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Paul M. Horowitz - One of the best experts on this subject based on the ideXlab platform.
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the aggregation state of Rhodanese during folding influences the ability of groel to assist reactivation
Journal of Biological Chemistry, 2001Co-Authors: Anusri Mitra Bhattacharyya, Paul M. HorowitzAbstract:Abstract The in vitro folding of Rhodanese involves a competition between formation of properly folded enzyme and off-pathway inactive species. Co-solvents like glycerol or low temperature, e.g. refolding at 10 °C, successfully retard the off-pathway formation of large inactive aggregates, but the process does not yield 100% active enzyme. These data suggest that mis-folded species are formed from early folding intermediates. GroEL can capture early folding intermediates, and it loses the ability to capture and reactivate Rhodanese if the enzyme is allowed first to spontaneously fold for longer times before it is presented to GroEL, a process that leads to the formation of unproductive intermediates. In addition, GroEL cannot reverse large aggregates once they are formed, but it could capture some folding intermediates and activate them, even though they are not capable of forming active enzyme if left to spontaneous refolding. The interaction between GroEL and Rhodanese substantially but not completely inhibits intra-protein inactivation, which is responsible for incomplete activation during unassisted refolding. Thus, GroEL not only decreases aggregation, but it gives the highest reactivation of any method of assistance. The results are interpreted using a previously suggested model based on studies of the spontaneous folding of Rhodanese (Gorovits, B. M., McGee, W. A., and Horowitz, P. M. (1998) Biochim. Biophys. Acta1382, 120–128 and Panda, M., Gorovits, B. M., and Horowitz, P. M. (2000) J. Biol. Chem. 275, 63–70).
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Rhodanese as a thioredoxin oxidase
The International Journal of Biochemistry & Cell Biology, 2000Co-Authors: Dhirendra L Nandi, Paul M. Horowitz, John WestleyAbstract:Abstract A major catalytic difference between the two most common isoforms of bovine liver mitochondrial Rhodanese (thiosulfate: cyanide sulfurtransferase, EC 2.8.1.1) has been observed. Both isoforms were shown to be capable of using reduced thioredoxin as a sulfur-acceptor substrate. However, only the less negative form in common with the recombinant mammalian Rhodanese expressed in E. coli, can also catalyze the direct oxidation of reduced thioredoxin evidently by reactive oxygen species. These activities are understood in terms of the established persulfide structure (R–S–SH) of the covalently substituted Rhodanese in the sulfurtransferase reaction and an analogous sulfenic acid structure (R–S–OH) when the enzyme acts as a thioredoxin oxidase. The observations suggest a role for one Rhodanese isoform in the detoxication of intramitochondrial oxygen free radicals.
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domain separation precedes global unfolding of Rhodanese
Journal of Biological Chemistry, 1999Co-Authors: Toru Shibatani, Gisela Kramer, Boyd Hardesty, Paul M. HorowitzAbstract:Abstract The enzyme Rhodanese was investigated for the conformational transition associated with its urea unfolding. When Rhodanese was treated with 0 or 3 m urea, the activity was not significantly affected. 4.25 m urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6m urea. The 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 m urea equilibrations, and the fluorescence was dramatically increased with 4.25 m urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 m urea equilibration, Rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 m urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact Rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of Rhodanese.
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Rhodanese folding is controlled by the partitioning of its folding intermediates
Biochimica et Biophysica Acta, 1998Co-Authors: Boris M Gorovits, William A Mcgee, Paul M. HorowitzAbstract:Rhodanese is used widely as a model for protein folding, since the enzyme as usually studied refolds poorly unless the process is assisted. Here, the influence of the partitioning of the folding intermediates of bovine Rhodanese on the efficiency of its refolding has been investigated. Metastable intermediates can be formed during unfolding of the enzyme. The stabilities of these intermediates and the native protein with respect to chemical unfolding can be greatly increased by high concentrations of glycerol. The concentration dependence of the protein folding kinetics indicates that associative processes occur during renaturation. It is suggested that, during enzyme refolding, Rhodanese undergoes fast collapse to an intermediate state I' which partitions to at least two other states (I" and I"'). One of these states (I"') is able to refold to the native enzyme, while the other state (I") is in equilibrium with I' and is prone to slow irreversible aggregation. Stabilization of I" against irreversible aggregation by glycerol results in increased yield of the protein refolding and a complex temperature dependence of the protein renaturation. The nature of the I" type intermediate has been investigated. Based on the fact that extensive hydrophobic surfaces are exposed during formation of the intermediates, it is suggested that partial dissociation of the two structural domains of Rhodanese is an early event in unfolding. Interactions of different folding intermediates of Rhodanese with the chaperonin GroEL were investigated, and the results suggest that the more extensively unfolded intermediates bind tighter than those that appear later on the Rhodanese refolding pathway.
