. Previous studies showed that the activity of the separately purified rhodanese‐like domain of MOCS3 displayed 1000‐fold lower activity in comparison to bovine rhodanese with thiosulfate as sulfur source. When the six amino acid active site loop of MOCS3 rhodanese‐like domain was exchanged with the loop found in bovine rhodanese, thiosulfate:cyanide sulfurtransferase activity was increased 165‐fold. Site‐directed mutagenesis of each individual residue of the active site loop of the MOCS3 rhodanese‐like domain showed that the charge of the last amino acid determines thiosulfate sulfurtransferase activity. Replacing Asp417 by threonine resulted in 90‐fold increased activity, whereas replacing it by arginine increased the activity 470‐fold. Using a fully defined in vitro system containing precursor Z, MOCS2A, E. coli MoaE, E. coli MoeB, Mg‐ATP, MOCS3 rhodanese‐like domain, and thiosulfate, it was shown that sulfur transfer to MOCS2A was also affected by the alterations, but not as drastically. Our studies revealed that in humans and most eukaryotes thiosulfate is not the physiologic sulfur donor for MOCS3, whereas in bacterial homologs, which have an arginine at the last position of the active site loop, thiosulfate can be used as a sulfur source for molybdenum cofactor biosynthesis. The phylogenetic analysis of MoeB homologs showed that eukaryotic homologs are of bacterial origin. Furthermore, it could be shown that an MoeB homolog named MoeZ, where the dual CXXC zinc‐binding motif of the MoeB domain is not present, arose independently several times during evolution.

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Section: Resultsmentioning

confidence: 98%

Section: Resultsmentioning

confidence: 99%

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Site‐directed mutagenesis of the active site loop of the rhodanese‐like domain of the human molybdopterin synthase sulfurase MOCS3

Krepinsky

Leimkühler

2007

The FEBS Journal

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“…It has been shown that reduced dithiols such as dithiothreitol (1,53), dihydrolipoate (50), and E. coli thioredoxin 1 (48) a Each cell fractionation procedure was performed at least twice. Reported results are from typical experiments.…”

Section: Resultsmentioning

confidence: 99%

Characterization of a 12-Kilodalton Rhodanese Encoded by glpE of Escherichia coli and Its Interaction with Thioredoxin

et al. 2000

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Rhodaneses catalyze the transfer of the sulfane sulfur from thiosulfate or thiosulfonates to thiophilic acceptors such as cyanide and dithiols. In this work, we define for the first time the gene, and hence the amino acid sequence, of a 12-kDa rhodanese from Escherichia coli. Well-characterized rhodaneses are comprised of two structurally similar ca. 15-kDa domains. Hence, it is thought that duplication of an ancestral rhodanese gene gave rise to the genes that encode the two-domain rhodaneses. The glpE gene, a member of the sn-glycerol 3-phosphate (glp) regulon of E. coli, encodes the 12-kDa rhodanese. As for other characterized rhodaneses, kinetic analysis revealed that catalysis by purified GlpE occurs by way of an enzyme-sulfur intermediate utilizing a double-displacement mechanism requiring an active-site cysteine. The K m s for SSO 3 2؊ and CN ؊ were 78 and 17 mM, respectively. The apparent molecular mass of GlpE under nondenaturing conditions was 22.5 kDa, indicating that GlpE functions as a dimer. GlpE exhibited a k cat of 230 s ؊1 . Thioredoxin 1 from E. coli, a small multifunctional dithiol protein, served as a sulfur acceptor substrate for GlpE with an apparent K m of 34 M when thiosulfate was near its K m , suggesting that thioredoxin 1 or related dithiol proteins could be physiological substrates for sulfurtransferases. The overall degree of amino acid sequence identity between GlpE and the active-site domain of mammalian rhodaneses is limited (ϳ17%). This work is significant because it begins to reveal the variation in amino acid sequences present in the sulfurtransferases. GlpE is the first among the 41 proteins in COG0607 (rhodanese-related sulfurtransferases) of the database Clusters of Orthologous Groups of proteins (http://www.ncbi.nlm.nih.gov/COG/) for which sulfurtransferase activity has been confirmed.Genes of known function belonging to the glp regulon of Escherichia coli encode proteins that are responsible for the metabolism of sn-glycerol 3-phosphate (glycerol-P) and its precursors, glycerol and glycerophosphodiesters (38). The genes comprising this regulon belong to five operons. Transcription of all but the glpEGR operon is negatively regulated by the glp repressor GlpR, a member of the DeoR family of transcriptional regulators (34,65,(69)(70)(71)(72). Operon glpACB, encoding the subunits of the anaerobic glycerol-P dehydrogenase, is located near min 51 of the E. coli genome (38). Divergently transcribed from glpACB is glpTQ. The genes glpT and glpQ encode glycerol-P permease and periplasmic glycerophosphodiesterase, respectively (38). The glpFKX operon, at min 89, encodes glycerol diffusion facilitator, glycerol kinase, and a fructose 1,6-bisphophatase (38; J. L. Donahue, J. L. Bownas, W. G. Niehaus, Jr., and T. J. Larson, unpublished data). The genes glpE and glpG, together with the gene encoding the transcriptional repressor, glpR, form a complex operon at min 77 that is divergently transcribed from glpD (71). The gene glpD encodes the aerobic glycerol-P dehydrogenase (38).Prior to...

