Báo cáo khoa học: A new sulfurtransferase from the hyperthermophilic bacterium Aquifex aeolicus Being single is not so simple when temperature gets high potx

A new sulfurtransferase from the hyperthermophilic bacterium Aquifex aeolicus Being single is not so simple when temperature gets high ´ ` Marie-Cecile Giuliani1, Pascale Tron1, Gisele Leroy1, Corinne Aubert1, Patrick Tauc2 and ´ ` Marie-Therese Giudici-Orticoni1 ´ ´ ´ ´ Laboratoire de Bioenergetique et Ingenierie des Proteines (BIP), IBSM-CNRS, Marseille, France ´ ´ ´ Laboratoire de Biotechnologie et de Pharmacologie Genetique Appliquee – ENS-CACHAN, Cachan, France Keywords Aquifex aeolicus; hyperthermophile; oligomerization; sulfurtranferase; thermostability Correspondence M.-T Giudici-Orticoni, Laboratoire de ´ ´ ´ ´ Bioenergetique et Ingenierie des Proteines, IBSM-CNRS, 31 chemin Joseph Aiguier, 13402 Marseille cedex 20, France Fax: +33 91 16 45 78 Tel: +33 91 16 45 50 E-mail: giudici@ibsm.cnrs-mrs.fr (Received 20 April 2007, revised July 2007, accepted 12 July 2007) Sulfur is a functionally important element of living matter Rhodanese is involved in the enzymatic production of the sulfane sulfur which has been suggested as the biological relevant active sulfur species Rhodanese domains are ubiquitous structural modules occurring in the three major evolutionary phyla We characterized a new single-domain rhodanese with a thiosulfate : cyanide transferase activity, Aq-477 Aq-477 can also use tetrathionate and polysulfide Thermoactivity and thermostability studies show that in solution Aquifex sulfurtranferase exists in equilibrium between monomers, dimers and tetramers, shifting to the tetrameric state in the presence of substrate We show that oligomerization is important for thermostability and thermoactivity This is the first characterization of a sulfurtransferase from a hyperthermophilic bacterium, which moreover presents a tetrameric organization Oligomeric Aq-477 may have been selected in hyperthermophiles because subunit association provides extra stabilization doi:10.1111/j.1742-4658.2007.05985.x Sulfur adds considerable functionality to a wide variety of biomolecules because of its unique properties: its chemical bonds are both easily made and easily broken, and sulfur serves as both an electrophile (e.g in disulfides) and a nucleophile (e.g as thiol) [1–3] For incorporation into biomolecules, sulfur must be reduced and ⁄ or activated, and sulfate or polysulfides are substrates for reductases that are widespread in nature The activated form of sulfur, the ‘sulfane sulfur’ (R-S-SH) was suggested as the biologically relevant active sulfur species in the early 1980s Sulfane sulfur is produced enzymatically with the IscS protein, the SufS protein and rhodanese being the most prominent biocatalysts [3] Rhodaneses (thiosulfate : cyanide sulfurtransferase or TSTs) are widespread enzymes that catalyse the transfer of a sulfane sulfur Thiosulfate is generally used as a substrate for rhodaneses in vitro assays, and cyanide is used as a sulfur acceptor to regenerate the covalent catalytic cysteinyl residue (Eqn 1a,b): SSO2À þ Rho-SH ! SO2À þ Rho-S-SH 3 ð1aÞ Rho-S-SH þ CNÀ ! Rho-SH þ SCNÀ ð1bÞ In addition to cyanide, other thiophilic acceptor compounds are also acceptable [4,5] Despite numerous studies, the physiological role of rhodaneses remains unclear and is still widely debated because the in vivo substrate has not been identified [6–13] The difficulties in establishing the in vivo functions of rhodaneses lie in the multiplicity of rhodanese modules and rhodanese Abbreviations BN, Blu native gel; MST, mercaptopyruvate sulfurtransferase; rec, recombinant; Rho, rhodanese; SR, sulfur reductase; ST, sulfurtransferase; Sud, sulfide dehydrogenase; TST, thiosulfate sulfurtransferase 4572 FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al activities However, the role of persulfide cysteine at the catalytic site has been demonstrated [14–17] A typical feature of the rhodanese superfamily is the modular structure of its various members [18] Rhodaneses or sulfurtransferases are ubiquitous enzymes found in many organisms from all three domains of life Even though the discovery of the hyperthermophiles has important ramifications, not only in microbial physiology and evolution, but also in biotechnology, no rhodanese has been characterized from these extremophilic organisms The prototypic enzyme, bovine liver rhodanese, consists of an N-terminal inactive rhodanese module (the catalytic cysteinyl residue is replaced) and a C-terminal catalytic module, each encompassing about 120 amino acids [19,20] This domain organization is also typical for many rhodanese sequences distributed in all kingdoms These two domains show weak sequence similarity [18] In addition to the two-domain rhodaneses, single-domain versions are known [5,21–23], with Escherichia coli GlpE protein as the prototype [5,16] Characterization of single-domain rhodanese indicates that the N-terminal domain in two-domain rhodaneses is not essential for catalysis Rhodanese modules may also be involved in processes beside sulfur transfer An interesting example is the rhodanese-homologous domain of the E coli YbbB protein, which is responsible for the exchange of sulfur for selenium in 2-thiouridine in vivo [12] In addition, characterization of sulfide dehydrogenase (Sud) from a mesophilic bacterium, Wolinella succinogenes revealed for the first time the direct intervention of rhodanese in energetic sulfur metabolism, because this protein is the sulfur donor for the terminal acceptor of respiratory chain sulfur reductase in W succinogenes Microorganisms with the remarkable property of growing at temperatures near and above 100 °C have been isolated from shallow submarine and deep-sea volcanic environments over the last 20 years The majority of these hyperthermophilic microorganisms are archaea and they are considered to represent the most slowly evolving forms of life [24–26] Numerous hyperthermophilic archaea are known, but very few hyperthermophilic bacteria have been discovered to date Most known hyperthermophilic bacteria are members of the genus Aquifex and have an optimal growth temperature of 85 °C [24,27] Aquifex is a hyperthermophilic, hydrogen-oxidizing, microaerophilic, obligate chemolithoautotrophic bacterium It obtains energy for growth from hydrogen, oxygen and sulfur (thiosulfate or elemental sulfur), and uses the reductive tricarboxylic acid cycle to fix CO2 Stimulated Oligomeric sulfurtransferase from Aquifex aeolicus by the exceptional phylogenetic and physiological properties of A aeolicus, as well as by its potential as a source of extremely stable enzymes, we undertook several studies on the energetic metabolism of this organism, whose genome has been completely sequenced [28] In particular, we studied its hydrogen ⁄ sulfur metabolism [29,30] Two rhodanese-coding genes, rhdA1 and rhdA2 are annotated in the A aeolicus genome Both belong to the two-domain family of rhodaneses Here, we describe the identification, purification and biochemical and biophysical characterization of a new single-domain rhodanese in A aeolicus, which was not identified by annotation of the genome The results shed light on some particularities of the protein which may be linked to the need for extremophiles and their macromolecules to develop molecular mechanisms adapted to extreme physicochemical conditions Its possible metabolic roles in A aeolicus are also discussed Results Evidence for sulfurtransferase (ST) activity in A aeolicus ST activity was routinely measured under an argon atmosphere in an assay mixture containing thiosulfate and cyanide The amount of SCN– produced was representative of the catalysis After centrifugation of the cell extract, ST activity was found to be associated with the soluble fraction, whereas the membrane fraction was inactive After cellular separation, 90% of the activity was present in the cytoplasmic extract and around 10% in the periplasmic extract As no lactate dehydrogenase activity could be detected in the periplamic extract, we propose that the enzymes required for sulfur transfer are present in A aeolicus periplasmic and cytoplasmic spaces We obtained evidence for the presence of sulfurtransferase activity in A aeolicus grown on elemental sulfur or thiosulfate However, extracts of cells grown with elemental sulfur showed a specific sulfurtransferase activity 5.5· the specific activity measured with cells grown with thiosulfate We thus decided to use cells cultured on H2 ⁄ S° medium to work on the ST enzymes Purification of a new ST A protein with ST activity was purified by Q-Sepharose, HTP and Superdex 200 gel-filtration chromatography from the cytoplasmic fraction of A aeolicus grown on H2 ⁄ S° medium (Table 1) At the final step, ST activity was detected in different fractions The FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4573 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al Table