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the importance of the n terminal segment for dnaj mediated folding of Rhodanese while bound to ribosomes as peptidyl trna
Journal of Biological Chemistry, 1995Co-Authors: Wieslaw Kudlicki, Gerald A. Merrill, Gisela Kramer, O. W. Odom, Boyd Hardesty, Paul M. HorowitzAbstract:Two lines of evidence indicate the importance of the N-terminal portion of Rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound Rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent Rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type Rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type Rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length Rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of Rhodanese is required for its chaperone-dependent folding on the ribosome.
Boyd Hardesty - One of the best experts on this subject based on the ideXlab platform.
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domain separation precedes global unfolding of Rhodanese
Journal of Biological Chemistry, 1999Co-Authors: Toru Shibatani, Gisela Kramer, Boyd Hardesty, Paul M. HorowitzAbstract:Abstract The enzyme Rhodanese was investigated for the conformational transition associated with its urea unfolding. When Rhodanese was treated with 0 or 3 m urea, the activity was not significantly affected. 4.25 m urea treatment led to a time-dependent loss of activity in 60 min. Rhodanese was completely inactivated within 2 min in 6m urea. The 1,1′-bi(4-anilino)naphthalene-5,5′-disulfonic acid fluorescence intensity was not significantly increased during 0, 3, and 6 m urea equilibrations, and the fluorescence was dramatically increased with 4.25 m urea, indicating that hydrophobic surfaces are exposed. After 0 and 3 m urea equilibration, Rhodanese was not significantly proteolyzed with trypsin. Treatment with 4.25 m urea led to simultaneous formation of major 12-, 15.9-, 17-, and 21.2-kDa fragments, followed by progressive emergence of smaller peptides. The N termini of the 17- and 21.2-kDa bands were those of intact Rhodanese. The N terminus of the 15.9-kDa band starts at the end of the interdomain tether. The 12-kDa band begins with either residue 183 or residue 187. The size and sequence information suggest that the 17- and 15.9-kDa bands correspond to the two domains. The 21.2- and 12-kDa bands appear to be generated through one-site tryptic cleavage. It is concluded that urea disrupts interaction between the two domains, increasing the accessibility of the interdomain tether that can be digested by trypsin. The released domains have increased proteolytic susceptibility and produce smaller peptides, which may represent subdomains of Rhodanese.
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Inhibition of the release factor-dependent termination reaction on ribosomes by DnaJ and the N-terminal peptide of Rhodanese.
Journal of Bacteriology, 1995Co-Authors: Wieslaw Kudlicki, Gerald A. Merrill, Gisela Kramer, O. W. Odom, Boyd HardestyAbstract:A peptide consisting of the 17 N-terminal amino acids of native bovine Rhodanese in combination with the chaperone DnaJ specifically inhibits release factor- and stop codon-dependent hydrolysis of N-formylmethionine from N(formyl)-methionyl-tRNA bound with AUG to salt-washed ribosomes. Neither the peptide nor DnaJ by itself causes this inhibition. The N-terminal peptide and DnaJ both singularly and combined do not affect the peptidyltransferase reaction per se. The total amount of Rhodanese synthesized in the cell-free coupled transcription-translation system is reduced by the peptide, with concomitant accumulation of full-length enzymatically inactive Rhodanese polypeptides on ribosomes. In combination with DnaJ, the N-terminal polypeptide inhibits the termination and release of full-length Rhodanese peptides that have accumulated on Escherichia coli ribosomes during the course of uninhibited coupled transcription-translation in the cell-free system. This inhibition appears to involve release factor 2-mediated termination at the UGA termination codon in the coding sequence for Rhodanese. It is suggested that the N-terminal peptide inhibits the binding of the release factor to ribosomes. These data appear to provide the first report of differential inhibition of the termination reaction on ribosomes without inhibition of the peptidyltransferase reaction and peptide elongation.
