Despite this classical view, recent computational, structural and biochemical studies have highlighted the critical role of Keywords 5¢-deoxy-5¢-methylthioadenosine phosphorylase; CXC mo
Trang 1Archaea require a CXC motif for stability and folding
Giovanna Cacciapuoti, Iolanda Peluso, Francesca Fuccio and Marina Porcelli
Department of Biochemistry and Biophysics ‘F Cedrangolo’, Second University of Naples, Italy
Introduction
In the fascinating field of protein biochemistry,
ther-mostability and folding comprise important factors
that are currently gaining wide attention Disulfide
bonds represent an important structural feature of
many proteins, especially extracellular ones They not
only stabilize protein structures by lowering the
entropy of the unfolded polypeptide, but also are
required for the proper folding and biological activity
of several proteins Disulfide bond formation occurs in
the endoplasmic reticulum and mitochondrial
inter-membrane space of eukaryotes and in the periplasm of
prokaryotes [1] Disulfide bonds are a typical feature
of secretory proteins and are considered to contribute significantly to their overall stability [2] By contrast,
in intracellular proteins from well-known organisms, and as a result of the reductive chemical environment inside the cells [3], the presence of these covalent links
is limited to proteins involved in the mechanism of the response to redox stress [4] or to proteins catalyz-ing oxidation–reduction processes [1,5] Despite this classical view, recent computational, structural and biochemical studies have highlighted the critical role of
Keywords
5¢-deoxy-5¢-methylthioadenosine
phosphorylase; CXC motif and oxidative
protein folding; disulfide bonds;
hyperthermophilic proteins; purine
nucleoside phosphorylase
Correspondence
G Cacciapuoti, Dipartimento di Biochimica e
Biofisica ‘F Cedrangolo’, Seconda Universita`
di Napoli, Via Costantinopoli 16,
80138 Napoli, Italy
Fax: +39 081 5667519
Tel: +39 081 5667519
E-mail: giovanna.cacciapuoti@unina2.it
(Received 5 June 2009, revised 22 July
2009, accepted 29 July 2009)
doi:10.1111/j.1742-4658.2009.07247.x
5¢-Deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfatari-cus (SsMTAPII) and purine nucleoside phosphorylase from Pyrococcus furiosus (PfPNP) are hyperthermophilic purine nucleoside phosphorylases stabilized by intrasubunit disulfide bonds In their C-terminus, both enzymes harbour a CXC motif analogous to the CXXC motif present at the active site of eukaryotic protein disulfide isomerase By monitoring the refolding of SsMTAPII, PfPNP and their mutants lacking the C-terminal cysteine pair after guanidine-induced unfolding, we demonstrated that the CXC motif is required for the folding of these enzymes Moreover, two synthesized CXC-containing peptides with the same amino acid sequences present in the C-terminal regions of SsMTAPII and PfPNP were found to act as in vitro catalysts of oxidative folding These small peptides are involved in the folding of partially refolded SsMTAPII, PfPNP and their CXC-lacking mutants, with a concomitant recovery of their catalytic activ-ity, thus indicating that the CXC motif is necessary to obtain complete reversibility from the unfolded state of the two proteins The two CXC-containing peptides are also able to reactivate scrambled RNaseA The data obtained in the present study represent the first example of how the CXC motif improves both stability and folding in hyperthermophilic proteins with disulfide bonds
Abbreviations
AdoMet, S-adenosylmethionine; GdnCl, guanidinium chloride; GSH, glutathione; GSSG, glutathione disulfide; MTA,
5¢-deoxy-5¢-methylthioadenosine; MTAP, 5¢-deoxy-5¢-methylthioadenosine phosphorylase; PDI, protein disulfide isomerase; PfCGC, NH 2 -RRCGCKD-COOH; PfPNP, purine nucleoside phosphorylase from Pyrococcus furiosus; PNP, purine nucleoside phosphorylase; sRNaseA, scrambled RNaseA; SsCSC, NH2-GSCSCCN-COOH; SsMTAPII, 5¢-deoxy-5¢-methylthioadenosine phosphorylase II from Sulfolobus solfataricus.