Yields and enrichments of the ST activity One unit of ST activity corresponds to the uptake of lmol of SCNỈmin)1 Sulfurtransferase activity Preparation Specific activity (mg)1) Total activity (U) Yield (%) Soluble proteins Q Sepharose (300 mM NaCl) HTP (300 mM phosphate) Superdex 200 134 306 550 850 35,160 7800 3000 1000 22 8.5 2.8 calibration curve shows that these fractions correspond roughly to a molecular mass of 50 to 15 kDa We divided this activity peak into three fractions corresponding to approximately 15, 25 and 50 kDa (Fig 1) N-Terminal sequence determination of these fractions led to the identification of these peaks as containing the same protein A search for this sequence in the Aquifex proteins database reveals one protein, Aq-477 This protein, encoded by aq477, was annotated as a protein of unknown function with a molecular mass of 12 804 Da as deduced from the amino acid sequence Mass spectra of each fraction demonstrated one major protein with molecular mass of 12 810 Da This suggests a possible oligomerization state of the protein in the dimer and tetramer, in line with the masses corresponding to the different activity fractions detected As the protein contains only one mAU ( ) 200 300 5, 5, 5, 4, 4, 4, 4, 4, 100 100 150 200 log (PM) Activity ( ) u/ml 50 1, 1, 2, 2, Ve/Vo 14 15.5 16.9 Elution volume (ml) Fig Size-exclusion chromatography of wild-type Aq-477 An S200 column (1 · 30 cm) was equilibrated in 100 mM Tris ⁄ HCl, 50 mM NaCl, pH 7.6 at 20 °C The protein (200 lL at mgỈmL)1) was detected by its absorbance (d) and its activity (m) (Inset) Calibration curve in 100 mM Tris ⁄ HCl, 50 mM NaCl, pH 7.6, flow rate 0.3 mLỈmin)1, sample volume 200 lL at mgỈmL)1 of rusticyanine (17 kDa); dihemic cytochrome c (21 kDa); ovalbumin (43 kDa); alcohol dehydrogenase (150 kDa) 4574 cysteine residue, Cys69, a disulfide bridge may (or may not) be formed between two monomers MS data demonstrate the absence, in the samples, of one or more possible interdisulfide bridges possibly involved in the oligomerization Aq-477 is a single-domain ST The product of aq477 purified from A aeolicus, is able to transfer sulfur from thiosulfate to cyanide According to the sequence deduced from the gene, no signal peptide is detected, which is in agreement with the purification of the protein from the cytoplasm of A aeolicus aq477 is flanked by genes panD and aq478 Because a single base pair separates the termination and initiation codons for panD and aq477, they appear to be organized as an operon panD encodes an aspartate decarboxylase which catalyses the decarboxilation of aspartate to produce b-alanine, a precursor of coenzyme A aq478 encodes a protein similar to proteins involved in signal transduction mechanisms The product of aq477 was annotated as a hypothetical protein related to a member of COG0607P, which regroup 168 rhodanese-related sulfurtransferases All these homologues belong to a a ⁄ b-fold protein domain found duplicated in the rhodanese proteins Each protein from this family contains at least one cysteine residue, which has been found to be essential for the protein’s function [18] Unlike classical two-domain rhodaneses, Aq-477 is composed of a single-domain rhodanese fold, the catalytic domain as it contains the characteristic catalytic cysteine Few single-domain rhodaneses have been characterized in detail The primary sequence of Aq-477 shows only slight similarity with other single-domain proteins of known 3D structure, i.e 21% homology with GlpE from E coli, 25% with Sud from W succinogenes and 27% with a TTHA0613 ORF from T thermophilus [21,31,32] However, structural alignment (Fig 2) shows the same global fold for all single-domain STs with a typical a ⁄ b topology and the extension and location of the regular secondary structure elements approximately coincide in all these proteins However, some differences are found The major difference is the presence of an extra a helix in Sud protein Moreover, the mesophilic ST (Sud and GlpE) have an insertion between b3 and a4 In addition, in Aq-477, the prediction proposes a shift of b5 through the C-terminus The a6 helix is absent in thermo ⁄ hyperthermophiles enzymes Despite the low overall sequence identity, a few residues are conserved in b strands b2 and b4, which FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al Oligomeric sulfurtransferase from Aquifex aeolicus Fig Structural alignment of Aq-477 from A aeolicus, GlpE from E coli, Sud from W succinogenes and TTHA0613 from T termophilus sequences Secondary structures are indicated, based on the 3D prediction (http://www.compbio.dundee.ac.uk/$www-jpred/) for A aeolicus and from the 3D structure for Glpe, Sud and TTHA0613 Residues involved in the a helix are boxed and those in b sheet are underlined Conserved residues involved in substrate binding are in bold The arrow indicates the active-site cysteine constitute the structural core of the protein In b2, the sequence XDXR (X being hydrophobic residues) is conserved In b4, a set of four hydrophobic residues preceding the position occupied by cysteine is conserved These residues are also conserved in twodomain rhodaneses We can conclude that these residues play an important role in the global folding processes of the mesophilic, themophilic and hyperthermophilic proteins The potential active site is located between a central b strand and a a helix In Aq-477, Cys69 is the first residue of loop 69–74 Moreover, the positive charges (R30, R70, R74) found in the substrate-binding pocket for the negative polysulfide chain are conserved In Sud protein R46 and E50 are involved in substrate binding [31] These residues are conserved in Aq-477 (R30, E34) Various proteins can catalyse sulfur transfer In addition to the two classical two-domain rhodanese proteins, annotated as rhdA1 and rhdA2 in the A aeolicus genome, a fourth gene exhibiting sequence similarity to the rhodaneses family [18] is detected in the genome using the catalytic motif for ST as query for a BLAST search This other protein, Aq-1599, is annotated as a protein of unknown function in the A aeolicus genome We identified it as single domain ST Aq-1599 is predicted to be periplasmic Cloning, heterologous production and purification of recombinant Aq-477 from A aeolicus The aq477 gene encoding A aeolicus ST, was amplified by PCR, inserted into a pET22 expression vector and expressed in E coli BL21-CodonPlus (DE3)-RIPL strain After induction, a high level of protein was detected in the soluble extract Ten milligrams of soluble protein were obtained from L of culture after two purification steps, as described in the Experimental procedures The N-terminal sequence was identical to the native protein purified from A aeolicus The reconstructed mass spectra of recombinant Aq-477 gave only one peak corresponding to a molecular mass of 12 813 Da This is consistent with the molecular mass calculated from the sequence and supports this protein corresponding to the mature enzyme Moreover, it demonstrates the absence of interdisulfide bridges in the enzyme Physicochemical and catalytic properties UV–Vis spectroscopy performed in the oxidized and reduced states showed no signal except at 280 nm, corresponding to the protein absorption Aq-477 does not contain prosthetic groups or heavy metal ions FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4575 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al As the active site of sulfurtransferases involves a cysteine residue, Aq-477 was incubated with a cysteinemodifying reagent, iodoacetamide to verify that Cys69 is required for ST activity All the activity disappeared at a : molar ratio (iodoacetamide ⁄ Aq-477) This demonstrates that Cys69 is: (a) involved in the catalysis, and (b) not involved in disulfide bond formation Several previously characterized rhodaneses are specifically inhibited by anions [33] As seen for GlpE, the only single-domain enzyme tested [5], slight inhibition by anions was observed for Aq-477 Addition of ammonium sulfate or ammonium acetate, at an ionic strength of 300 mm, resulted in 25% inhibition of rhodanese activity Various compounds were tested as sulfur donors Cysteine, dithiothreitol and b-mercaptopyruvate were unable to replace thiosulfate as the sulfur donor These results show that Aq-477 is not a mercaptopyruvate sulfurtransferase Kinetics analysis was carried out with thiosulfate, tetrathionate and polysulfide as sulfur donors In contrast to Sud from W succinogenes or GlpE from E coli, Aq-477 was active with the three substrates All the kinetics show a Michaelis–Menten behaviour and the parameters are summarized in Table Clearly, it appears that polysulfide sulfur was a very efficient sulfur donor, indicating that this sulfur compound is probably the real substrate H2S production was also tested Because of the high unspecific reaction with tetrathionate and polysulfide in the presence of dithiothreitol, the test was performed in presence of NaBH4 instead of