The rhodanese from Azotobacter vinelandii RhdA is a sulfurtransferase which catalyzes in vitro the production of thiocyanate, transferring the sulfane sulfur atom from thiosulfate to cya
Trang 1Azotobacter vinelandii rhodanese
Selenium loading and ion interaction studies
Sonia Melino1, Daniel O Cicero1,2, Maria Orsale1, Fabio Forlani3, Silvia Pagani3and Maurizio Paci1,2
1
Dipartimento di Scienze e Tecnologie Chimiche and2INFM, Sez B, University of Rome ‘Tor Vergata’, Italy;
3
Dipartimento di Scienze Molecolari Agroalimentari, University of Milan, Italy
Rhodanese is a sulfurtransferase which in vitro catalyzes the
transfer of a sulfane sulfur from thiosulfate to cyanide Ionic
interactions of the prokaryotic rhodanese-like protein from
Azotobacter vinelandii were studied by fluorescence and
NMR spectroscopy The catalytic Cys230 residue of the
enzyme was selectively labelled using [15N]Cys, and changes
in1H and15N NMR resonances on addition of different ions
were monitored The results clearly indicate that the sulfur
transfer is due to a specific reaction of the persulfurated Cys
residue with a sulfur acceptor such as cyanide and not to the
presence of the anions Moreover, the1H-NMR spectrum of
a defined spectral region is indicative of the status of the
enzyme and can be used to directly monitor sulfur loading even at low concentrations Selenium loading by the addition
of selenodiglutathione was monitored by fluorescence and NMR spectroscopy It was found to involve a specific interaction between the selenodiglutathione and the catalytic cysteine residue of the enzyme These results indicate that rhodanese-like proteins may function in the delivery of reactive selenium in vivo
Keywords: 15N-NMR; Azotobacter vinelandii; rhodanese; selenodiglutathione; sulfurtransferase
The rhodanese from Azotobacter vinelandii (RhdA) is a
sulfurtransferase which catalyzes in vitro the production of
thiocyanate, transferring the sulfane sulfur atom from
thiosulfate to cyanide, by a double displacement mechanism
(thiosulfate–cyanide sulfurtransferase, EC 2.8.1.1) [1–3]
The best studied rhodanese is that from bovine liver
(Rhobov) Studies on its catalytic mechanism in vitro have
shown that, during the transfer of sulfane sulfur from
thiosulfate to cyanide, this enzyme cycles between two stable
intermediates, a sulfur-loaded (ES) and a sulfur-free form
(E) Physical properties of these intermediates have been
demonstrated to be different by a variety of solution
methods [4–6], but crystallographic data do not appear to
show appreciable flexibility in the rhodanese when ES
crystals are soaked with cyanide [7,8] Thermodynamic
calculations [9] on the two forms of Rhobov reveal that the
ES form is about 8 calÆmol)1more stable than the E form It
has been suggested that the conformational changes in
rho-danese may form the basis of its activity The physiological
role of this class of enzyme is still unclear, but its wide distribution among eukaryotes and prokaryotes suggests that it is involved in essential metabolic pathways The proposed roles include cyanide detoxification [3], restoration
of iron-sulfur centres in Fe-S proteins such as ferredoxin [10,11], and sulfur metabolism [3,12] It has recently been found to be involved in selenium trafficking [13]; selenium uptake in the persulfide position of the bovine enzyme is achieved by reaction with selenodiglutathione (SDG), the primary metabolite of selenite The selenium-loaded enzyme (ESe) has been proposed to be the carrier of selenium for selenophosphate synthase It has been hypothesized that a rhodanese-like enzyme may behave as a transferase for the regulation of selenium concentration in vivo [13]
Recombinant RhdA is one of the most recently expressed prokaryotic enzymes [14], and its 3D structure has been elucidated [8] In contrast with Rhobov, which has four cysteine residues, RhdA has only one (Cys230), which is the residue involved in the catalytic mechanism This is a fundamental advantage in the study of rhodanese-like proteins
The interconversion between the ES and E form has been studied by NMR spectroscopy in parallel with fluorescence methods [4] Selective [15N]Cys labelling of RhdA was performed in order to investigate, by NMR spectroscopy changes in the status of the active site when the enzyme cycles between the two forms Analysis of high-resolution
1H-NMR spectra of the ES and E form has revealed some differences that are diagnostic of the two forms In this work, we propose the use of an alternative method, 1D NMR spectroscopy, to investigate the interconversion between the ES and E form in solution and to monitor the state of the enzyme by addition of substrates or inhibitors
Correspondence to M Paci, Dipartimento di Scienze e Tecnologie
Chimiche, Universita` di Roma ‘Tor Vergata’, Via della Ricerca
Scientifica, 00133-Rome, Italy.