Trang 2these covalent links in the structural stabilization of
intracellular proteins in some hyperthermophilic
Archaeaand Bacteria [6–12]
The abundance of disulfides observed across
hyper-thermophilic organisms also has stimulated research
into the identification of the biochemical mechanisms
related to disulfide maintenance It was recently
dem-onstrated that specific protein disulfide
oxidoreducta-ses, which are structurally and functionally related to
protein disulfide isomerase (PDI) [13], play a key role
in intracellular disulfide shuffling in hyperthermophilic
proteins [14]
Two enzymes, 5¢-deoxy-5¢-methylthioadenosine
pho-sphorylase II from Sulfolobus solfataricus (SsMTAPII)
[10,11] and purine nucleoside phosphorylase from
Pyrococccus furiosus (PfPNP) [12], have been isolated
and characterized from hyperthermophilic Archaea
These enzymes are members of purine nucleoside
phos-phorylases (PNP), comprising ubiquitous enzymes of
purine metabolism that function in the salvage
path-way of the cells [15] SsMTAPII is a homohexamer
(subunit 30 kDa) characterized by extremely high
affinity towards 5¢-deoxy-5¢-methylthioadenosine
(MTA), a natural sulfur-containing nucleoside formed
by S-adenosylmethionine (AdoMet) mainly through
polyamine biosynthesis [16] SsMTAPII shares 51%
identity with human 5¢-deoxy-5¢-methylthioadenosine
phosphorylase (MTAP) and is able to recognize
adeno-sine [10], in contrast to human MTAP which is strictly
specific for MTA [17] PfPNP displays a much higher
similarity with MTAP than with PNP family members
[12] Similar to human PNP [18], PfPNP shows an
absolute specificity for inosine and guanosine [12]
SsMTAPII and PfPNP show features of exceptional
thermophilicity and thermostability and are
character-ized by stabilizing disulfide bonds Two pairs of
intra-subunit disulfide bridges have been revealed by the
crystal structure of SsMTAPII [11] and three pairs of
intrasubunit disulfide bridges have been assigned to
PfPNP by integrating biochemical methodologies with
MS [12]
It is interesting to note that SsMTAPII and PfPNP
contain, in their C-terminal region, an unusual CXC
motif (i.e cysteines separated by one neighbouring
amino acid X) as a typical feature, and that the
substi-tution of these two cysteines with serines significantly
affects both their thermodynamic and kinetic stability
[10,12], suggesting the involvement of the cysteine pair
in the thermal stabilization of both enzymes
In the present study, we demonstrate that the CXC
motif of SsMTAPII and PfPNP plays an important
functional role in the oxidative folding of these
enzymes and that two short CXC-containing peptides
with amino acid sequences corresponding to those present in the C-terminal region of SsMTAPII and PfPNP, respectively, act in vitro as functional mimics
of PDI The presence of a CXC motif in hyperthermo-philic proteins with multiple disulfide bonds, such as SsMTAPII and PfPNP, represents the first example of
a new molecular strategy adopted by these enzymes to improve their stability and to preserve their folded state under extreme conditions
Results and Discussion
SsMTAPII and PfPNP require a CXC motif to preserve their folded state
SsMTAPII and PfPNP are highly thermoactive, with
an optimum temperature of 120C, and extremely thermostable, with apparent Tm values of 112C and
110 C, respectively These enzymes are also character-ized by a remarkable kinetic stability and resistance to many chemicals, including SDS and guanidinium chlo-ride (GdnCl) [10,12] As demonstrated by structural and biochemical studies, SsMTAPII and PfPNP utilize multiple intrasubunit disulfide bridges as a principal mechanism to achieve superior levels of stability [11,12] A striking structural feature of these two enzymes is the presence in their C-terminal region of a cysteine pair, organized in an unusual CXC sequence motif This structural motif plays an important role in thermal stability because the substitution of cysteine with serine results in a significant lowering of the thermodynamic and kinetic stability parameters of SsMTAPII and PfPNP [10,12] The stabilizing role of the CXC disulfide appears almost intriguing Indeed, disulfide bonds are expected to make a higher contri-bution to thermostability when they join residues far apart in the primary structure Instead, in SsMTAPII and PfPNP, they are separated by a single residue of small size (i.e serine and glycine, respectively) Therefore, the stability imparted to the native protein structure could be almost marginal On the basis of these observations, it is possible to hypothesize that CXC plays an important functional role in stabilizing the protein through an oxidative folding mechanism involving the other structural disulfides of the enzyme Although rare in nature, few examples of oxidized CXC have been reported in the literature, including CSC in the Mengo virus coat protein [19], CSC in the L40C mutant of NADH peroxidase [20], CDC in AK1 protease from Bacillus sp [21], CTC in chaperone Hsp33 from Escherichia coli [22], CPC in redox-regu-lated import receptor Mia40 [23,24] and CGC in MTAP from P furiosus [9], which is highly
