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

Rhodaneses thiosulfate : cyanide sulfurtransferase or TSTs are widespread enzymes that catalyse the transfer of a sulfane sulfur.. The purified Aq-477 was active, with thiosulfate or poly

bacterium Aquifex aeolicus

Being single is not so simple when temperature gets high

Marie-Ce´cile Giuliani1, Pascale Tron1, Gise`le Leroy1, Corinne Aubert1, Patrick Tauc2and

Marie-The´re`se Giudici-Orticoni1

1 Laboratoire de Bioe´nerge´tique et Inge´nierie des Prote´ines (BIP), IBSM-CNRS, Marseille, France

2 Laboratoire de Biotechnologie et de Pharmacologie Ge´ne´tique Applique´e – ENS-CACHAN, Cachan, France

Sulfur adds considerable functionality to a wide variety

of biomolecules because of its unique properties: its

chemical bonds are both easily made and easily

bro-ken, 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

sul-fur’ (R-S-SH) was suggested as the biologically

rele-vant active sulfur species in the early 1980s Sulfane

sulfur is produced enzymatically with the IscS protein,

the SufS protein and rhodanese being the most

promi-nent 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):

SSO23 þ Rho-SH ! SO23 þ Rho-S-SH ð1aÞ

Rho-S-SHþ CN! Rho-SH þ SCN ð1bÞ

In addition to cyanide, other thiophilic acceptor com-pounds 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

Keywords

Aquifex aeolicus; hyperthermophile;

oligomerization; sulfurtranferase;

thermostability

Correspondence

M.-T Giudici-Orticoni, Laboratoire de

Bioe´nerge´tique et Inge´nierie des Prote´ines,

IBSM-CNRS, 31 chemin Joseph Aiguier,

13402 Marseille cedex 20, France

Fax: +33 4 91 16 45 78

Tel: +33 4 91 16 45 50

E-mail: giudici@ibsm.cnrs-mrs.fr

(Received 20 April 2007, revised 6 July

2007, accepted 12 July 2007)

doi:10.1111/j.1742-4658.2007.05985.x

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

Abbreviations

BN, Blu native gel; MST, mercaptopyruvate sulfurtransferase; rec, recombinant; Rho, rhodanese; SR, sulfur reductase; ST, sulfurtransferase; Sud, sulfide dehydrogenase; TST, thiosulfate sulfurtransferase.

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]

Rhoda-neses or sulfurtransferases are ubiquitous enzymes

found in many organisms from all three domains of

life Even though the discovery of the

hyperthermo-philes has important ramifications, not only in

biotechnology, no rhodanese has been characterized

from these extremophilic organisms

The prototypic enzyme, bovine liver rhodanese,

con-sists 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

king-doms These two domains show weak sequence

simi-larity [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

responsi-ble for the exchange of sulfur for selenium in

2-thio-uridine in vivo [12]

In addition, characterization of sulfide

dehydroge-nase (Sud) from a mesophilic bacterium, Wolinella

succinogenesrevealed for the first time the direct

inter-vention of rhodanese in energetic sulfur metabolism,

because this protein is the sulfur donor for the

termi-nal acceptor of respiratory chain sulfur reductase in

W succinogenes

Microorganisms with the remarkable property of

growing at temperatures near and above 100C 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 85C [24,27] Aquifex is a

hyperthermophilic, hydrogen-oxidizing,

microaero-philic, 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

by the exceptional phylogenetic and physiological prop-erties of A aeolicus, as well as by its potential as a source of extremely stable enzymes, we undertook sev-eral studies on the energetic metabolism of this organ-ism, whose genome has been completely sequenced [28]

In particular, we studied its hydrogen⁄ sulfur metabo-lism [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 macro-molecules to develop molecular mechanisms adapted to extreme physicochemical conditions Its possible meta-bolic 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 rep-resentative 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 peri-plamic extract, we propose that the enzymes required for sulfur transfer are present in A aeolicus periplas-mic and cytoplasperiplas-mic spaces