KCN as described by Table Steady-state kinetic parameters of Aq-477 from A aeolicus Values were obtained from direct experimental measurements fitted to the Michaelis–Menten equation Apparent Km values for polysulfide refer to polysulfide sulfur concentration nd, no data Activity Vm (s)1) Apparent Km (mM) Thiosulfate rhodanese sulfurtransferase Thiosulfate sulfurtransferase Tetrathionate rhodanese sulfurtransferase Polysulfide sulfur rhodanese sulfurtransferase Polysulfide sulfur sulfurtransferase Thioredoxin sulfurtransferase 3-Mercaptopyruvate rhodanese sulfurtransferase 7865 ± 200 no activity 5.7 ± 0.9 (S2O32–); 2.1 ± 0.37 (CN–) nd 8802 ± 500 6.9 ± (S4O6) 165,000 < 0.05 (Sn2–) 36 nd 72 nd no activity nd 4576 Table Temperature dependence of thiosulfate rhodanese sulfurtransferase and polysulfide sulfur rhodanese sulfurtransferase activities of Aq-477 from A aeolicus nd, no data Temperature (°C) Thiosulfate rhodanese sulfurtransferase activity (s)1) Polysulfide sulfur rhodanese sulfurtransferase activity (s)1) 85 60 37 25 8000 5000 2500 1000 nd 165000 85000 32000 Klimmeck et al [34] To detect H2S production, 100fold more enzymes were needed compared with the kinetics of thiocyanate production, suggesting that this reaction was not physiological It has been shown that reduced E coli thioredoxin serves as a sulfur-acceptor substrate for GlpE from E coli [5] To test thioredoxin as a sulfur acceptor substrate for Aq-477, we adapted the assay method described for GlpE at 50 °C (see Experimental procedures for the basis for this assay) Aq-477 may also be able to utilize dithiol proteins such as thioredoxin as a sulfur acceptor However, owing to the amount and stability of the proteins (thioredoxin and thioredoxin reductase) needed in this test at 50 °C, determination of the kinetic parameters was not possible The purified Aq-477 was active, with thiosulfate or polysulfide as a sulfur donor and cyanide as sulfur acceptor, over a large range of temperatures (25– 80 °C) (Table 3) Aq-477 showed a temperature optimum at 80 °C and only 8% of the activity was observed at 25 °C Aq-477 showed a pH optimum at and no sulfurtransferase activity was observed at pH values < Oligomerization state of Aq-477 Four single-domains ST have been described to date For two of them, i.e GlpE from E coli and Sud from W succinogenes, biochemical characterization has shown a homodimeric organization [5,34] Gel-filtration chromatography was used to determine the apparent molecular mass of recombinant Aq-477 One peak is obtained, corresponding to a mix of the tetrameric, dimeric and monomeric forms as for the wildtype protein (Fig 3, trace A) When each fraction was concentrated and run through the column, the same elution profile was obtained Use of Superose 12 instead of an S200 column did not enable us to obtain a more homogeneous form This indicates that Aq-477 forms soluble oligomers that are in equilibrium FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al Oligomeric sulfurtransferase from Aquifex aeolicus B A 15 Elution volume (ml) Fig Size-exclusion chromatography of recombinant Aq-477 An S200 (1 · 30 cm) column was equilibrated in (A) 100 mM Tris ⁄ HCl, 100 mM NaCl, pH 7.6 or (B) 100 mM Tris ⁄ HCl, 100 mM NaCl, 10 mM Na2S2O3 pH 7.6 at 20 °C Profile A: injection of 50 lL of rec-Aq-477 at 2.6 mgỈmL)1; profile B: injection of 50 lL of recAq-477 at 2.6 mgỈmL)1 in the presence of 10 mM Na2S2O3 The protein was detected by its absorbance at 280 nm with the monomer and the energy barriers for the interconversion are not very high, as judged by the ease of interconversion Electrophoretic migration of rec-Aq-477, under denaturing conditions, shows two bands, at 25 and 12 kDa (Fig 4A, lane 2) After transfer on a poly(vinylidene difluoride) membrane, automated Edman degradation yielded the same N-terminal sequence up to the 10th cycles for the two bands In a similar way, western blotting after SDS ⁄ PAGE of each fraction from the S200 column (1.5 mgỈmL)1) showed two bands at 12 and 25 kDa for the recombinant enzyme (Fig 4A, lane 3) The same pattern was observed in the presence or absence of dithiothreitol (data not shown) confirming the absence of a disulfide bridge These results show that, even under denaturing conditions, the dimeric form was detected This suggests a tight interaction between the two subunits and a possible physiological role for the oligomeric form of Aq-477 With the native enzyme from A aeolicus, two bands are detected by antibodies at 12 and 50 kDa, suggesting a tetrameric organization of the enzyme (Fig 4B, lane 3) When the samples are boiled in the presence of higher SDS concentrations (2%) the higher molecular mass bands tend to disappear (data not shown) Only one band around 50 kDa was detected by western blotting performed after denaturing electrophoresis of soluble crude extract (Fig 4B, lane 2) The same experiment performed with crude extract from A aeolicus cultivated on H2 ⁄ Na2SO3 shows that Aq-477 was less abundant under these growth conditions than with S° as the sulfur electron acceptor (Fig 4B, lane 4) These results demonstrate that: (a) the oligomeric state of the enzyme contains at least four subunits in vivo, and (b) the high stability of the oligomeric state under these experimental conditions as a high SDS concentration was necessary to destabilize the subunit interactions The oligomeric state of Aq-477 influenced by substrate, protein concentration and salt The previous experiments suggest the existence of an equilibrium reaction between monomer, dimer and at least tetramer First, we tested the effect of the sulfur donor on the Aq-477 oligomeric state Gel-filtration chromatography was realized in presence of 10 mm Na2S2O3 or mm polysulfide (Fig 3, trace B) Compared with the same experiment without substrate (Fig 3, trace A) the peak was more homogenous and the molecular mass was 50 kDa This result demonstrates the tetrameric organization of Aq-477 in the presence of substrate For a more defined correlation between the concentration and the different states of oligomerization, a stock solution of mgỈmL)1 wild-type or recombinant Aq-477 was diluted to 0.4 and 0.04 mgỈmL)1 at 25 °C in 50 mm Tris ⁄ HCl, 100 mm NaCl, pH 7.6 and same amount of protein was loaded onto 10–20% Blue native (BN) gel The gel patterns were different because the diluted enzyme (1 ⁄ 100) presents a lower molecular mass than the enzyme without dilution or if diluted at ⁄ 10 (Fig 5, lanes A and B) When the same experiment was carried out in the absence of salt in the protein sample, the monomeric form was observed (Fig 5, lane C) The presence of 100 mm NaCl in the sample induced oligomerization of the enzyme, suggesting hydrophobic interactions between the subunits In the same way, the presence of thiosulfate at 10 mm in the sample stabilizes the oligomeric form (Fig 5, lane D) Time-resolved fluorescence anisotropy experiments confirmed most of the results obtained by size-exclusion chromatography and BN gel Fluorescence anisotropy measurements are based on the depolarization of light that occurs during the rotational diffusion of macromolecules or biological complexes The extent of light FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4577 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al A B 67 80 43 50 40 30 20,1 14,4 30 20 Fig SDS polyacrylamide gel of the purified Aq-477 from A aeolicus (A) SDS polyacrylamide gel of the purified recombinant Aq-477 Lane 1, molecular mass markers (in kDa); lane 2, lg of recombinant Aq-477; lane 3, immunoblotting experiments of recombinant Aq-477, lg of recombinant Aq-477 was loaded onto the gel before detection by immunoblotting using anti-(Aq-477) sera (B) SDS polyacrylamide gel of Aq-477 from A aeolicus Lane 1, molecular mass markers (in kDa); lane 2, immunoblotting experiments of soluble crude extract from A aeolicus cultivated on H2 ⁄ So medium Protein (1 lg) was loaded onto the gel before detection by immunoblotting using anti-(Aq-477) sera Lane 3, immunoblotting experiments of soluble crude extract from A aeolicus cultivated on H2 ⁄ NaS2O3 medium Protein (1 lg) was loaded onto the gel before detection by immunoblotting using anti-(Aq-477) sera Lane 4, immunoblotting experiments of enriched fraction of Aq-477 from A aeolicus Protein (100 ng) was loaded onto the gel before detection by immunoblotting using anti-(Aq-477) sera A B C Table Comparison of lifetime and long rotational correlation times of Aq-477 for different protein concentrations, in the absence or presence of substrate at 25 °C Correlations times were measured by monitoring tryptophan fluorescence (kex ¼ 295 nm; kem ¼ 330 nm) as indicated in the Experimental procedures The normalized values of the correlation times (reference temperature 20 °C) were obtained using the Perrin equation D Aq-477 concentration (lM) 0.4 Fig BN gel of purified recombinant Aq-477 Lane A, 20 lg of Aq-477 at 0.4 mgỈmL)1; lane B, 20 lg of Aq-477 at 0.04 mgỈmL)1; lane C, 20 lg of