Trang 3homo-logous to SsMTAPII and PfPNP It is interesting to
note that a CGC motif at the C-terminus of the yeast
thiol oxidase Erv2p was found to be involved in the
exchange of the de novo synthesized disulfide bridge
with substrate protein [25] Moreover, it was
demon-strated that a synthesized CGC peptide and a CGC
motif in a mutant of E coli thioredoxin were
func-tional mimics of PDI [26] Taken together, these data
allowed us to speculate that the CXC motif in
SsM-TAPII and PfPNP could act as a redox reagent and
exert its stabilizing role by rescuing, in analogy with
PDI, any possible damage of the other disulfide bonds
of the protein To demonstrate the active role of CXC
motif in the oxidative folding process, we carried out
the unfolding of SsMTAPII, PfPNP and their
CXC-lacking mutants by incubation for 22 h at 25C with
6 m GdnCl in 20 mm Tris⁄ HCl (pH 7.4), containing
30 mm dithiothreitol The reversibility of the
GdnCl-induced unfolding was then examined by assaying
the catalytic activity after complete removal of the
denaturant
As shown in Fig 1, SsMTAPII and PfPNP are able
to refold with a recovery of catalytic activity of 59%
and 90%, respectively, compared to their control
enzymes By contrast, lower values of reactivation were
observed for SsMTAPIIC259S⁄ C261S and PfPNPC
254S⁄ C256S, the two CXC-lacking mutants (25% and 46% activity, respectively) These results demonstrate that the CXC motif is necessary to obtain almost com-plete reversibility from the unfolded state, and suggest that the cysteine pair is able to act as redox reagent in the rearrangement of scrambled disulfide bonds, thus contributing to the recovery of native and biologically active enzyme The observation that, in analogy with Erv2p [25], the CXC motif is part of a flexible C-termi-nal segment in SsMTAPII and in PfPNP [10,12], and that the CXC motif is very close to the two pairs of disulfide bridges in SsMTAPII [11], further strengthens this hypothesis
The data obtained in the present study represent the first example of how the CXC motif improves stability and folding in hyperthermophilic disulfide-containing proteins and could provide useful information with respect to engineering stable proteins and enzymes for therapeutic and industrial applications
From the sequence comparison of the C-terminal region of several PNPs present in databases, it appears that, despite their remarkable amino acid sequence identity, the CXC motif is conserved only in hyper-thermophilic enzymes, whereas it is absent in their mesophilic counterparts (Fig 2) This observation suggests a specific role of this structural motif in the stability of hyperthermophilic PNPs against extreme temperature and allows the hypothesis that the CXC disulfide could represent an additional aspect (i.e besides protein disulfide oxidoreductase protein) of the complex system involved in disulfide bond mainte-nance in hyperthermophilic organisms
NH2-GSCSCCN-COOH (SsCSC) and
NH2-RRCGCKD-COOH (PfCGC) act as in vitro catalysts of oxidative protein folding
PDI, the most efficient known catalyst of oxidative folding, is a multifunctional eukaryotic enzyme that utilizes the active site motif CGHC to catalyze the for-mation of native disulfides and the rearrangement of incorrect disulfide bonds, especially those within kineti-cally trapped, structured folding intermediates [13] In recent years, interest in protein folding in vitro has expanded rapidly, mainly focusing on the production,
in bacteria, of disulfide-containing proteins with poten-tial biotechnological applications Therefore, increased attention has been paid to the design and synthesis of novel, small-molecule reagents that could improve the efficiency of the oxidative folding process Recently, on the basis of the physical properties of PDI, a variety
of CXXC peptides have been synthesized and assayed [27] The active site of PDI has also been modeled as a
SsMTAPII
SsMTAPII C259S/C261S
PfPNP PfPNP C254S/C256S
100
60
80
SsMTAPII C259S/C261S PfPNP C254S/C256S
0
20
40
Fig 1 Refolding of SsMTAPII, PfPNP and their CXC-lacking
mutants after induced unfolding The reversibility of
Gdn-induced unfolding of MTAPII, PfPNP and their respective mutants
was started by a 20-fold dilution of the unfolding mixture and
extensive dialysis until complete removal of GdnCl Refolding was
analyzed by catalytic activity measurements performed under
stan-dard conditions U ⁄ R indicates SsMTAPII, PfPNP and their mutants
refolded after GdnCl-induced unfolding The activity of control
enzymes was expressed as 100% Each value is the average of
three separate experiments.