We obtained evidence for the presence of sulfur-transferase activity in A aeolicus grown on elemental sulfur or thiosulfate However, extracts of cells grown with elemental sulfur showed a specific sulfurtrans-ferase 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-Sepha-rose, HTP and Superdex 200 gel-filtration chromato-graphy 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

calibration curve shows that these fractions

corres-pond 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

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 cyto-plasm 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 cata-lyses the decarboxilation of aspartate to produce b-alanine, a precursor of coenzyme A aq478 encodes

a protein similar to proteins involved in signal trans-duction mechanisms The product of aq477 was annotated as a hypothetical protein related to a member of COG0607P, which regroup 168 rhoda-nese-related sulfurtransferases All these homologues belong to a a⁄ b-fold protein domain found dupli-cated 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 rhodane-ses, Aq-477 is composed of a single-domain rhoda-nese fold, the catalytic domain as it contains the characteristic catalytic cysteine Few single-domain rhodaneses have been characterized in detail The pri-mary 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 approxi-mately coincide in all these proteins However, some differences are found The major difference is the presence of an extra a helix in Sud protein More-over, 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⁄ hyper-thermophiles enzymes

Despite the low overall sequence identity, a few residues are conserved in b strands b2 and b4, which

Table 1 Yields and enrichments of the ST activity One unit of ST

activity corresponds to the uptake of 1 lmol of SCNÆmin)1.

Preparation

Sulfurtransferase activity

Yield (%)

Specific activity (UÆmg)1)

Total activity (U)

Q Sepharose (300 m M NaCl) 306 7800 22

HTP (300 m M phosphate) 550 3000 8.5

Ve/Vo

1, 1, 2, 2,

4, 4, 4, 4, 4, 5, 5, 5,

Elution volume (ml)

14 15.5 16.9

1 2 3

0

50

100

150

200

mAU ( )

Activity

( ) u/ml

100

200

300

Fig 1 Size-exclusion chromatography of wild-type Aq-477 An

S200 column (1 · 30 cm) was equilibrated in 100 m M Tris ⁄ HCl,

50 m M NaCl, pH 7.6 at 20 C The protein (200 lL at 4 mgÆmL)1)

was detected by its absorbance (d) and its activity (m) (Inset)

Cali-bration curve in 100 m M Tris ⁄ HCl, 50 m M NaCl, pH 7.6, flow rate

0.3 mLÆmin)1, sample volume 200 lL at 2 mgÆmL)1of rusticyanine

(17 kDa); dihemic cytochrome c (21 kDa); ovalbumin (43 kDa);

alco-hol dehydrogenase (150 kDa).

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

two-domain rhodaneses We can conclude that these

residues play an important role in the global folding

processes of the mesophilic, themophilic and

hyper-thermophilic 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

More-over, the positive charges (R30, R70, R74) found in

the substrate-binding pocket for the negative

polysul-fide 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

aeo-licus genome, a fourth gene exhibiting sequence

simi-larity 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 purifica-tion 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 solu-ble protein were obtained from 1 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 recon-structed 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, cor-responding to the protein absorption Aq-477 does not contain prosthetic groups or heavy metal ions

Fig 2 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 Con-served residues involved in substrate binding are in bold The arrow indicates the active-site cysteine.

As the active site of sulfurtransferases involves a

cys-teine residue, Aq-477 was incubated with a cyscys-teine-

cysteine-modifying reagent, iodoacetamide to verify that Cys69

is required for ST activity All the activity disappeared

at a 1 : 1 molar ratio (iodoacetamide⁄ Aq-477) This

demonstrates that Cys69 is: (a) involved in the

cataly-sis, and (b) not involved in disulfide bond formation

Several previously characterized rhodaneses are

spe-cifically 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

rho-danese 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 2 Clearly, it appears that polysulfide sulfur was

a very efficient sulfur donor, indicating that this sulfur

compound is probably the real substrate H2S

produc-tion was also tested Because of the high unspecific

reaction with tetrathionate and polysulfide in the

sence of dithiothreitol, the test was performed in

pre-sence of NaBH4 instead of KCN as described by

Klimmeck et al [34] To detect H2S production, 100-fold 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 1 serves as a sulfur-acceptor substrate for GlpE from

E coli[5] To test thioredoxin as a sulfur acceptor sub-strate for Aq-477, we adapted the assay method described for GlpE at 50C (see Experimental proce-dures 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–

80C) (Table 3) Aq-477 showed a temperature opti-mum at 80C and only 8% of the activity was observed at 25C Aq-477 showed a pH optimum at 9 and no sulfurtransferase activity was observed at pH values < 7

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-filtra-tion chromatography was used to determine the apparent molecular mass of recombinant Aq-477 One peak is obtained, corresponding to a mix of the tetra-meric, dimeric and monomeric forms as for the wild-type 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