Aq-477 at 0.4 mgỈmL)1 without salt; lane D, 20 lg of Aq-477 at 0.04 mgỈmL)1+ S2O3 10 mM depolarization between excitation and emission times is then related to the molecular size of the macromolecule Analysis of the time-resolved fluorescence anisotropy data displays the distribution of rotational correlation times (h), which are related to the hydrodynamics volumes Aq-477 contains one tryptophan residue and it was studied using intrinsic tryptophan fluorescence Excitation at 295 nm resulted in an emission spectrum with one maximum at 330 nm In the presence of a saturating concentration of thiosulfate, an increase of 30% was observed without a significant shift of the maximum (data not shown) This behaviour has previously been seen with rhodanese from A vinelandi [35] Between 40 and 0.4 lm, Aq-477 displayed different rotational correlation time (Table 4), confirming that its oligomeric state is strongly dependent upon the protein concentration At low concentrations and 25 °C, the rotational correlation time was ns, which corresponds to a globular protein of around 14 kDa This demonstrates that at low concentrations the major part of the enzyme was 4578 Thiosulfate 10 mM Lifetime (ns) Long rotational correlation time (ns) – 1.66 + 1.41 11 – 1.8 14 40 + 1.57 21 – 1.11 20 + 1.16 25 monomeric The equilibrium was shifted to the dimeric form (14 ns) then to a trimeric or tetrameric form (20 ns) when the protein concentration was increased (Table 3) The same measurements were done with Aq-477 sample freshly prepared in the presence of a saturating concentration of thiosulfate At low protein concentrations, instead of the monomer, the dimeric form was detected with a longer correlation time of 11 ns In a similar way, at higher concentrations, the trimer and tetrameric forms were detected in the presence of substrate Over the concentration range used, there is equilibrium between monomer, dimer, trimer and tetramer Our results show that the initial step in the oligomerization process is the formation of dimers This is in agreement with the result obtained by electrophoresis under denaturing conditions The dimeric form is an active intermediate structural conformation that evolves to at least a tetrameric form Our results FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al Oligomeric sulfurtransferase from Aquifex aeolicus confirm that: (a) a dilution step generates the monomeric form, and (b) the substrate induces the oligomerization state and a more homogenous state were carried out with a monomeric enzyme sample freshly prepared in the presence of 500 lm polysulfide or mm Na2S2O3 The spectrum obtained (Fig 6B) was similar to that of the oligomeric form, indicating a role for the substrate in the stabilization or induction of the more stable form To better understand the stability of the various forms of Aq-477, its activity was measured for the oligomeric and monomeric forms preincubated at different temperatures from to 80 °C Independent of the incubation temperature (4, 25 or 80 °C), the activity of the monomeric form of Aq-477 decreased to 10% with a similar time-course of inactivation over the whole temperature range (Fig 7A) Addition of substrate after 40 or 60 of incubation did not induce any modification in the traces This indicates the irreversibility of the inactivation process The same experiments were carried out with the oligomeric enzyme (Fig 7B) Independent of the temperature incubation used, the activity at 80 °C was stable and 95% activity was still present after 150 The corresponding half-life of irreversible inactivation increased from 19 for the monomeric form to 320 for the oligomeric form As shown previously by fluorescence and CD experiments, deactivation of the Aq-477 monomer is prevented by the presence of a sulfur donor As shown in Fig 7C, addition of or 10 mm S2O3 or 500 lm polysulfide sulfur in a freshly prepared monomeric form, results in $ 80% stabilization of the activity at 80 °C These results demonstrate that: (a) the monomeric form is unstable, (b) monomer inactivation is irreversible, (c) the substrate prevents inactivation, and (d) temperature alone does not induce the active form Stability and activity of Aq-477 Relative CD Intensity at 222 nm (%) We generated monomeric and oligomeric forms of Aq-477 using a dilution step We first verified the global fold of the protein by CD experiments in the far-UV Independent of the degree of polymerization, the enzyme was correctly folded Deconvolution of the spectra gives 28% a helix and 23% b sheet This was in the same range as obtained using various secondary prediction software (around 30% a helix and 23% b sheet) These experiments show the absence of denaturation of the enzyme directly after the dilution step at room temperature As the enzyme presents activity at 80 °C and no activity at 25 °C, it was interesting to study the effect of temperature on the structural stability by CD ellipticity at 222 nm [36] Changes in helical content of the various forms of Aq-477 after thermal treatment are shown in Fig Decrease in ellipticity at 222 nm was observed for the monomeric form of Aq-477 indicating a loss of protein secondary structure at high temperature (Fig 6A, black circles) Compared with the oligomeric form (Fig 6A, black triangles), the monomeric form is less stable with a transition around 60 °C Denaturation of the monomer of Aq-477 appears to be an irreversible process as the initial signal is not recovered at 25 °C after thermal treatment The heat tolerance of the oligomeric Aq-477 indicates a considerable contribution of the oligomerization process to the thermal stability The same experiments A 100 B 100 80 80 60 60 40 40 20 20 30 40 50 60 70 80 90 30 40 50 60 70 80 90 Temperature (°C) Fig Changes in relative CD intensity at 222 nm for monomeric (d) or oligomeric (m) Aq-477 (A) Relative CD intensity at 222 nm at various temperatures for oligomeric or freshly prepared monomeric form of Aq-477 (B) Relative CD intensity at 222 nm for freshly prepared monomeric form of Aq-477 prepared in the presence of mM Na2S2O3 (d) or 500 lM polysulfide (.) The degree of polymerization was controlled by BN gel FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4579 Residual activity (%) Oligomeric sulfurtransferase from Aquifex aeolicus 120 M.-C Giuliani et al 120 A 100 120 B 100 C 100 80 80 80 60 60 60 40 40 40 20 20 20 0 20 40 60 80 100 120 0 50 100 150 200 50 100 150 Time (min) Fig Thermal inactivation of freshly prepared monomer or oligomer of Aq-477 (A) Time course of irreversible inactivation of a freshly prepared monomeric form of Aq-477 at °C (d), 25 °C (.), and 80 °C (j) (B) Time course of irreversible inactivation of a freshly prepared monomeric form of Aq-477 (.) or oligomeric form (d) at 25 °C (C) Time course of irreversible inactivation of a freshly prepared monomeric form of Aq-477 at 25 °C without (d) and in the presence of 500 lM (r) polysulfide sulfur in the buffer.The degree of polymerization was controlled by BN gel The ability of proteins to adopt different quaternary structures is essential for many biological processes such as signal transduction, cell-cycle regulation and enzyme catalysis We tested the impact of the oligomerization state on the kinetic of ST to determine whether oligomerization can promote regulation of the kinetic behaviour Steady-state kinetics were measured with monomeric enzyme and compared with values obtained with oligomeric enzyme Lineweaver–Burk plots indicated that whatever the enzyme forms there was no cooperativity between the different active sites of the oligomeric enzyme However, the apparent Vm was fourfold smaller for the monomeric enzyme than for oligomeric enzyme The K app values were similar (5.48 ± mm) m This is typically observed in the case of an irreversible inactivation of the enzyme as the amount of active enzyme in the test became smaller In conclusion, these results demonstrate: (a) the absence of kinetic regulation, such as cooperativity, in the oligomeric enzyme; and (b) that the oligomeric form is the active form of the enzyme Discussion organization which is controlled and induced by the substrate In recent years, a considerable number of proteins with a rhodanese homology fold have been detected The rhodanese fold was first observed in the crystal structure of bovine mitochondrial rhodanese [19] and later in the crystal structures of TTHA0613 from Thermus thermophilus HB8 and At5g66040.1 from Arabidopsis thaliana [32,37] This domain was found in the Cdc25 class of protein phosphatases and in a variety of proteins such as sulfite dehydrogenase, in certain stress proteins and in cyanide and arsenate resistance proteins [13] Genome sequencing has shown that ORFs coding for rhodanese or the mercaptopyruvate sulfurtransferase (MST) homologue are present in most eubacteria, archaea and eukaryota [38,39] Often, several genes encoding for distinct ‘rhodanese-like’ proteins are found in the same genome, suggesting that