Fax: + 39 0672594328, Tel.: + 39 0672594446,
E-mail: paci@uniroma2.it
Abbreviations: RhdA, rhodanese of Azotobacter vinelandii; E,
sulfur-free form of rhodanese; ES, sulfur-loaded form of rhodanese;
ESe, selenium-loaded form of rhodanese; HSQC, heteronuclear
single quantum coherence; Rhobov, bovine rhodanese;
SDG, selenodiglutathione.
Enzyme: rhodanese; thiosulfate–cyanide sulfurtransferase (EC 2.8.1.1).
(Received 9 July 2003, revised 25 August 2003,
accepted 5 September 2003)
Trang 2Materials and methods
Preparation of the protein
The plasmid pQER1 [14], containing the gene coding for
RhdA with an N-terminal His-tag, was used to transform
the BL-21(DE3)[pREP4] Escherichia coli strain [14], and
overexpression of the recombinant protein was induced by
addition of isopropyl thio-b-D-galactoside to a
mid-expo-nential culture RhdA was purified by chromatography on
a Ni/nitrilotriacetate/agarose column (Qiagen) The
His-tagged protein was eluted by addition of 200 mMimidazole
and precipitated in 75% saturated ammonium sulfate The
protein concentration was determined using A0.1%
280¼ 1.3 [2], and the molecular mass of 31 kDa was estimated by
SDS/PAGE Rhodanese activity was measured by the
discontinuous colorimetric assay described by So¨rbo [15]
The presence of the His-tag did not affect enzymatic
activity The sulfur-free form (E) was prepared by adding a
10-fold molar excess of cyanide to ES rhodanese in 50 mM
Tris/HCl (pH 7.4)/0.3M NaCl followed by a 10-min
incubation at room temperature Excess cyanide and
thiocyanate were removed by loading the protein solution
on to a Centricon-3 (3000 molecular mass cut-off; Amicon)
As a control, ES (to which no cyanide was added) was
analogously treated The conversion of ES into E was
monitored by measuring the increase in fluorescence
quantum yield that accompanies the removal of the
persulfide sulfur [4]
Production of [15N]Cys-containing RhdA
His-tagged RhdA protein labelled with [15N]Cys was
expressed by growing the transformed BL21[pREP4] E coli
strain in medium containing: 2 mgÆmL)1 succinic
acid; 0.9 mgÆmL)1 magnesium acetate tetrahydrate;
10 mgÆmL)1 K2HPO4; 2 mgÆmL)1 sodium acetate
trihy-drate; 1 mgÆmL)1 ammonium chloride; 0.01 mgÆmL)1
CaCl2; 0.004 mgÆmL)1FeCl2; 0.05 mgÆmL)1nicotinic acid;
0.05 mgÆmL)1thiamin; 0.1 lgÆmL)1biotin; 0.125 mgÆmL)1
guanosine, cytosine and uracil; 0.08 mgÆmL)1 thymine;
0.4 mgÆmL)1 L-alanine, L-glutamic acid, L-glutamine,
L-arginine and glycine; 0.25 mgÆmL)1 L-aspartic acid;
0.1 mgÆmL)1 L-asparagine, L-histidine, L-isoleucine,
L-lysine, L-proline, L-threonine, L-tyrosine and L-valine;
0.25 mgÆmL)1 L-methionine; 1.6 mgÆmL)1 L-serine;
1 mgÆmL)1 L-leucine; 0.05 mgÆmL)1 L-tryptophan, L
-cys-tine, L-phenylalanine and L-cysteine; 1% glycerol;
0.1 mgÆmL)1 ampicillin; 0.025 mgÆmL)1 kanamycin A
10-mL volume of BL21[pREP4]/pQER1 culture grown
overnight in Luria–Bertani medium was added to 500 mL
expression medium (filtered through 0.2-lm nylon filter),
and incubated (for 6 h) at 37°C in an orbital shaker to
A600¼ 1 The culture was then induced with 1 mM
isopropyl thio-b-D-galactoside, followed by a 10-min
incu-bation The cell suspension was harvested by centrifugation,
washed twice with 500 mL 0.9 mgÆmL)1NaCl, and
resus-pended in the presence of 1 mM isopropyl thio-b-D
-galactoside, in 500 mL of the expression medium in which
both cystine and cysteine were replaced by 15N-labelled
cystine (0.2 mgÆmL)1) (Isotec, Sigma-Aldrich, UK) After
2.5 h of induction at 37°C, cells were harvested by
centrifugation and stored at )80 °C The procedure used for purification of the labelled protein was as described above After purification, the enzyme was assayed as previously described [14] The procedure used for the expression and purification of the uniformly 15N-labelled protein will be reported elsewhere The sample obtained gives well-resolved and intense 15N-NMR spectra; the results will be reported elsewhere
Preparation of the selenium-substituted rhodanese RhdA in the E form was prepared by adding KCN to the enzyme solution, with a molar ratio of E to KCN of 1 : 10,