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the importance of the n terminal segment for dnaj mediated folding of Rhodanese while bound to ribosomes as peptidyl trna
Journal of Biological Chemistry, 1995Co-Authors: Wieslaw Kudlicki, Gerald A. Merrill, Gisela Kramer, O. W. Odom, Boyd Hardesty, Paul M. HorowitzAbstract:Two lines of evidence indicate the importance of the N-terminal portion of Rhodanese for correct folding of the nascent ribosome-bound polypeptide. A mutant gene lacking the codons for amino acids 1-23 of the wild-type protein is expressed very efficiently by coupled transcription/translation on Escherichia coli ribosomes; however, the mutant protein that is released from the ribosomes is enzymatically inactive. The mutant protein does not undergo the reaction that is promoted by the bacterial chaperone, DnaJ, which appears to be essential for folding of ribosome-bound Rhodanese into the native conformation. The effect of DnaJ is monitored by fluorescence from coumarin cotranslationally incorporated at the N terminus of nascent Rhodanese. Secondly, a synthetic peptide corresponding to the N-terminal 17 amino acids of the wild-type protein interferes with the synthesis of wild-type Rhodanese but has much less effect on the synthesis of the N-terminal deletion mutant. The N-terminal peptide inhibits the effect of DnaJ on the nascent wild-type Rhodanese and blocks the chaperone-mediated release and activation of ribosome-bound full-length Rhodanese polypeptides that accumulate during in vitro synthesis. The results lead to the hypothesis that the N-terminal segment of Rhodanese is required for its chaperone-dependent folding on the ribosome.
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chaperone dependent folding and activation of ribosome bound nascent Rhodanese analysis by fluorescence
Journal of Molecular Biology, 1994Co-Authors: Wieslaw Kudlicki, Gisela Kramer, O. W. Odom, Boyd HardestyAbstract:Fluorescently labeled Rhodanese was synthesized by coupled transcription/translation in a cell-free Escherichia coli system. A derivative of coumarin was co-translationally incorporated at the N terminus of the polypeptide. Molecules released from the ribosomes during the incubation are enzymatically active; however, continued incubation results in accumulation of enzymatically inactive full-length Rhodanese polypeptides on the ribosomes. These can be activated and released in the presence of the added chaperones, DnaJ, DnaK, GrpE, GroEL, GroES and ATP. Fluorescence parameters (quantum yield, anisotropy and the emission maximum) of ribosome-bound coumarin-labeled Rhodanese are affected differentially by addition of the chaperones individually or sequentially. Rhodanese released from the ribosomes in the presence of all chaperones (enzymatically active) differs in fluorescence properties from Rhodanese released by GroES or DnaK only or by puromycin (enzymatically inactive) indicating a difference in conformation. Using sparsomycin, an inhibitor of the peptidyl transferase reaction, full-length Rhodanese can be trapped on the ribosomes. A ribosome-bound intermediate formed by DnaJ or DnaJ plus DnaK was demonstrated by the effect of these chaperones on fluorescence spectra resulting from binding of anticoumarin antibodies to the N terminus of newly synthesized Rhodanese. The results support the hypothesis that folding of nascent proteins can take place on the ribosome.