Trang 4CGC peptide, a molecule that, upon oxidation, forms
a strained 11-membered disulfide ring, representing a
good oxidizing agent [26] This peptide shows a
disul-fide reduction potential close to that of PDI and its
first thiol pKa is less than that of the natural redox
reagent glutathione Therefore, this CXC peptide is
able to function as an efficient catalyst of disulfide
isomerization [26] On the basis of these observations,
and in analogy with PDI, it is possible to hypothesize
a nucleophilic attack of the thiolate from CXC on an
incorrect protein disulfide followed by a thiol–disulfide
interchange within the substrate, leading in turn to a
native disulfide Two CXC-containing peptides, namely
SsCSC and PfCGC, whose amino acid sequences are
identical to those present in the C-terminal region of
SsMTAPII and PfPNP, respectively, have been
synthe-sized and their disulfide isomerase activity has been
assayed utilizing the partially refolded forms of
SsMTAPII, PfPNP and their CXC-lacking mutants as
substrates As shown in Fig 3, both peptides are
involved in the oxidative folding of these enzymes with
a concomitant recovery of their catalytic activity
Indeed, after 22 h of incubation in the presence of
SsCSC and PfCGC, the enzymatic activity of
SsMTAPII and its mutant, expressed as a percentage
of their control enzymes, reaches 86.8% and 51.8%, and 68% and 49.3%, respectively (Fig 3A) Similar results were obtained when the unfolded⁄ refolded forms of PfPNP and its mutant were assayed under the same experimental conditions (Fig 3B) It is inter-esting to note that, although PfPNP and its mutant show a higher reactivation values than SsMTAPII and its mutant (Fig 3), the ratio of these values with respect of their control enzymes is similar, thus indicat-ing that the efficiency of the process is comparable These data demonstrate that the CXC motif of SsM-TAPII and PfPNP is active, even when isolated from the proteins, and that it is able to induce the in vitro oxidative folding of these enzymes
To further confirm the ability of SsCSC and PfCGC
to function as efficient catalysts of oxidative protein folding, the disulfide isomerase activity of these CXC-containing peptides was assayed by monitoring the reactivation of scrambled RNaseA (sRNaseA) As shown in Fig 4, after 210 min of incubation in the presence of SsCSC and PfCGC, inactive sRNaseA shows a 5.5-fold and 3.6-fold activation, respectively, compared to the 8.9-fold activation observed in the presence of PDI These results demonstrate that the two CXC-containing peptides can act in the same way
Fig 2 Multiple sequence alignment of C-terminal regions of hyperthermophilic and mesophilic PNPs The CXC motif is shown in white lettering on a black background.
100
SsMTAPII C259S/C261S
PfPNP PfPNP C254S/C256S
20
40
60
80
0
U|R
U/R + PDI
U/R + SsCSC U/R + PfCGC
U|R U/R + PDI U/R + SsCS C
U/R + PfCGC
Fig 3 Effect of CXC-containing peptides on the reactivation of refolded SsMTAPII, PfPNP and their mutants SsMTAPII, PfPNP and their mutants, partially refolded after GdnCl-induced unfolding, (U ⁄ R), were incu-bated for 22 h at 30 C in the presence of various oxidative folding catalysts (reactiva-tion assay) The catalytic activity of (A) SsM-TAPII and SsMSsM-TAPIIC259S ⁄ C261S and (B) PfPNP and PfPNPC254S ⁄ C256S was then measured under standard assay conditions The activity of control enzymes was expressed as 100% Each value is the average of three separate experiments.