in 50 mM Tris/HCl buffer, pH 7.4 After the reaction, the protein solution was dialyzed at 4°C for 12 h The selenium-loaded RhdA, E–Se, was prepared from the persulfide-free enzyme by reaction with a solution of selenite and glutathione (GSH), in a molar ratio of 1 : 4, respect-ively, in 50 mMTris/HCl, pH 7.4, containing 1 mMEDTA,
as described previously [13]
NMR spectroscopy NMR measurements were performed at an RhdA concen-tration of 0.1–0.4 mM in 50 mM Tris/HCl (pH 7.25)/ 0.3M NaCl and at 20°C on a Bruker AVANCE instru-ment, operating at a proton frequency of 700 MHz equipped with a z-gradient triple resonance probe Data were processed and analysed on an IRIS O2 work station (Silicon Graphics) usingNMRPIPE[16] andNMRVIEW
[17]
Fluorescence measurements All fluorescence measurements were made using an LS50 Perkin–Elmer spectrofluorimeter equipped with a thermo-statically controlled stirrer cell holder The temperature was maintained at 23°C, and the protein concentration was kept constant at 6 lM The excitation and emission bandwidths were 5 and 3 nm, respectively The excitation wavelength was set at 286 nm, and the spectra were recorded from 300 to 400 nm The changes in fluorescence intensity at 336 nm (Fobs) are given as DF %:
DF%¼ abs½ðFobs F0Þ=F0 100 where F0is the original fluorescence intensity of RhdA
ES and E were used as references Fluorescence meas-urements were made in the presence of different fixed ion concentrations with an enzyme concentration of 6 lMin
50 mMTris/HCl, pH 7.2
Results and Discussion
15N labelling of Cys230 of RhdA and15N-NMR spectroscopy
Incorporation of [15N]Cys in the expression of RhdA protein was estimated to be 10% from1H-15N hetero-nuclear single quantum coherence (HSQC) spectra, com-paring the15N-filtered spectrum of [15N]Cys-RhdA with the unfiltered enzyme at a fixed delay and also with the1H-15N HSQC spectra of the uniformly 15N-labelled RhdA
Trang 3(unpublished) The low level of [15N]Cys incorporation is
probably due to transamination reactions and to the use of a
nonauxotrophic strain of E coli In previous fluorescence
experiments and crystallographic investigations, native
RhdA was prepared as the persulfurated form at Cys230
[8,18] We observed that RhdA is obtained as a mixture of
ES and E In fact, two different cross-peaks were observed
in the 1H-15N HSQC spectrum (Fig 1A) of the
over-expressed [15N]Cys230–RhdA when protein purification
was performed without addition of thiosulfate We
evalu-ated that, after purification, 30% of the overexpressed
RhdA was in sulfur-loaded form and 70% in the sulfur-free
form (Fig 1A) The fluorescence and MALDI-TOF data from the same NMR samples confirm the estimate of the ratio between the two forms of RhdA made from NMR (data not shown) Addition of a thiosulfate and cyanide excess, respectively, allowed identification of the 1H-15N correlation peaks corresponding, respectively, to the ES and
E form (Fig 1B,C) In both cases, the samples were dialyzed before the NMR experiments to remove the excess reagents The observed changes in chemical shift were 0.26 and 1.0 p.p.m for1H and15N, respectively Additional analysis
of1H-15N HSQC spectra from a uniformly labelled sample
of RhdA showed that about 20 peaks out of the 230 observed signals show different chemical shifts between the two forms of the protein (unpublished) These changes may reflect the conformational changes associated with sulfur loading, probably of residues located near the active site
1
H-NMR spectroscopy
1H-NMR spectra were obtained for the ES and E forms They show a characteristic1H resonance and, in particular, differences can be seen in the region typical of the indolyl protons of tryptophan and imidazolyl protons of histidine Water presaturation was performed before data acquisition, making it difficult to detect the fast exchanging NHs of histidines Moreover, all resonances in this region showed
15N resonances at 128–131 p.p.m (data not shown), typical
of the NH group of the side chain of tryptophans These two observations led us to tentatively assign those reso-nances as belonging to tryptophans Figure 2 shows the selected regions that are diagnostic of the different state of
Fig 1 1 H- 15 N NMR HSQC spectra of [ 15 N]Cys230-labelled RhdA.