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activation and release of enzymatically inactive full length Rhodanese that is bound to ribosomes as peptidyl trna
Journal of Biological Chemistry, 1994Co-Authors: Wieslaw Kudlicki, Gisela Kramer, O. W. Odom, Boyd HardestyAbstract:Synthesis of Rhodanese in a cell-free coupled transcription/translation system derived from Escherichia coli leads to an accumulation of full-length Rhodanese protein on the ribosomes as well as to enzymatically active protein that is released from the ribosomes into the supernatant fraction. The ribosome-bound protein is enzymatically inactive but can be activated and released from the ribosomes without additional protein synthesis by subsequent incubation in the presence of the added chaperones DnaJ, DnaK, GrpE, GroEL, and GroES plus ATP. Efficient activation requires that all of the chaperones are present together during incubation which yields fully active Rhodanese. Incubation in the presence of DnaJ only inhibits release whereas incubation with only GroES or DnaK promotes the release of enzymatically inactive protein. Incubation of the ribosome with puromycin leads to the release of enzymatically inactive protein whereas release and activation in the presence of all of the chaperones is blocked by sparsomycin. The effect of these antibiotics provides very strong evidence that enzymatically inactive, full-length Rhodanese is bound to the ribosomes as peptidyl-tRNA and that the peptidyl transferase reaction is required for its release. Considered together, the data indicate that chaperone-mediated late stages of Rhodanese folding into the enzymatically active, native conformation are intimately associated with the process of termination and release that occurs as part of the reaction cycle of protein synthesis.
Silvia Pagani - One of the best experts on this subject based on the ideXlab platform.
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mobilization of sulfane sulfur from cysteine desulfurases to the azotobacter vinelandii sulfurtransferase rhda
Amino Acids, 2011Co-Authors: Francesca Cartini, Silvia Pagani, Jutta Papenbrock, William Remelli, Patricia Dos C Santos, Fabio ForlaniAbstract:Mobilization of the L-cysteine sulfur for the persulfuration of the Rhodanese of Azotobacter vinelandii, RhdA, can be mediated by the A. vinelandii cysteine desulfurases, IscS and NifS. The amount of cysteine was higher in mutant strains lacking rhdA (MV474) than in wild type. The diazotrophic growth of MV474 was impaired. Taking into account the functional results about Rhodanese-like proteins and RhdA itself, it is suggested that RhdA-dependent modulation of L-cysteine levels must deal with a redox-related process.
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the lack of Rhodanese rhda affects the sensitivity of azotobacter vinelandii to oxidative events
Biochemical Journal, 2009Co-Authors: Angelo Cereda, Aristodemo Carpen, Gianluca Picariello, Gabriella Tedeschi, Silvia PaganiAbstract:The rhdA gene of Azotobacter vinelandii codes for RhdA, a Rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the Rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous Rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe–S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe–S clusters, in the present study we analysed the defects of the rhdA -null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe–S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner–Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe–4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys 230 thiol.
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the lack of Rhodanese rhda affects the sensitivity of azotobacter vinelandii to oxidative events
Biochemical Journal, 2009Co-Authors: Angelo Cereda, Aristodemo Carpen, Gianluca Picariello, Gabriella Tedeschi, Silvia PaganiAbstract:The rhdA gene of Azotobacter vinelandii codes for RhdA, a Rhodanese-domain protein with an active-site loop structure which has not currently been found in proteins of the Rhodanese-homology superfamily. Considering the lack of information on the functional role of the ubiquitous Rhodaneses, in the present study we examined the in vivo functions of RhdA by using an A. vinelandii mutant strain (MV474), in which the rhdA gene was disrupted by deletion. Preliminary phenotypic characterization of the rhdA mutant suggested that RhdA could exert protection over Fe–S enzymes, which are easy targets for oxidative damage. To highlight the role of RhdA in preserving sensitive Fe–S clusters, in the present study we analysed the defects of the rhdA -null strain by exploiting growth conditions which resulted in enhancing the catalytic deficiency of enzymes with vulnerable Fe–S clusters. We found that a lack of RhdA impaired A. vinelandii growth in the presence of gluconate, a carbon source that activates the Entner–Doudoroff pathway in which the first enzyme, 6-phosphogluconate dehydratase, employs a 4Fe–4S cluster as an active-site catalyst. By combining proteomics, enzymatic profiles and model systems to generate oxidative stress, evidence is provided that to rescue the effects of a lack of RhdA, A. vinelandii needed to activate defensive activities against oxidative damage. The possible functionality of RhdA as a redox switch which helps A. vinelandii in maintaining the cellular redox balance was investigated by using an in vitro model system that demonstrated reversible chemical modifications in the highly reactive RhdA Cys230 thiol. Abbreviations: AhpC, alkyl hydroperoxide reductase; 2-DE, two-dimensional PAGE; DTT, dithiothreitol; ED, Entner–Doudoroff; MALDI-TOF, matrix-assisted laser-desorption ionization–time-of-flight; PHB, polyhydroxybutyrate; PMS, phenazine methosulfate; TST, thiosulfate–cyanide sulfurtransferase; %V, relative volume
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The "Rhodanese" Fold and Catalytic Mechanism of 3-Mercaptopyruvate Sulfurtransferases: Crystal Structure of SseA from Escherichia coli
Journal of Molecular Biology, 2004Co-Authors: Andrea Spallarossa, Aristodemo Carpen, Fabio Forlani, Silvia Pagani, Martino Bolognesi, Andrea Armirotti, Domenico BordoAbstract:3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the Rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic Rhodaneses. In particular, conformational variation of the Rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.