the encoded proteins may have distinct biological functions In A aeolicus, two genes encode two multidomain rhodaneses rhdA1 and rhdA2 Besides Aq-477, we have detected only one other ORF that potentially encodes a protein with a rhodanese fold Aq-1599 Characterization of this protein is in progress Aq-477 is a single-domain rhodanese We purified and characterized a protein from A aeolicus annotated as a hypothetical protein In vitro, it catalyses the transfer of sulfane sulfur from thiosulfate to cyanide to form thiocyanate According to this activity and its amino acid sequence, Aq-477 belongs to the rhodanese (or sulfurtransferase) family It is the first single-domain sulfurtransferase to be characterized from hyperthermophilic bacteria Moreover, it is the only one-domain STS to present at least a tetrameric thermoactive and thermostable 4580 Aq-477 is a thermostable and thermoactive tetramer ST Few single-domain rhodaneses have been characterized in detail Resolution of the 3D structure of Sud, sulfurtransferase from W succinogenes, shows a dimeric organization [28] and this enzyme probably functions as a dimer in solution GlpE was also described as a dimeric enzyme but the 3D structure did not confirm this [7] Dimerization in Sud occurs via the a1 helix which is absent in Aq-477 and GlpE The two last FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al structures solved were those of T thermophilus and Ar thaliana rhodaneses [29,33] These two enzymes were monomeric However, no data are available on their organization in solution, active form or physiological role Dimerization of RhdA from A vinelandii has also been shown, but only when mutations were introduced into the catalytic loop inducing an interdisulfide bridge [16] or when the enzyme was considerably overexpressed [17] However, in all cases, the active enzyme was the monomeric form [16,17] Our results on Aq-477 from A aeolicus show that this enzyme exists at least as a monomer, dimer and tetramer at 25 °C Western blotting on crude extracts revealed one major band around 50 kDa, suggesting that the active form in vivo is the oligomeric form Aq-477 is the only single-domain rhodanese characterized to date as a thermoactive and thermostable tetramer The crystal structures of many proteins from hyperthermophiles have been solved, and several factors responsible for their extreme thermostability have been proposed, including an increase in the number of ion pairs and hydrogen bonds, core hydrophobicity and packing density, as well as the oligomerization of several subunits and an entropic effect due to the relatively shorter surface loops and peptide chains [40] Protein stability arises from a combination of many factors, which each contribute to various extents in different proteins It seems that there is no single dominating factor, even in hyperthermophilic proteins [41] Comparative examination of the primary structure of ST did not point to any obvious features that could explain the high thermostability of Aq-477 from A aeolicus except for a decrease in the number of asparagine residues (4 versus 12 in Sud), a diminution in glycine residues and an increase in the number of hydrophobic residues (53 versus 48%) The same features were observed in TTHA0613 from T thermophilus HB8 We have shown that: (a) the monomeric form was less stable than the oligomers, and (b) the concentration and ⁄ or substrate induce the dimerization and the tetramerization Few enzymes from hyperthermophilic organisms are higher-order oligomers than their counterparts in mesophilic organisms and potential stabilizing role of increased subunit interactions via oligomerization has been suggested [41–44] Moreover, the oligomeric organization of proteins, and especially of enzymes, provides an additional level of complexity and plays an important role in numerous biological processes In the simplest case of homodimers, the intersubunit interface can provide an additional shared binding site for noncompetitive ligands, and ⁄ or mediate conformational changes [45] Oligomeric sulfurtransferase from Aquifex aeolicus The kinetic behaviour of Aq-477 does not present any cooperativity processes As a consequence, oligomerization of the enzyme was not in line with allosteric regulation of the activity but more probably with thermal stability Thus, protein stability and not efficiency has been selected for in the evolution of this oligomer and assembly of identical subunits to noncovalently associated oligomers is thought to ensure their survival in hyperthermophiles This is also the case for other hyperthermophily enzymes such as phosphoribosylanthranilate isomerase from Thermotoga maritima [46], and formyltransferase from the hyperthermophile Methanopyrus kaudleris [47] One of the major driving forces for protein oligomerization originates from shape complementarity between the associating molecules, brought about by a combination of hydrophobic and polar interactions (e.g hydrogen bonds and salts bridges) [48] Our results show the probable role of hydrophobic interactions between subunits because dissociation occurs in the absence of salt Functional role of Aq-477 Like all enzymes belonging to the rhodanese family, the function of the single-domain enzymes in vivo is seriously debated When mercaptopyruvate was used as the sulfur donor, no activity was detected with Aq-477, suggesting that this protein was not a MST This is in agreement with the amino acid composition of the active site loop which is different from the characteristic motif of MST i.e CG[S ⁄ T]GVT with no charged residues in the loop [18] In the same way, the Cd25 phosphatase domain and arsenate resistance role were excluded as in these enzymes an elongated seven amino acid active-site loop was present The Aq-477 amino acid loop presents the motif of the catalytic domain of thiosulfate cyanide sulfurtransferase (TST) which is distributed among bacteria, archaea and eukaryotes Aq-477 catalyses sulfur transfer from thiosulfate, tetrathionate and polysulfide To date, this is the only enzyme in which use of these different sulfur donors has been demonstrated in vitro, because Sud is inactive with thiosulfate [34] and the polysulfide sulfurtranferase activity of GlpE has not been demonstrated [5] We propose that aq477 encodes a monomeric rhodanese with polysulfide sulfurtranferase activity and, therefore to rename this gene rhdB1 Members of the genus Aquifex were obtained from marine hydrothermal systems [27] where sulfur is the predominant compound Therefore, it is not surprising to find numerous genes that encode putative proteins involved in sulfur metabolism in the A aeolicus genome A supercomplex from A aeolicus involved in FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4581 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al sulfur reduction in the cytoplasmic space has been characterized by our group [30] In Acidithiobacillus ferrooxidans, proteomic experiments have shown the induction of ST when sulfur was used as the electron donor, and the role of this enzyme as a sulfur carrier for various energetic complexes has been suggested [49] The well-studied Wolinella system proposes also a direct interaction between Sud and the polysulfur reductase Experiments are currently being developed by our group to determine if Aq-477 is the physiological partner of the sulfur reductase Experimental procedures All restriction enzymes were obtained from Promega (Madison WI) PCR was carried out using PWO DNA polymerase from Roche (Mannheim, Germany) DNA ligase was obtained from Roche Cloning oligonucleotides were purchased from MWG and DNA sequencing was performed by Genome Express (Marseilles, France) Bacterial strains and plasmids The bacterial strains and plasmids used are listed in Table BL21 (DE3)-RIPL (Stratagene, La Jolla, CA) harbouring peT22aq477 was used to overproduce Aq-477 for purification To construct peT22aq477, the aq477 gene was amplified by PCR with the primers 5¢-GGCATATGTTTA TGAACGTTCCGG-3¢ and 5¢-GGGTCGACTTAAGATT TAGCAGGT-3¢, where the underlined letters indicate the restriction sites for, respectively, NdeI and SalI After cleavage with NdeI and SalI, the amplified product was cloned into peT22 to create peT22aq477 Growth conditions for overproduction and purification of recombinant Aq-477 E coli BL21-CodonPlus (DE3)-RIPL was transformed with the peT22aq477 plasmid A culture (0.5 L) of recombinant E coli was grown at 37 °C to D600 ¼ 0.6 and then induced with mm isopropyl thio-b-d-thiogalactoside for h at Table Bacterial strains and DNA vectors Strain or vector E coli BL21-CodonPlus (DE3)-RIPL Aquifex aeolicus pET-22b(+) pET22Aq477 4582 Genotype, comments and ⁄ or reference E coli B F– ompT hsdS(rB– mB–) dcm+ Tetr gal k(DE3) endA Hte [argU proL Camr] [argU ileY leuW Strep ⁄ Specr] VF5 (Deckert) 63744–3 Novagen Novagen aq477 gene from A aeolicus in NdeI-SalI fragment of pET-22b(+) 37 °C Cells were harvested by centrifugation and disrupted