Trang 5as PDI, catalyzing the rearrangement of incorrect
disulfide bonds in a protein substrate It is interesting
to note that SsCSC displays a higher oxidative folding
activity than PfCGC (Figs 3 and 4), suggesting that
the presence in SsCSC of a third thiol in the sequence
CXCC could most likely enhance the reactivity toward
disulfide bonds
In conclusion, the results obtained in the present
study provide insight into the variety of molecular
mechanisms utilized for stabilizing folded proteins under
extreme thermal environments and allow us to speculate
that disulfide bonds and the CXC motif could combine
together to provide a novel stabilization strategy
Experimental procedures
Materials
Glutathione disulfide (GSSG), glutathione (GSH), bovine
liver PDI and bovine pancreatic sRNaseA were obtained
from Sigma GdnCl and dithiothreitol were obtained from
Applichem (Darmstadt, Germany) [methyl-14C]AdoMet
(50–60 mCiÆmmol)1 was supplied by the Radiochemical
Centre (Amersham Bioscience, Little Chalfont, UK) MTA
and 5¢-[methyl-14
C]MTA were prepared from unlabeled and
labeled AdoMet [10] Specifically synthesized
CXC-contain-ing peptides, PfCGC and SsCSC, were obtained from
PRIMM (Milan, Italy) All reagents were of the purest
commercial grade
Expression and purification of SsMTAPII, PfPNP
and their mutant forms
SsMTAPII, PfPNP and their CXC-lacking mutants (i.e
SsMTAPIIC259S⁄ C261S and PfPNPC254S ⁄ C256S) utilized
for these studies were expressed and purified as previously described [10,12] Manipulations of DNA and E coli were carried out using standard protocols [10,12,28] Protein concentration was determined by the Bradford assay [29]
Assays of enzyme activity
PNP activity was determined by monitoring the formation
of purine base from the corresponding nucleoside by HPLC using a Beckman System Gold apparatus (Beckman Coul-ter, Fullerton, CA, USA) The assay was carried out as described previously [12]
MTAP activity was determined by monitoring the forma-tion of [methyl-14C]5-methylthioribose-1-phosphate from 5¢-[methyl-14C]MTA [10] In all enzymatic assays, the amount of the protein was adjusted so that no more than 10% of the substrate was converted to product and the reaction rate was strictly linear as a function of time and protein concentration
GdnCl-induced unfolding and refolding
SsMTAPII, PfPNP and their respective CXC-lacking mutants (final concentration 0.4 mgÆmL)1) were incubated for 22 h at 25C in the presence of 6 m GdnCl in 20 mm Tris⁄ HCl (pH 7.4) containing 30 mm dithiothreitol Unfold-ing was probed by recordUnfold-ing the intrinsic fluorescence emis-sion To test the reversibility of the process, the refolding was started by a 20-fold dilution of the unfolding mixture
in Tris⁄ HCl 20 mm (pH 7.4) The refolded enzyme, after extensive dialysis against Tris⁄ HCl 20 mm (pH 7.4) until complete removal of GdnCl, was analyzed by catalytic activity measurements performed under standard condi-tions
Reactivation assay of SsMTAPII, PfPNP and their CXC-lacking mutants
The activity of SsCSC and PfCGC as catalysts of oxidative protein folding was tested by the ability to reactivate SsMTAPII, PfPNP, SsMTAPIIC259S⁄ C261S and PfPNPC254S⁄ C256S after GdnCl-induced unfolding and refolding
SsCSC and⁄ or PfCGC were first reduced with a five-fold excess of dithiothreitol in 50 mm Tris⁄ HCl (pH 7.4) for
10 min at 30C and then incubated at 30 C for 22 h in a reactivation mixture containing (in a final volume of
50 lL): 50 mm Tris⁄ HCL (pH 7.4), 2 mm EDTA, a gluta-thione redox buffer (1 mm GSH, 0.2 mm GSSG), 540 lm CXC peptide, and the protein to be reactivated (3 lg; final concentration 2 lm) After the incubation, SsMTAPII or PfPNP activity was measured under standard conditions The activity of control enzymes was expressed as 100% The positive control was represented by the reactivation of
0.8
0.4
0
Time (min)
Fig 4 Time course for the reactivation of sRNaseA by various
oxi-dative folding catalysts - - -, None; , PDI; d, SsCSC; , PfCGC.