Table 2 Steady-state kinetic parameters of Aq-477 from A

aeoli-cus 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(m M )

Thiosulfate rhodanese

sulfurtransferase

7865 ± 200 5.7 ± 0.9 (S 2 O 32–);

2.1 ± 0.37 (CN – ) Thiosulfate

sulfurtransferase

no activity nd Tetrathionate rhodanese

sulfurtransferase

8802 ± 500 6.9 ± 2 (S4O6) Polysulfide sulfur

rhodanese

sulfurtransferase

165,000 < 0.05 (S n2–)

Polysulfide sulfur

sulfurtransferase

Thioredoxin

sulfurtransferase

3-Mercaptopyruvate

rhodanese

sulfurtransferase

no activity nd

Table 3 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)

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

dena-turing 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

blot-ting after SDS⁄ PAGE of each fraction from the S200

column (1.5 mgÆmL)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 blot-ting performed after denaturing electrophoresis of solu-ble 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 demon-strate that: (a) the oligomeric state of the enzyme con-tains 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 1 mm polysulfide (Fig 3, trace B) Com-pared with the same experiment without substrate (Fig 3, trace A) the peak was more homogenous and the molecular mass was 50 kDa This result demon-strates the tetrameric organization of Aq-477 in the presence of substrate

For a more defined correlation between the concen-tration and the different states of oligomerization, a stock solution of 4 mgÆmL)1 wild-type or recombinant Aq-477 was diluted to 0.4 and 0.04 mgÆmL)1 at 25C

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

1⁄ 10 (Fig 5, lanes A and B) When the same experi-ment was carried out in the absence of salt in the pro-tein 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 macro-molecules or biological complexes The extent of light

Elution volume (ml)

15

A B

Fig 3 Size-exclusion chromatography of recombinant Aq-477 An

S200 (1 · 30 cm) column was equilibrated in (A) 100 m M Tris ⁄ HCl,

100 m M NaCl, pH 7.6 or (B) 100 m M Tris ⁄ HCl, 100 m M NaCl,

10 m M Na2S2O3pH 7.6 at 20 C Profile A: injection of 50 lL of

rec-Aq-477 at 2.6 mgÆmL)1; profile B: injection of 50 lL of

rec-Aq-477 at 2.6 mgÆmL)1 in the presence of 10 m M Na2S2O3. The

protein was detected by its absorbance at 280 nm.

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

vol-umes 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

satu-rating 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

corre-lation time (Table 4), confirming that its oligomeric

state is strongly dependent upon the protein

concentra-tion At low concentrations and 25C, the rotational

correlation time was 7 ns, which corresponds to a

globu-lar protein of around 14 kDa This demonstrates that at

low concentrations the major part of the enzyme was

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 sat-urating concentration of thiosulfate At low protein con-centrations, 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 sub-strate Over the concentration range used, there is equi-librium between monomer, dimer, trimer and tetramer Our results show that the initial step in the oligo-merization process is the formation of dimers This is

in agreement with the result obtained by electrophore-sis under denaturing conditions The dimeric form is

an active intermediate structural conformation that evolves to at least a tetrameric form Our results

A

14,4

43 30 20,1

67

B

20

80 50 40 30

Fig 4 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, 1 lg of recombinant Aq-477; lane 3, immunoblotting experiments of recombinant Aq-477,

1 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⁄ S o 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 H 2 ⁄ NaS 2 O 3 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.

Fig 5 BN gel of purified recombinant Aq-477 Lane A, 20 lg of

Aq-477 at 0.4 mgÆmL)1; lane B, 20 lg of Aq-477 at 0.04 mgÆmL)1;

lane C, 20 lg of Aq-477 at 0.4 mgÆmL)1without salt; lane D, 20 lg

of Aq-477 at 0.04 mgÆmL)1+ S 2 O 3 10 m M

Table 4 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 mea-sured by monitoring tryptophan fluorescence (kex¼ 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.