by French press in 50 mm Tris ⁄ HCl, pH 7.6 containing proteases inhibitors cocktail from Roche Crude extract was centrifuged at 14 000 g for 10 and immediately heated to 80 °C for 40 to precipitate heat-labile E coli proteins Following centrifugation at 4000 g for 15 min, the supernatant containing the overproduced protein was concentrated using Centriprep concentrators (Amicon France, Epernon, France) with YM-10 membranes and was loaded onto a Superdex S200 high-resolution column (1 · 30 cm, FPLC apparatus, Amersham Pharmacia Biotech, Piscataway, NJ) equilibrated in buffer 100 mm Tris ⁄ HCl, 50 mm NaCl pH 7.6 and eluted in the same buffer (0.3 mLỈmin)1) Active fractions were concentrated, frozen in liquid nitrogen and stored at )20 °C All steps were performed at room temperature Growth conditions and purification of Aq-477 wild-type A aeolicus was cultivated in 1.4 Nm)2 bottles under 1.4 bar H2 ⁄ CO2 (80:20) in SME medium modified according to Guiral et al [30] at pH 6.8 in the presence of thiosulfate (1 gỈL)1) or S° (7.5 gỈL)1) and harvested in the late exponential growth phase Typical yields were $ 400 mg of cell material per L of culture After centrifugation (30 3700 g, °C), the pellet was frozen and stored at )80 °C Periplasmic extraction was performed as described by Brugna et al [50] Lactate dehydrogenase activity was measured to show whether the extract was contaminated by cytoplasmic proteins After periplasmic extraction, cell material (50 g) was resuspended in 50 mm Tris ⁄ HCl, mm EDTA, 10 lgỈmL)1 DNase, 5% glycerol and proteases inhibitors (pH 7.6) under argon, and broken in French press cell Debris and unbroken cells were removed by centrifugation (10 000 g, 15 min) Membrane and soluble proteins were separated by ultracentrifugation (45 min, 15 000 g, rotor 45 Ti, Beckman) After dialysis the supernatant containing the soluble proteins was loaded onto a Q-Sepharose (1.6 · 10 cm, FLPC) equilibrated in 50 mm Tris ⁄ HCl (pH 7.6) buffer The absorbed proteins were eluted by a linear gradient of NaCl (0–1 m) in the same buffer Sulfurtransferase activity was found in the 300 mm NaCl fraction The fraction was loaded onto a HTP column (1 · 10 cm biogel; Bio-Rad, Hercules, CA) equilibrated in 50 mm Tris ⁄ HCl, 300 mm NaCl (pH 7.6) The column was washed with the same buffer and proteins were eluted by a linear gradient of potassium phosphate (0–1 m), pH 7.6 The 300 mm phosphate fraction contained the major part of sulfurtransferase activity After concentration using Centriprep concentrators (Amicon) with YM-10 membranes, the last step of purification was done with a Superdex 200 (1 · 30 cm) high-resolution column (FPLC apparatus, Amersham Pharmacia Biotech) equilibrated in 100 mm Tris ⁄ HCl, 50 mm NaCl pH 7.6 and eluted in the FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al same buffer (0.3 mL min)1) Active fractions were concentrated, frozen in liquid nitrogen and stored at )20 °C All steps were performed at room temperature Sulfurtransferase activity assays All buffers used for activity assays were preincubated under argon, all assays were done at 80 °C One unit of ST activity corresponds to the production of lmol of thiocyanate or H2S per minute Thiosulfate rhodanese sulfurtransferase S2 O2 ỵ CN : SCN ỵ SO2 ị 3 Thiosulfate rhodanese sulfurtransferase activity was determined by measuring SCN formation as the red Fe(SCN)3 complex from cyanide and thiosulfate [34] The reaction mixture contained 100 mm Tris ⁄ HCl, pH 9.0, 10 mm KCN, mm mercaptoethanol, and enzyme extract and was initiated by addition of 10 mm Na2S2O3 This was realized in presence and in absence of 2.5 mm dithiothreitol After incubation at 85 °C for 10 the reaction was stopped by addition of 200 lL acidic iron reagent (FeCl3, 50 gỈl1; 65% HNO3, 200 mLỈL1) After centrifugation at 13 000 g for the absorption was read at 460 nm Spontaneous rates of thiocyanate formation were determined by omitting the crude extract from the reaction mixture Amounts of product formation were quantified using a standard curve done with NaSCN Test without enzyme was done as control S2O3 stability at 80 °C and linearity of SCN– production up to 20 has been verified Steady state kinetics were done as described in [5] The final concentration of Aq-477 was 30 nm 3-Mercaptopyruvate rhodanese sulfurtransferase (HSCH2COCOO)+CN):CH3COCOO)+SCN)) The assay mixture consisted of 100 mm Tris ⁄ HCl, pH 9.0, 10 mm KCN, 2.5 mm dithiothreitol and enzyme extract and was initiated by mm 3-mercaptopyruvate Assays were incubated and treated as described above The final concentration of Aq-477 was 30 nm Thiosulfate sulfurtransferase S2 O2 ỵ BH : HS ỵ SO2 þ BH3 Þ 3 Assay mixtures (1 mL) contained 100 mm Tris ⁄ HCl, pH 9.0, 2.5 mm dithiothreitol and protein extracts as stated above, and were started by adding 200 lm sodium thiosulfate Reactions were incubated for 20 at 37 °C The amount of H2S developed during the reaction was fixed by adding 100 lL 30 mm FeCl3 dissolved in 1.2 m HCl and 100 lL 20 mm N,N¢-dimethyl-p-phenylene-diamine dissolved in 7.2 m HCl Samples were kept in the dark for Oligomeric sulfurtransferase from Aquifex aeolicus 20 min, centrifuged and the absorption of methylene blue formed was measured at 670 nm For quantification, standard curves were prepared or the molar extinction coefficient of 15 · 106Ỉcm)1Ỉm)1 was used The final concentration of Aq-477 was lm 3-Mercaptopyruvate sulfurtransferase HSCH2 COCOO ỵ BH : HS ỵ BH3 þ CH3 COCOỒ Þ Enzyme assays were performed as described for thiosulfate sulfurtransferase with the exception of adding 3-mercaptopyruvate, rather than thiosulfate, as a substrate at a final concentration of mm Tetrathionate rhodanese sulfurtransferase S4 O2 ỵ CN : SCN ỵ S3 O2 ị 6 Enzyme assays were carried out as described for thiosulfate sulfurtransferase with the exception of adding tetrathionate, rather than thiosulfate, as a substrate at a final concentration of 10 mm The final concentration of Aq-477 was 30 nm Tetrathionate sulfurtransferase S4 O2 ỵ BHỵ : HS ỵ BH3 ỵ S3 O2 ị 6 In this case NaBH4 (5 mm) replaced KCN in the mixture assay as described by Klimmek et al [34] H2S production was measured as described for thiosulfate sulfurtransferase The final concentration of Aq-477 was lm Polysulfide rhodanese sulfurtransferase S2 ỵ CN : SCN ỵ S2 ị n nÀ1 Polysulfide sulfur was generated as described by Klimmeck et al [34] and the polysulfide assay was carried out following polysulfide consumption directly by measuring ˚ A360 (a ¼ 0.38 mm)1.cm)1 polysulfide sulfur) at 60 °C as described previously [34] No dithiothreitol was added to the medium The final concentration of Aq-477 was nm Polysulde sulfurtransferase S2 ỵ BH4 : HS ỵ Sn1 ỵ BH3 ị n In this case, NaBH4 (5 mm) replaced KCN in the mixture assay as described by Klimmek et al [34] H2S production was measured following polysulfide consumption directly ˚ by measuring A360 (a ¼ 0.38 mm)1.cm)1) at 60 °C The final concentration of Aq-477 was lm Thioredoxin sulfurtransferase We used an NADPH-coupled assay with thioredoxin reductase to show whether reduced thioredoxin is an effective FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4583 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al sulfur acceptor substrate for Aq-477 NADPH, thioredoxin f and thioredoxin reductase f stability has been verified at various temperatures The system was not stable at 85 °C, so the activity was measured at 50 °C Each assay (final volume, 0.5 mL) contained 50 mm Tris ⁄ HCl (pH 7.6), 0.25 mL)1 thioredoxin reductase, 100 lm NADPH, and 16 lm thioredoxin Cuvettes containing all reagents except NADPH were used as blanks NADPH was added, and the reaction mixtures were allowed to equilibrate Measurements of the absorbance at 340 nm were used to ensure that the mixtures had reached equilibrium After equilibrium had been reached, purified Aq-477 (160 nm), thiosulfonate (30 mm), or both were added For steady-state kinetics studies, thiosulfate, tetrathionate and polysulfide were used as sulfur donor and cyanide as sulfur acceptor The final concentration of Aq-477 was 35 nm Steady-state kinetics experimental measurements were fitted to the Michaelis–Menten equation using sigma-plot The temperature dependence of the thiosulfate sulfurtransferase activity as well as polysulfide rhodanese sulfurtransferase was measured between 20 and 80 °C in 100 mm Tris ⁄ HCl, pH 9.0 pH dependence of the thiosulfate sulfurtransferase activity was carried out in 100 mm Mes (pH and 6.5), 100 mm Mops (pH 7.1), 100 mm Hepes (pH 7.1, 7.6 and 8), 100 mm Tris ⁄ HCl (pH and 9) or 100 mm glycine (pH 9.2 and 10) protein concentration was 0.05 mgỈmL)1 in 20 mm Tris ⁄ HCl buffer, pH 7.6 All CD spectra were baseline corrected by buffer and analysed using CD spectroscopy deconvolution cdnn 2.1 software for determining the secondary-structure classes and K2d algorithm [51] CD spectra were also monitored at 222 nm at various temperatures (20–90 °C at °CỈs)1) for the monomeric and the oligomeric