Each value is the average of three separate experiments.
Trang 6the enzymes catalyzed by PDI (final concentration 0.11 lm)
under the same experimental conditions
Reactivation of sRNaseA
Disulfide isomerase activity was assayed as described
previ-ously [30] by monitoring the reactivation of sRNaseA, a
fully oxidized protein containing a random distribution of
its four disulfide bonds SsCSC and PfCGC were first
reduced with a five-fold excess of dithiothreitol in 50 mm
Tris⁄ HCl (pH 7.4) for 10 min at 30 C and then incubated
for 1 h at 30C in a reactivation mixture containing 0.1 m
Tris-acetate buffer (pH 8.0), 2 mm EDTA, a glutathione
redox buffer (1 mm GSH, 0.2 mm GSSG), 2 mm
CXC-pep-tide, and sRNaseA (0.5 mgÆmL)1in 10 mm acetic acid; final
concentration 19.6 lm) cCMP at a final concentration of
4 mm was then added and A296, as a result of
RNase-cata-lyzed cCMP hydrolysis, was monitored continuously for
210 min at 30C The positive control was the reactivation
of sRNaseA catalyzed by PDI (final concentration 4 lm)
The control for the non-enzymatic reactivation of sRNaseA
was represented by the same mixture without the addition
of any oxidative folding catalyst
References
1 Riemer J, Bulleid N & Herrmann JN (2009) Disulfide
formation in the ER and mitochondria: two solutions
to a common process Science 324, 1284–1287
2 Matsumara M, Signor G & Mathews BW (1989)
Susb-stantial increase of protein stability by multiple disulfide
bonds Nature 342, 291–293
3 Gilbert HF (1990) Molecular and cellular aspects of
thiol-disulfide exchange Adv Enzymol Relat Areas Mol
Biol 63, 69–172
4 Aslund F & Beckwith J (1999) Bridge over troubled
waters: sensing stress by disulfide bond formation Cell
96, 751–753
5 Prinz WA, Aslund F, Holmgren A & Beckwith J (1997)
The role of the thioredoxine and glutaredoxine
path-ways in reducing protein disulfide bonds in the
Escheri-chia colicytoplasm J Biol Chem 272, 15661–15667
6 Toth EA, Worby C, Dixon JE, Goedken ER, Marqusee
S & Yeates TO (2000) The crystal structure of
adenylo-succinate lyase from Pyrobaculum aerophilum reveals an
intracellular protein with three disulfide bonds J Mol
Biol 301, 433–450
7 Appleby TC, Mathews II, Porcelli M, Cacciapuoti G &
Ealick SE (2001) Three-dimensional structure of a
hyperthermophilic 5¢-deoxy-5¢-methylthioadenosine
phosphorylase from Sulfolobus solfataricus J Biol Chem
42, 39232–39242
8 Meyer J, Clay MD, Johnson MK, Stubna A, Munch E,
Higgins C & Wittung-Stafshede P (2002) A
hypertherm-ophilic plant-type 2Fe-2S ferredoxin from Aquifex
aeoli-cusis stabilized by a disulfide bond Biochemistry 41, 3096–3108
9 Cacciapuoti G, Moretti MA, Forte S, Brio A, Camard-ella L, Zappia V & Porcelli M (2004) Methylthioadeno-sine phosphorylase from the archaeon Pyrococcus furiosus.Mechanism of the reaction and assignment of disulfide bonds Eur J Biochem 271, 4834–4844
10 Cacciapuoti G, Forte S, Moretti MA, Brio A, Zappia V
& Porcelli M (2005) A novel hyperthermostable 5¢-deoxy-5¢-methylthioadenosine phosphorylase from the archaeon Sulfolobus solfataricus FEBS J 272, 1886–1899
11 Zhang Y, Porcelli M, Cacciapuoti G & Ealick SE (2006) The crystal structure of 5¢-deoxy-5¢-methylthio-adenosine phosphorylase II from Sulfolobus solfataricus,