Aq-477 concentration (l M )

Lifetime (ns) 1.66 1.41 1.8 1.57 1.11 1.16 Long rotational

correlation time (ns)

confirm that: (a) a dilution step generates the

mono-meric form, and (b) the substrate induces the

oligomer-ization state and a more homogenous state

Stability and activity of Aq-477

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

dena-turation of the enzyme directly after the dilution step

at room temperature As the enzyme presents activity

at 80C and no activity at 25 C, it was interesting to

study the effect of temperature on the structural

stabil-ity by CD ellipticstabil-ity at 222 nm [36] Changes in helical

content of the various forms of Aq-477 after thermal

treatment are shown in Fig 6 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

60C Denaturation of the monomer of Aq-477

appears to be an irreversible process as the initial

sig-nal is not recovered at 25C 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

were carried out with a monomeric enzyme sample freshly prepared in the presence of 500 lm polysulfide

or 3 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 sta-bility of the various forms of Aq-477, its activity was measured for the oligomeric and monomeric forms preincubated at different temperatures from 4 to

80C Independent of the incubation temperature (4, 25 or 80C), 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 min of incubation did not induce any modification in the traces This indicates the irreversibility of the inactiva-tion process The same experiments were carried out with the oligomeric enzyme (Fig 7B) Independent of the temperature incubation used, the activity at 80C was stable and 95% activity was still present after

150 min The corresponding half-life of irreversible inactivation increased from 19 min for the monomeric form to 320 min for the oligomeric form As shown previously by fluorescence and CD experiments, deacti-vation of the Aq-477 monomer is prevented by the presence of a sulfur donor As shown in Fig 7C, addi-tion of 5 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 mono-meric form is unstable, (b) monomer inactivation is irreversible, (c) the substrate prevents inactivation, and (d) temperature alone does not induce the active form

Temperature (°C)

Relative CD Intensity at 222 nm (%) 20

40

60

80

100

20 40 60 80

100

Fig 6 Changes in relative CD intensity at 222 nm for monomeric (d) or oligomeric (m) Aq-477 (A) Relative CD intensity at 222 nm at vari-ous 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 3 m M Na 2 S 2 O 3 (d) or 500 l M polysulfide (.) The degree of polymerization was con-trolled 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

oligo-merization 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

oligo-meric 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 Vmwas fourfold

smal-ler for the monomeric enzyme than for oligomeric

enzyme The Kappm values were similar (5.48 ± 1 mm)

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

Aq-477 is a single-domain rhodanese

We purified and characterized a protein from A

aeo-licus annotated as a hypothetical protein In vitro, it

catalyses the transfer of sulfane sulfur from

thiosul-fate to cyanide to form thiocyanate According to

this activity and its amino acid sequence, Aq-477

belongs to the rhodanese (or sulfurtransferase)

fam-ily 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

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 Ther-mus thermophilus HB8 and At5g66040.1 from Arabid-opsis 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 sulfurtransfer-ase (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 rhoda-neses rhdA1 and rhdA2 Besides Aq-477, we have detected only one other ORF that potentially encodes

a protein with a rhodanese fold Aq-1599 Characte-rization of this protein is in progress

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, sul-furtransferase 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

120 100 80 60 40 20

0

Residual activity (%) 0

20

40

60

80

100

120

0 20 40 60 80 100 120

0 20 40 60 80 100

120

Time (min)

50

B

Fig 7 Thermal inactivation of freshly prepared monomer or oligomer of Aq-477 (A) Time course of irreversible inactivation of a freshly pre-pared monomeric form of Aq-477 at 4 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 l M (r) polysulfide sulfur in the buffer.The degree of polymerization was controlled by BN gel.

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

physio-logical role Dimerization of RhdA from A vinelandii

has also been shown, but only when mutations were

introduced into the catalytic loop inducing an

interdis-ulfide 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

25C 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

aeo-licusexcept 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

hydropho-bic 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

concentra-tion and⁄ or substrate induce the dimerization and the

tetramerization

Few enzymes from hyperthermophilic organisms are

higher-order oligomers than their counterparts in

me-sophilic 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

homodi-mers, the intersubunit interface can provide an

additional shared binding site for noncompetitive

ligands, and⁄ or mediate conformational changes [45]

The kinetic behaviour of Aq-477 does not present any cooperativity processes As a consequence, oligo-merization of the enzyme was not in line with allosteric regulation of the activity but more probably with ther-mal 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 phosphoribosyl-anthranilate 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 interac-tions 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 seri-ously debated When mercaptopyruvate was used as the sulfur donor, no activity was detected with Aq-477, sug-gesting that this protein was not a MST This is in agree-ment 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 bac-teria, 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 sulfurtran-ferase 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 gen-ome A supercomplex from A aeolicus involved in