form Samples were prepared in the absence of substrate in 20 mm Tris ⁄ HCl buffer, pH 7.6 or in 20 mm Tris ⁄ HCl buffer, pH 7.6 with mm thiosulfate or 500 lm polysulfide N-Terminal sequence determination N-Terminal amino acid sequences were determined from soluble protein or after SDS ⁄ PAGE After electrophoresis on 12% polyacrylamide gel under denaturing conditions, proteins were transferred onto a poly(vinylidene difluoride) membrane for 45 at a current intensity of 0.8 mcm)2 using a semidry electrophoretic transfer unit Sequence determinations were carried out with an Procise 494 microsequencer (Applied Biosystems, Foster City, CA) Quantitative determination of phenylthiohydantoin derivates was done by high-pressure liquid chromatography (Waters, Manchester, UK) monitored by a data and chromatography control station (Waters) Molecular mass determination Inhibition and inactivation studies Anion inhibition studies were carried out using ammonium acetate (50–400 mm) and ammonium sulfate (50–400 mm) in thiosulfate rhodanese sulfurtransferase assay Inactivation of cysteine was performed on purified Aq-477 by incubation in 100 mm Tris ⁄ HCl pH 7.6 with iodoacetamide reagent in : 0.5, : and : molecular ratios, for h at room temperature The remaining ST activity was determined and compared with that of Aq-477 incubated without iodoacetamide Stability studies measurements were performed incubating monomeric or oligomeric form of Aq-477 at °C, 25 °C and 80 °C Activity was measured at various times The same experiments were carried out with enzyme prepared with various substrate concentrations (1–20 mm) The enzymatic assay was carried out with Na2S2O3 10 mm final or polysulfide sulfur at 500 lm Stability studies experimental measurements were fitted to a decreasing exponential equation using sigma-plot CD studies CD spectra were recorded on a Jasco J-715 spectropolarimeter equipped with a Peltier-type temperature control system (model PTC-348WI) in the far-UV (195–250 nm) Experimental conditions were 0.2 nmỈmin)1; temperature 20 or 80 °C Spectra were averaged from five acquisitions The 4584 MALDI-MS was performed on a reflectron time-of-flight mass spectrometer equipped with delayed extraction (Voyager DE-RP, Perspective Biosystem Inc., Paris, France) The sample (0.7 lL) was mixed directly on the support with an equal volume of matrix (saturated solution of sinapinic acid in 40% acetonitrile and 60% water made 0.1% in trifluoroacetic acid) Analytical procedures Native gel electrophoresis Purified enzyme was loaded onto a native 4% polyacrylamide stacking ⁄ 10% running gel (Mini-Protean II, Bio-Rad) or on 12.5% polyacrylamide Phast Gels with Phast Gel native buffer strips (Phast System; Pharmacia, Uppsala, Sweden) BN gels (10–20%) were used as described by Schagger & von Jagow [52] Oligomeric Aq-477 (20 lg) was loaded onto the gel, and dilution shock was carried out or not on the protein (in the same buffer) To study the effect of salt or substrate, Aq-477 was incubated for 10 at 80 °C with 100 mm NaCl or and 10 mm thiosulfate, before migration Denaturing gel electrophoresis Purified enzyme (1 lg) was incubated for at 90 °C with a sample loading buffer containing SDS 2% and FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al Oligomeric sulfurtransferase from Aquifex aeolicus 20 mm dithiothreitol, and loaded onto a 4% polyacrylamide stacking ⁄ 12% running SDS gel (Mini-Protean II, Bio-Rad) or on 12.5% polyacrylamide Phast Gels with SDS buffer strips (Phast System; Pharmacia) After migration, the gel was stained as described previously [53] Immunoblotting After migration, western blotting was performed using standard procedures Anti-(Aq-477) sera against A aeolicus recombinant Aq-477 were used and the detection reaction was performed using goat peroxidase-conjugated anti-(rabbit IgG) serum (Sigma, St Louis, MO) and SuperSignal West Pico chemiluminescent substrate reagents (Pierce, Rockford, IL) Time-resolved fluorescence experiments Time-resolved fluorescence parameters (lifetimes and correlation times) were obtained from the two polarized fluorescence decays I^(t) and I//(t), using time-correlated single-photon counting technique The instrumentation setup was essentially similar to those previously described [54,55] with modifications Briefly, a time-correlated singlephoton counting SPC-430 card (Becker-Hickl GmbH, Paris, France) was used for the acquisition; the time scaling was 19.63 ps per channel and 4096 channels were used The excitation light pulse source was a Ti-sapphire femtosecond laser (Millenia-pumped Tsunami, Spectra Physics), the repetition rate of the laser was set down at MHz, associated with a third harmonic generator tuned to 300 nm Fluorescence emission was detected through a monochromator (ARC SpectraPro-150) set at 345 nm (Dk ¼ 15 nm) by a microchannel plate photomultiplier (Hamamatsu R1564U-06) connected to an amplifier Phillips Scientific 6954 at 25 °C The two polarized components of the fluorescence decay were collected alternately over a period of 30 s until the total count of the I// component reached $ 15 · 106 The microcell was thermostated with a Haake type-F3 circulating nbath Analysis of the decay curves was performed using the quantified maximum entropy method [56] The anisotropy decay is described by the following equation: rtị ẳ with n P iẳ1 n X III tị GI ? tị ẳ qi et=hi III tị ỵ 2GI ? tị iẳ1 2ị qi ẳ q0 where G is the G-factor, the hi are the individual correlation times and the qi are the associated fractional amplitudes Normalization of h for a given temperature was performed using: h¼ gðTÞ V kT ð3Þ where g is the viscosity, V is the volume of the rotating unit, k the Bolzmann constant, and T the temperature (K) Sequence analysis Multiple sequence alignments were performed using clustal w [57] Sequences were retrieved via the NCBI server (http://www.ncbi.nlm.nih.gov) Secondary structure topology was predicted after multiple sequence alignment at http://pbil-univlyon1.fr Acknowledgements We gratefully acknowledge Marielle Bauzan (Fermentation Plant Unit IBSM., Marseilles, France) for ´ growing the bacteria, Regine Lebrun (Proteomic Analysis Center, Marseilles, France) for N-terminal sequencing and mass determination, and Marianne Guiral, Wolfgang Nitschke, Mari Luz Cardenas, ` Athel Cornish-Bowden and Mireille Bruschi for helpful discussions References Beinert H (2000) A tribute to sulfur Eur J Biochem 267, 5657–5664 Beinert H (2000) Iron–sulfur proteins: ancient structures, still full of surprises J Biol Inorg Chem 5, 2–15 Kessler D (2006) Enzymatic activation of sulfur for incorporation into biomolecules in prokaryotes FEMS Microbiol 30, 825–840 Cianci M, Gliubich F, Zanotti G & Berni R (2000) Specific interaction of lipoate at the active site of rhodanese Biochim Biophys Acta 1481, 103–108 Ray WK, Zeng G, Potters MB, Mansuri AM & Larson TJ (2000) Characterization of a 12-kilodalton rhodanese encoded by glpE of Escherichia coli and its interaction with thioredoxin J Bacteriol 182, 2277–2284 Cerletti P (1986) Seeking a better job for an underemployed enzyme: rhodanese Trends Biochem Sci 11, 369–372 Leimkuhler S & Rajagopalan KV (2001) A sulfurtransă ferase is required in the transfer of cysteine sulfur in the in vitro synthesis of molybdopterin from precursor Z in Escherichia coli J Biol Chem 276, 22024–22031 Lauhon CT, Skovran E, Urbina HD, Downs DM & Vickery LE (2004) Substitutions in an active site loop of Escherichia coli IscS result in specific defects in Fe–S cluster and thionucleoside biosynthesis in vivo J Biol Chem 279, 19551–19558 Lauhon C & Kambampati R (2000) The iscS gene in Escherichia coli is required for the biosynthesis of 4thiouridine, thiamin, and NAD J Biol Chem 275, 20096–20103 10 Lauhon CT (2002) Requirement for IscS in biosynthesis of all thionucleosides in Escherichia coli J Bacteriol 184, 6820–6829 FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4585 Oligomeric sulfurtransferase from Aquifex aeolicus M.