(A) 1 H- 15 N HSQC spectrum of 0.2 m M [ 15 N]Cys230-labelled RhdA in
50 m M Tris/HCl (pH 7.2)/0.3 M NaCl, after purification at 20 °C (B)
Spectrum of [15N]Cys230-labelled RhdA after treatment with 2 m M
thiosulfate (ES form) (C) Spectrum of [15N]Cys230-labelled RhdA
after treatment with 2 m M KCN (E form).
Fig 2 Selected regions of1H-NMR spectrum of RhdA The two characteristic1H spectra of the ES and E forms of the enzyme are shown in (A) and (B), respectively The transition between the two forms can be observed as a shift in the peaks (the NH of the indole ring of a Trp, from 11.6 to 12.1 ppm) on conversion of the enzyme from the E to the ES form on addition of thiosulfate ions (E/thiosulfate, 1 : 10) (C).
Trang 4the enzyme The shift in resonance from 11.6 to 12.1 p.p.m.
is a sensitive check of the transition from the ES to the E
form (Fig 2A,B) Moreover, during this transition, a
high-field shift of the resonance of a methyl group was also
observed, i.e the peak at 0.6 p.p.m disappeared and a new
peak appeared at 1.25 p.p.m Figure 2C shows the
beha-viour of the same methyl resonance upon reconversion of
E into ES by addition of thiosulfate Identification of these
resonances requires extensive assignment work, but some
educated guesses may be made before the assignment is
complete It has been reported that addition of cyanide in
soaking experiments on RhdA crystal results in the removal
of the persulfide S atom bound to Cys230, and this reaction
induces conformational changes in the Cys230 and Trp195
side chains, which disrupts the Arg235 side chain [8] Close
inspection of the crystal structure reveals that the methyl
groups of Leu238 and Leu180 face Arg235 and Trp195, the
residues affected by the conformational changes around the
active site, thus these are likely candidates to be affected by
the change in the persulfurated state of the protein
Previous studies have shown that a number of residues
surrounding the catalytic Cys230 are able to generate a
strong positive electrostatic field which reaches an estimated
value of 18 kTÆe)1under standard physiological conditions
(pH 7.5, ionic strength 0.15M) [19] Therefore we studied
the interaction of the active site of RhdA with negative ions
by 1H-NMR spectroscopy, monitoring whether there is
transition between the forms of the enzyme after addition of
these ions Figure 3C,D shows the spectrum of the ES form
after addition of phosphate and hypophosphite ions No changes in chemical shift were observed up to a molar ratio of RhdA (ES) to ion of 1 : 10 at pH 7.2 The results indicate that, in solution, the catalytic Cys230 residue was not affected
by the presence of these ions, up to the concentrations used Fluorescence experiments
As also observed for Rhobov, RhdA shows an intrinsic fluorescence with a maximum at 336 nm, resulting from six tryptophan residues present in the polypeptide chain [4,5,20] Fluorescence spectroscopy is particularly useful in the study of rhodanese as it can report on modifications of the active site cysteine In fact, formation of a persulfide group in the active site quenches the intrinsic fluorescence
of the protein without affecting its shape (Fig 4A) This has been attributed to local perturbation or long-range energy transfer [20] Therefore we carried out a fluores-cence quenching study to monitor the change in confor-mation of RhdA The results before and after addition of hypophosphite ions are shown in Fig 4B,C There was a small effect of quenching on the E form after addition of a very high concentration of hypophosphite ions (Fig 4C)
It is probable that these anions are electrostatically attracted by the positively charged side chains of the residues around the active site and bind in their proximity, influencing the intensity of the fluorescence of the trypto-phan residues and resulting in a fluorescence quenching effect
Fig 3 Selected regions of the1H-NMR spectrum of RhdA of the two forms of the enzyme, ES (A) and E (B), with1H-NMR spectra of RhdA (ES) after the addition of phosphate (C) and hypophosphite ions (D) A molar ratio of RhdA (ES) to ion of 1 : 10 in 50 m M Tris/HCl, pH 7.2 was used for (C,D).