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the crystal structure of a sulfurtransferase from azotobacter vinelandii highlights the evolutionary relationship between the Rhodanese and phosphatase enzyme families
Journal of Molecular Biology, 2000Co-Authors: Domenico Bordo, Aristodemo Carpen, Silvia Pagani, Rita Colnaghi, Daniela Deriu, Martino BolognesiAbstract:Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a Rhodanese. The crystal structure of the A. vinelandii Rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine Rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii Rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii Rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.
Domenico Bordo - One of the best experts on this subject based on the ideXlab platform.
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The "Rhodanese" Fold and Catalytic Mechanism of 3-Mercaptopyruvate Sulfurtransferases: Crystal Structure of SseA from Escherichia coli
Journal of Molecular Biology, 2004Co-Authors: Andrea Spallarossa, Aristodemo Carpen, Fabio Forlani, Silvia Pagani, Martino Bolognesi, Andrea Armirotti, Domenico BordoAbstract:3-Mercaptopyruvate sulfurtransferases (MSTs) catalyze, in vitro, the transfer of a sulfur atom from substrate to cyanide, yielding pyruvate and thiocyanate as products. They display clear structural homology with the protein fold observed in the Rhodanese sulfurtransferase family, composed of two structurally related domains. The role of MSTs in vivo, as well as their detailed molecular mechanisms of action have been little investigated. Here, we report the crystal structure of SseA, a MST from Escherichia coli, which is the first MST three-dimensional structure disclosed to date. SseA displays specific structural differences relative to eukaryotic and prokaryotic Rhodaneses. In particular, conformational variation of the Rhodanese active site loop, hosting the family invariant catalytic Cys residue, may support a new sulfur transfer mechanism involving Cys237 as the nucleophilic species and His66, Arg102 and Asp262 as residues assisting catalysis.
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the Rhodanese cdc25 phosphatase superfamily sequence structure function relations
EMBO Reports, 2002Co-Authors: Domenico Bordo, Peer BorkAbstract:Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla. They are found as tandem repeats, with the C-terminal domain hosting the properly structured active-site Cys residue, as single domain proteins or in combination with distinct protein domains. An increasing number of reports indicate that Rhodanese modules are versatile sulfur carriers that have adapted their function to fulfill the need for reactive sulfane sulfur in distinct metabolic and regulatory pathways. Recent investigations have shown that Rhodanese domains are also structurally related to the catalytic subunit of Cdc25 phosphatase enzymes and that the two enzyme families are likely to share a common evolutionary origin. In this review, the Rhodanese/Cdc25 phosphatase superfamily is analyzed. Although the identification of their biological substrates has thus far proven elusive, the emerging picture points to a role for the amino-acid composition of the active-site loop in substrate recognition/specificity. Furthermore, the frequently observed association of catalytically inactive Rhodanese modules with other protein domains suggests a distinct regulatory role for these inactive domains, possibly in connection with signaling.