-C Giuliani et al 11 Bui BT, Escalettes F, Chottard G, Florentin D & Marquet A (2000) Enzyme-mediated sulfide production for the reconstitution of [2Fe)2S] clusters into apo-biotin synthase of Escherichia coli Sulfide transfer from cysteine to biotin Eur J Biochem 267, 2688–2694 12 Wolfe MD, Ahmed F, Lacourciere GM, Lauhon CT, Stadtman TC & Larson TJ (2004) Functional diversity of the rhodanese homology domain: the Escherichia coli ybbB gene encodes a selenophosphate-dependent tRNA 2-selenouridine synthase J Biol Chem 279, 1801–1809 13 Cereda A, Carpen A, Picariello G, Iriti M, Faoro F, Ferranti P & Pagani S (2007) Effects of the deficiency of the rhodanes-like RhdA in Azotobacter vinelandii FEBS Lett 581, 1625–1630 14 Bordo D, Deriu D, Colnaghi R, Carpen A, Pagani S & Bolognesi M (2000) The crystal structure of a sulfurtransferase from Azotobacter vinelandii highlights the evolutionary relationship between the rhodanes and phosphatase enzyme families J Mol Biol 298, 691–704 15 Bordo D, Forlani F, Spallarossa A, Colnaghi R, Carpen A, Bolognesi M & Pagani S (2000) A persulfate cysteine promotes active site reactivity in Azotobacter vinelandii rhodanese Biol Chem 382, 1245–1252 16 Forlani F, Carpen A & Pagani S (2003) Evidence that elongation of the catalytic loop of the Azotobacter vinelandii rhodanese changed selectivity from sulphur- to phosphate-containing substrates Protein Eng 16, 515–519 17 Cereda A, Forlani F, Lametti S, Bernhardt R, Ferranti P, Picariello G, Pagani S & Bonomi F (2003) Molecular recognition between Azotobacter vinelandii rhodanese and sulphur acceptor protein Biol Chem 384, 1473–1481 18 Bordo D & Bork P (2002) The rhodanese ⁄ Cdc25 phosphatase superfamily Sequence–structure–function relations EMBO Reports 3, 729–746 19 Ploegman JH, Drent G, Kalk KH, Hol WG, Heinrikson RL, Keim P, Weng L & Russell J (1978) Structure of bovine liver rhodanese I Structure determination at ˚ 2.5 A resolution and a comparison of the conformation and sequence of its two domains Nature 273, 124–129 20 Ploegman JH, Drent G, Kalk KH, Hol WG, Heinrikson RL, Keim P, Weng L & Russell J (1979) The structure of bovine liver rhodanese II The active site in the sulfur-substituted and the sulfur-free enzyme J Mol Biol 127 (2), 149–162 21 Spallarossa A, Donahue JL, Larson TJ, Bolognesi M & Bordo D (2001) Escherichia coli GlpE is a prototype sulfurtransferase for the single-domain rhodanese homology superfamily Structure 9, 1117–1122 22 Adams H, Teertstra W, Koster M & Tommassen J (2002) PspE (phage-shock protein E) of Escherichia coli is a rhodanese FEBS Lett 518, 173–176 4586 23 Kleerebezem M, Crielaard W & Tommassen J (1996) Involvement of stress protein PspA (phage shock protein A) of Escherichia coli in maintenance of the protonmotive force under stress conditions EMBO J 15, 162–171 24 Kelly RM & Adams MWW (1994) Metabolism in hyperthermophilic microorganisms Antonie V Leeuwenhoek 66, 247–270 25 Adams MW (1994) Biochemical diversity among sulfurdependent, hyperthermophilic microorganisms FEMS Microbiol Rev 15, 261–277 26 Stetter KO (1999) Extremophiles and their adaptation to hot environments FEBS Lett 452 (1–2), 22–25 27 Olsen GJ, Woese CR & Overbeek R (1994) The winds of (evolutionary) change: breathing new life into microbiology J Bacteriol 176, 1–6 28 Deckert G, Warren PV, Gaasterland T, Young WG, Lenox AL, Graham DE, Overbeek R, Snead MA, Keller M, Aujay M et al (1998) The complete genome of the hyperthermophilic bacterium Aquifex aeolicus Nature 392, 353–358 29 Brugna-Guiral M, Tron P, Nitschke W, Stetter KO, Burlat B, Guigliarelli B, Bruschi M & Giudici-Orticoni MT (2003) [NiFe] hydrogenases from the hyperthermophilic bacterium Aquifex aeolicus: properties, function, and phylogenetics Extremophiles 7, 145–157 30 Guiral M, Tron P, Aubert C, Gloter A, Iobbi-Nivol C & Giudici-Orticoni MT (2005) A membrane-bound multienzyme, hydrogen-oxidizing, and sulfur-reducing complex from the hyperthermophilic bacterium Aquifex aeolicus J Biol Chem 280, 42004–42015 31 Lin YJ, Dancea F, Lohr F, Klimmek O, PfeifferMarek S, Nilges M, Wienk H, Kroger A & Ruterjans H (2004) Solution structure of the 30 kDa polysulfide-sulfurtransferase homodimer from Wolinella succinogenes Biochemistry 43, 1418–1424 32 Hattori M, Mizohata E, Tatsuguchi A, Shibata R, Kishishita S, Murayama K, Terada T, Kuramitsu S, Shirouzu M & Yokoyama S (2006) Crystal structure of the single-domain rhodanese homologue TTHA0613 from Thermus thermophilus HB8 Proteins 64, 284–287 33 Alexander K & Volini M (1987) Properties of an Escherichia coli rhodanese J Biol Chem 262, 6595–6604 34 Klimmek O, Kreis V, Klein C, Simon J, Wittershagen A & Kroger A (1998) The single cysteine residue of the Sud protein is required for its function as a polysulfidesulfurtransferase in Wolinella succinogenes Eur J Biochem 253, 263–269 35 Finazzi Agro A, Federici G, Giovagnoli C, Cannella C & Cavallini D (1972) Effect of sulfur binding on rhodanese fluorescence Eur J Biochem 28, 89–93 36 Greenfield NJ (1996) Methods to estimate the conformation of proteins and polypeptides from circular dichroism data Anal Biochem 235, 1–10 FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS M.-C Giuliani et al 37 Cornilescu G, Vinarov DA, Tyler EM, Markley JL & Cornilescu CC (2006) Solution structure of a singledomain thiosulfate sulfurtransferase from Arabidopsis thaliana Protein Sci 15, 2836–2841 38 Schultz J, Milpetz F, Bork P & Ponting CP (1998) MART, a simple modular architecture research tool: identification of signaling domains Proc Natl Acad Sci USA 95, 5857–5864 39 Mueller EG (2006) Trafficking in persulfides: delivering sulfur in biosynthetic pathways Nat Chem Biol 1, 185–194 40 Petsko GA (2001) Structural basis of thermostability in hyperthermophilic proteins, or ‘there’s more than one way to skin a cat’ Methods Enzymol 334, 469–478 41 Opitz U, Rudolph R, Jaenicke R, Ericsson L & Neurath H (1987) Proteolytic dimers of porcine muscle lactate dehydrogenase Characterization, folding, and reconstitution of the truncated and nicked polypeptide chain Biochemistry 26, 1399–1406 42 Walden H, Taylor GL, Lorentzen E, Pohl E, Lilie H, Schramm A, Knura T, Stubbe K, Tjaden B & Hensel R (2004) Structure and function of a regulated archaeal triosephosphate isomerase adapted to high temperature J Mol Biol 342, 861–875 43 Vieille C & Zeikus GJ (2001) Hyperthermophilic enzymes: sources, uses, and molecular mechanisms for thermostability Microbiol Mol Biol Rev 65, 1–43 44 Tanaka Y, Tsumoto K, Yasutake Y, Umetsu M, Yao M, Fukada H, Tanaka I & Kumagai I (2004) How oligomerization contributes to the thermostability of an archaeon protein Protein 1-isoaspartyl-O-methyltransferase from Sulfolobus tokodaii J Biol Chem 279, 32957–33267 45 Traut TW (1994) Dissociation of enzyme oligomers: a mechanism for allosteric regulation CRC Crit Rev Biochem 29, 125–163 46 Thoma R, Hennig M, Sterner R & Krischner K (2000) Structure and function of mutationally generated monomers of dimeric phosphoribosylanthranilate isomerase from Thermotoga maritima Structure 8, 265–276 47 Shima S, Thauer RK, Ermler U, Durchschlag H, Tziatzios C & Schubert D (2000) A mutation affecting the association equilibrium of formyltransferase from the Oligomeric sulfurtransferase from Aquifex aeolicus 48 49 50 51 52 53 54 55 56 57 hyperthermophilic Methanopyrus kandleri and its influence on the enzyme’s activity and thermostability Eur J Biochem 267, 6619–6623 Jones S & Thornton JM (1996) Principles of protein– protein interactions Proc Natl Acad Sci USA 93, 13–20 Acosta M, Beard S, Ponce J, Vera M, Mobarec JC & Jerez CA (2005) Identification of putative sulfurtransferase genes in the extremophilic Acidithiobacillus ferrooxidans ATCC 23270 genome: structural and functional characterization of the proteins OMICS 9, 13–29 Brugna M, Giudici-Orticoni MT, Spinelli S, Brown K, Tegoni M & Bruschi M (1998) Kinetics and interaction studies between cytochrome c3 and Fe-only hydrogenase from Desulfovibrio vulgaris Hildenborough Proteins 33, 590–600 Andarde MA, Chacon P, Merelo JJ & Moran F (1993) Evaluation of secondary structure of proteins from UV circular dichroism spectra using an unsupervised learning neural network Protein Eng 6, 383–390 Schagger H & von Jagow G (1991) Blue native electrophoresis for isolation of membrane protein complexes in enzymatically active form Anal Biochem 199, 223–231 Mortz E, Krogh TN, Vorum H & Gorg A (2001) Improved silver staining protocols for high sensitivity protein identification using matrix-assisted laser desorption ⁄ ionization-time of flight analysis Proteomics 1, 1359–1363 Deprez E, Tauc P, Leh H, Mouscadet JF, Auclair C, Hawkins ME & Brochon JC (2001) DNA binding induces dissociation of the multimeric form of HIV-1 integrase: a time-resolved fluorescence anisotropy study Proc Natl Acad Sci USA 98, 10090–10095 Deprez E, Tauc P, Leh H, Mouscadet JF, Auclair C & Brochon JC (2000) Oligomeric states of the HIV-1 integrase as measured by time-resolved fluorescence anisotropy Biochemistry 39, 9275–9284 Brochon JC (1994) Maximum entropy method of data analysis in time-resolved spectroscopy Methods Enzymol 240, 262–311 Thompson JD, Higgins DG & Gibson TJ (1994) CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice Nucleic Acids Res 22, 4673–4680 FEBS Journal 274 (2007) 4572–4587 ª 2007 The Authors Journal compilation ª 2007 FEBS 4587 ... which is in agreement with the purification of the protein from the cytoplasm of A aeolicus aq477 is flanked by genes panD and aq478 Because a single base pair separates the termination and initiation... codons for panD and aq477, they appear to be organized as an operon panD encodes an aspartate decarboxylase which catalyses the decarboxilation of aspartate to produce b-alanine, a precursor of coenzyme... Characterization of this protein is in progress Aq-477 is a single- domain rhodanese We purified and characterized a protein from A aeolicus annotated as a hypothetical protein In vitro, it catalyses