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“…Rhodanese is able to produce inorganic sulfide in the presence of its putative biological substrate, thiosulfatc, and of suitable dithiols, such as dihydrolipoate [I 71 or dithiothreitol [18]. This reaction was found to depend on the presence of an iron-sulfur protein (e.g.…”

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confidence: 99%

Enzymic synthesis of the iron‐sulfur cluster of spinach ferredoxin

Pagani¹,

Bonomi²,

Cerletti³

1984

European Journal of Biochemistry

103

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A biologically active spinach ferredoxin was reconstituted from the apoprotein by incubation with catalytic amounts of the sulfurtransferase rhodanese in the presence of thiosulfate, reduced lipoate and ferric ammonium citrate. Analytical and spectroscopical features of the reconstituted ferredoxin were identical to those of the native one; yield of the reconstitution reaction was 80 7". Yields and kinetic parameters of the enzymic and chemical reconstitution were also compared. The higher efficiency of the enzymic system is ascribed to a productive interaction between rhodanese and apoferredoxin favouring the process of cluster build-up and insertion. The physiological relevance of this synthetic activity is discussed.Important progress has been made over the last few years in the field of iron-sulfur proteins. However, little still is known about the assembly of iron-sulfur clusters within the cell and their insertion into the various apoproteins.Attempts to reconstitute several iron-sulfur proteins by chemical methods have been more or less successful [1,2]. Because of the toxic nature of the reagents employed [3] and the non-specific chemical reactions [4], the physiological relevance of such models is doubtful.Evidence was also reported on the possible involvement of sulfurtransferascs, an ubiquitous class of enzymes [5,6], in the biosynthesis of iron-sulfur clusters [7,8]. 3-Mercaptopyruvate sulfurtransferase activity was found in both mitochondria and cytosol, but its involvement in the formation of the iron-sulfur cluster of adrenodoxin requires cysteine transaminase activity which is present almost only in the soluble fraction [7]. Rhodanese (thiosulfate-cyanide sulfurtransferase) activity was specifically found in mitochondrial fraction [9] and in chloroplasts where its activity appears to be related to active sulfur metabolism [lo].Rhodanese can restore chemical and functional properties, which have becn lost as a consequence of the alteration of the iron-sulfur cluster(s), in some iron-sulfur proteins such as mitochondrial succinate dehydrogenase [I 11 and NADH dehydrogenase [I21 as well as in the ferredoxins from either Clostridiumpasteurianum or spinach chloroplasts [I 31. A small amount of sulfur from radioactive thiosulfate was found inserted in the iron-sulfur protein as acid-labile [35S]sulfide [14,15]. The reducing equivalents which are necessary for the reduction of the sulfane sulfur of thiosulfate to sulfide were derived from the oxidation of sulfhydryl groups either on the iron-sulfur protein or on the sulfurtransferase itself [14 -161. In the latter case, a strong inactivation of rhodanese occurred. These results proved that rhodanese exerts a protective action on iron-sulfur proteins but they still do not provide direct evidence for an involvement of the sulfurtransferase in the exn o w synthesis of iron-sulfur clusters.Rhodanese is able to produce inorganic sulfide in the presence of its putative biological substrate, thiosulfatc, and of suitable dithiols, such as dihydrolipoate [I 71 o...

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Reaction of rhodanese with dithiothreitol

Cited by 25 publications

References 14 publications

Site‐directed mutagenesis of the active site loop of the rhodanese‐like domain of the human molybdopterin synthase sulfurase MOCS3

Site‐directed mutagenesis of the active site loop of the rhodanese‐like domain of the human molybdopterin synthase sulfurase MOCS3

Characterization of a 12-Kilodalton Rhodanese Encoded by glpE of Escherichia coli and Its Interaction with Thioredoxin

Enzymic synthesis of the iron‐sulfur cluster of spinach ferredoxin

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