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escherichia coli glpe is a prototype sulfurtransferase for the single domain Rhodanese homology superfamily
Structure, 2001Co-Authors: Andrea Spallarossa, Martino Bolognesi, Janet L. Donahue, Timothy J. Larson, Domenico BordoAbstract:Abstract Background: Rhodanese domains are structural modules occurring in the three major evolutionary phyla. They are found as single-domain proteins, as tandemly repeated modules in which the C-terminal domain only bears the properly structured active site, or as members of multidomain proteins. Although in vitro assays show sulfurtransferase or phosphatase activity associated with Rhodanese or Rhodanese-like domains, specific biological roles for most members of this homology superfamily have not been established. Results: Eight ORFs coding for proteins consisting of (or containing) a Rhodanese domain bearing the potentially catalytic Cys have been identified in the Escherichia coli K-12 genome. One of these codes for the 12-kDa protein GlpE, a member of the sn -glycerol 3-phosphate ( glp ) regulon. The crystal structure of GlpE, reported here at 1.06 A resolution, displays α/β topology based on five β strands and five α helices. The GlpE catalytic Cys residue is persulfurated and enclosed in a structurally conserved 5-residue loop in a region of positive electrostatic field. Conclusions: Relative to the two-domain Rhodanese enzymes of known three-dimensional structure, GlpE displays substantial shortening of loops connecting α helices and β sheets, resulting in radical conformational changes surrounding the active site. As a consequence, GlpE is structurally more similar to Cdc25 phosphatases than to bovine or Azotobacter vinelandii Rhodaneses. Sequence searches through completed genomes indicate that GlpE can be considered to be the prototype structure for the ubiquitous single-domain Rhodanese module.
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the crystal structure of a sulfurtransferase from azotobacter vinelandii highlights the evolutionary relationship between the Rhodanese and phosphatase enzyme families
Journal of Molecular Biology, 2000Co-Authors: Domenico Bordo, Aristodemo Carpen, Silvia Pagani, Rita Colnaghi, Daniela Deriu, Martino BolognesiAbstract:Rhodanese is an ubiquitous enzyme that in vitro catalyses the transfer of a sulfur atom from suitable donors to nucleophilic acceptors by way of a double displacement mechanism. During the catalytic process the enzyme cycles between a sulfur-free and a persulfide-containing form, via formation of a persulfide linkage to a catalytic Cys residue. In the nitrogen-fixing bacteria Azotobacter vinelandii the rhdA gene has been identified and the encoded protein functionally characterized as a Rhodanese. The crystal structure of the A. vinelandii Rhodanese has been determined and refined at 1.8 A resolution in the sulfur-free and persulfide-containing forms. Conservation of the overall three-dimensional fold of bovine Rhodanese is observed, with substantial modifications of the protein structure in the proximity of the catalytic residue Cys230. Remarkably, the native enzyme is found as the Cys230-persulfide form; in the sulfur-free state the catalytic Cys residue adopts two alternate conformations, reflected by perturbation of the neighboring active-site residues, which is associated with a partly reversible loss of thiosulfate:cyanide sulfurtransferase activity. The catalytic mechanism of A. vinelandii Rhodanese relies primarily on the main-chain conformation of the 230 to 235 active-site loop and on a surrounding strong positive electrostatic field. Substrate recognition is based on residues which are entirely different in the prokaryotic and eukaryotic enzymes. The active-site loop of A. vinelandii Rhodanese displays striking structural similarity to the active-site loop of the similarly folded catalytic domain of dual specific phosphatase Cdc25, suggesting a common evolutionary origin of the two enzyme families.
Changlin Tian - One of the best experts on this subject based on the ideXlab platform.
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fast conformational exchange between the sulfur free and persulfide bound Rhodanese domain of e coli ygap
Biochemical and Biophysical Research Communications, 2014Co-Authors: Wei Wang, Peng Zhou, Ying Xiong, Changlin TianAbstract:Abstract Rhodanese domains are abundant structural modules that catalyze the transfer of a sulfur atom from thiolsulfates to cyanide via formation of a covalent persulfide intermediate that is bound to an essential conserved cysteine residue. In this study, the three-dimensional structure of the Rhodanese domain of YgaP from Escherichia coli was determined using solution NMR. A typical Rhodanese domain fold was observed, as expected from the high homology with the catalytic domain of other sulfur transferases. The initial sulfur-transfer step and formation of the Rhodanese persulfide intermediate were monitored by addition of sodium thiosulfate using two-dimensional 1 H– 15 N correlation spectroscopy. Discrete sharp signals were observed upon substrate addition, indicting fast exchange between sulfur-free and persulfide-intermediate forms. Residues exhibiting pronounced chemical shift changes were mapped to the structure, and included both substrate binding and surrounding residues.