Trang 5Previous studies also showed that fluorescence changes in
RhdA seem to be modulated by phosphate anions, when the
protein was purified in phosphate buffer at pH 6.0 [19] In
phosphate buffer, recovery of the intrinsic fluorescence after
the addition of KCN, to produce sulfur-free RhdA, was significantly lower than in the presence of Tris/HCl (18% vs 46%) [19] The strong positive electrostatic field of the active site may be decreased in intensity by a large excess of phosphate ions, resulting in a decrease in the stability of the persulfide bond Crystallographic studies of the ES form of RhdA after the addition of 5 mMphosphate or hypophos-phite anions reported that these compounds completely remove the persulfide sulfur atom from Cys230 and in particular the hypophosphite anion was observed in the catalytic pocket In contrast, no phosphate anions were observed near the active site [19] An explanation for these different results may be found in the different behaviour of the protein in solution We used these different experimental conditions, i.e the molar ratio of ions, pH and incubation times, because our goal was to determine the behaviour of different anions compared with cyanide to evaluate the different affinities for the protein A large excess of, and long exposure to, phosphate, as used in the previous study [19], may have a different effect on the stability of the S-S bond Previously characterized rhodaneses, including the bovine liver enzyme [9] and the enzyme from E coli [21], are typically inhibited reversibly and competitively with respect
to thiosulfate by most anions (acetate, sulfate and phos-phate anions) at very high concentration Our results clearly indicate that the removal of the persulfide group from Cys230 is due to a selective reaction with a sulfane sulfur acceptor, such as cyanide, in conditions close in pH and ionic strength to physiological, and not to the simple presence of anions However, the limited survey of anions performed in the present study does not allow us to rule out the possibility that low molecular mass mimics of active site groups of normal protein acceptors may also be able to replace cyanide
Reaction of RhdA with SDG Selenium uptake in the persulfide position of RhdA was monitored by fluorescence and NMR spectroscopy after reaction with SDG This compound was prepared by the reaction of GSH with selenite as previously reported [13] and based on earlier studies [22,23], suggesting that SDG and its subsequent reduction to glutathionyl selenide anion [24,25] are key intermediates in the selenium metabolic pathway It has been observed that in vitro the labile SDG may react with Rhobov at neutral pH to generate an ESe form [13] The intrinsic fluorescence of RhdA before and after addition of the GSH/selenite solution leads to the selenium-loaded form of RhdA (ESe) (Fig 5) In fact, quenching of the intrinsic fluorescence corresponding to DF
of 22% at 336 nm was observed after incubation of RhdA (E) with SDG solution [RhdA (E)/SeO3-2/GSH, 1 : 5 : 20]
at 37°C for 10 min, whereas a DF of 11% was observed on addition of a 10-fold molar excess of thiosulfate with the same E form (Fig 5) No further changes in intrinsic fluorescence of the ESe form were observed after addition of
an excess of thiosulfate, confirming the presence of the loaded form of the enzyme (data not shown) On the other hand, fluorescence experiments with the ES form of RhdA showed no changes after addition of SDG Intrinsic fluorescence was measured by adding selenite or GSH to RhdA [at a molar ratio of RhdA (E) to SeO2–of 1 : 5 and
Fig 4 Fluorescence spectra of RhdA in the presence of anions (A) The
sulfur-loaded state of 6 l M RhdA in 50 m M Tris/HCl, pH 7.2, (solid
line), and the sulfur-free form of RhdA (dashed line) with thiosulfate
(E/thiosulfate, 1 : 100) (dotted line); (B) ES form (solid line) in the
presence of hypophosphite ions with a molar ratio of RhdA to ion of
1 : 1; 1 : 10; 1 : 20; 1 : 50; 1 : 100; (C) E form in the presence of
hypophosphite ions at the molar ratio used in (B).