a thermophilic enzyme stabilized by intramolecular disulfide bonds J Mol Biol 357, 252–262
12 Cacciapuoti G, Gorassini S, Mazzeo MF, Siciliano RA, Carbone V, Zappia V & Porcelli M (2007) Biochemical and structural characterization of mammalian-like purine nucleoside phosphorylase from the archaeon Pyrococcus furiosus FEBS J 274, 2482–2495
13 Wilkinson B & Gilbert HF (2004) Protein disulfide isomerase Biochim Biophys Acta 1699, 35–44
14 Pedone E, Limauro D & Bartolucci S (2008) The machinery for oxidative protein folding in thermophiles Antioxid Redox Signal 10, 157–169
15 Bzowska A, Kulikowska E & Shugar D (2000) Purine nucleoside phosphorylases: properties, functions and clinical aspects Pharmacol Ther 88, 349–425
16 Williams-Ashman HG, Seidenfeld J & Galletti P (1982) Trends in the biochemical pharmacology of 5¢-deoxy-5¢-methylthioadenosine Biochem Pharmacol 31, 277–288
17 Appleby TC, Erion MD & Ealick SE (1999) The structure of human 5¢-deoxy-5¢-methylthioadenosine phosphorylase at 1.7 A˚ resolution provides insights into substrate binding and catalysis Structure 7, 629–641
18 Mao C, Cook WJ, Zhou M, Federov AA, Almo SC & Ealick SE (1998) Calf spleen purine nucleoside phos-phorylase complexed with substrates and substrate analogues Biochemistry 37, 7135–7146
19 Krishnaswamy S & Rossmann MG (1990) Structural refinement and analysis of Mengo virus J Mol Biol
211, 803–844
20 Miller H, Mande SS, Parsonage D, Sarfaty SH, Hol
WG & Claiborne A (1995) An L40C mutation converts the cysteine-sulfenic acid redox center in enterococcal NADH peroxidase to a disulfide Biochemistry 34, 5180–5190
21 Smith CA, Toogood HS, Baker HM, Daniel RM & Baker EN (1999) Calcium-mediated thermostability in the subtilisin superfamily: the crystal structure of Bacil-lusAk.1 protease at 1.8 A˚ resolution J Mol Biol 294, 1027–1040
Trang 722 Jakob U, Muse W, Eser M & Bardwell JC (1999)
Chaperone activity with a redox switch Cell 96, 341–
352
23 Banci L, Bertini I, Cefaro C, Ciuffi-Baffoni S, Gallo A,
Martinelli M, Sideris DP, Katrakili N & Tokatiidis M
(2009) MIA40 is an oxidoreductase that catalyzes
oxida-tive protein folding in mitochondria Nat Struct Mol
Biol 16, 198–206
24 Terziyska N, Grumbt B, Kozany C & Hell K (2009)
Structural and functional roles of the conserved cysteine
residues of the redox-rehulated import receptor Mia40
in the intermembrane space of mitochondria J Biol
Chem 284, 1353–1363
25 Gross E, Sevier CS, Vala A, Kaiser CA & Fass D
(2002) A new FAD-binding fold and intersubunit
disul-fide shuttle in the thiol oxidase Erv2p Nat Struct Biol
9, 61–67
26 Woycechowsky KJ & Raines RT (2003) The CXC motif: a functional mimic of protein disulfide isomerase Biochemistry 42, 5387–5394
27 Lees WJ (2008) Small-molecule catalysts of oxidative protein folding Curr Opin Chem Biol 12, 740–745
28 Sambrook J, Fritsch EF & Maniatis T (1989) Molecular Cloning: A Laboratory Manual Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY
29 Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding Anal Biochem 72, 248–254
30 Lyles MM & Gilbert HF (1991) Catalysis of the oxida-tive folding of ribonuclease A by protein disulfide iso-merase: pre-steady-state kinetics and the utilization of the oxidizing equivalents of the isomerase Biochemistry
30, 619–625