Trang 6RhdA (E) to GSH of 1 : 20], and the quenching (DF)
observed was 4% for both (data not shown) This indicates
that the labile SDG compound, produced by reaction
between selenite and GSH, reacts with RhdA at neutral pH
to generate an ESe rhodanese The quenching of intrinsic
fluorescence for selenium loading of RhdA is higher than
that observed after treatment with thiosulfate because of the
higher quenching properties of selenium than of the
persulfide bond These results are in agreement with those
of Cannella et al [20], who prepared a selenium derivative
of Rhobov by using the synthetic substrate selenosulfate
and examined its spectroscopic properties Selenium binding
to the protein was also detected by NMR spectroscopy
Experiments on15N-Cys-labelled RhdA were performed
under the same conditions as for the fluorescence
experi-ments Figure 6 shows the HSQC spectra of the [15
N]Cys-labelled RhdA (E) in the presence of GSH/selenite The
spectrum indicates the formation of a new form of the Cys
residue The1H-15N cross-peak of the catalytic cysteine was
shifted (15N, 118.5 p.p.m.;1H, 8.56 p.p.m.) upon reaction
with the SDG Moreover, no changes in the1H and15N
resonances of the [15N]Cys230 were observed after the
addition of the selenite/GSH mixture to the sulfur-loaded
RhdA (data not shown)
Figure 7 shows the 1H-NMR spectral regions highly
indicative of the persulfurated and sulfur-free states of the
enzyme after addition of GSH alone, selenite alone, or
selenite/GSH equivalent to adding SDG The 1H-NMR
spectra of RhdA show that no loading occurred on addition
of either GSH or selenite alone to the E form (Fig 7B,C) In
contrast, the addition of selenite/GSH to the E form induces
changes in the1H-NMR resonance pattern similar to those
observed by addition of thiosulfate
No specific interactions with the catalytic site of the
enzyme were found in presence of the GSH or selenite ions
alone, indicating that the loading of selenium to the ESe
form occurs by a specific reaction with SDG These results
confirm the hypothesis that rhodanese-like proteins may function as components of the delivery system for reactive selenium in vivo
Prospective studies over the last few years have suggested that Se intake may protect against cancer [26,27] Several mechanisms have been proposed to explain the anticarci-nogenic effects of Se compounds [26] One hypothesis is that
Se compounds induce apoptosis in initiated premalignant cells, i.e SDG induces p53 [28] Furthermore, Ghose et al [29] recently reported that SDG induces apoptosis in oral cell cultures Induction of apoptosis has been attributed to SDG because of the observation that it alters the redox status of the cell by manipulating the level of a cellular reducing agent, such as thioredoxin, which has been implicated in growth control in other contexts and is overexpressed in many tumours [30] In fact, SDG has been shown to be a specific oxidant of reduced thioredoxin and inhibitor of thioredoxin reductase in a cell-free system [31,32] It has been shown that Rhobov has an affinity that
is 1000-fold higher for the reduced form of thioredoxin than
Fig 6 1 H- 15 N-NMR HSQC spectrum of the selectively [ 15 N]Cys230-labelled RhdA spectrum after addition of SDG The [15N]Cys ESe was obtained by addition of the SDG solution at 0.1 m M [ 15 N]Cys230-RhdA (E) [N]Cys230-RhdA (E)/SeO 32–/GSH, 1 : 10 : 40) in 20 m M Tris/HCl/ 0.3 M NaCl, pH 7.4.
Fig 5 Fluorescence changes induced by SDG on RhdA Sulfur-free
form (E) of 5 l M RhdA in 50 m M Tris/HCl (pH 7.4)/1 m M EDTA
(solid line); E form after addition of thiosulfate 50 l M (dotted line) or
SDG solution (to a final concentration of 25 l M SeO 3
2–
and 100 l M
GSH) (dashed line).
Trang 7for cyanide [33], so it seems reasonable to suppose that SDG
may have an indirect effect in vivo on the thioredoxin system
through the rhodanese system Moreover, the
rhodanese-like proteins may participate in detoxification of molecules
such as thiosulfate, selenite and SDG, raising interest about
the biological role of these proteins
Our results show that a simple1H-NMR spectrum can be
used as a sensitive and fast monitor of sulfur or selenium
loading of RhdA Although a protein of the size of RhdA
(31 kDa) gives hundreds of proton signals, the1H-NMR
spectrum shows two regions that are sufficiently well
resolved to follow the change in chemical shift induced by
the protein conversion of protons attached to nitrogen
(probably an indolyl proton of a tryptophan) and methyl
protons Thus this NMR experiment can be performed
without the need of isotope labelling, and, given the
sensitivity of modern high-field spectrometers, protein
concentrations of 10–50 lM and experimental times of a
few minutes are sufficient to obtain information on the state
of the enzyme
Acknowledgements
The technical assistance of Fabio Bertocchi is gratefully acknowledged.
This research was supported by MURST PRIN project
‘Sulfotrans-ferasi procariotiche’ (1999–2001 and 2002–03) and the target Project of
Italian CNR ‘Biotecnologie’ and the project FIRB of Italian MIUR.
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