[7] Protein Disulfide oxidoreductase from Pyrococcus furiosus: Biochemical properties

62

[71

REDOX AND THIOL-DEPENDENT PROTEINS

those of the dimeric enzyme of D. gigas. 6 Interestingly, under comparable assay conditions at 25-30 °, P. furiosus NROR is much more active (290 units/mg) 1 than either of these mesophilic enzymes (_<50 U/mg). 3'4 Such a comparison serves to emphasize the remarkable activity of the hyperthermophilic NROR, and perhaps adds some credence to its proposed physiological role at temperatures more than 70 ° below that which supports growth of P. furiosus. Acknowledgment This research was supported by grants from the Department of Energy.

[7] Protein Disulfide Oxidoreductase from

Pyrococcusfuriosus: Biochemical Properties By SIMONETTA BARTOLUCCI, DONATELLA DE PASCALE, a n d

Mos~ R o s s I

Introduction Protein disulfide oxidoreductases (PDI) are redox enzymes that catalyze dithiol-disulfide exchange reactions in eukaryotic and bacterial cells. These enzymes have a redox-active disulfide/dithiol group in the conserved motif CXXC. The two cysteine residues in the active site sequence can undergo reversible oxidation-reduction. Several proteins belonging to this superfamily have been identified and characterized; thioredoxin, glutaredoxin, protein disulfideisomerase (PDI), disulfide bond forming (DsbA), and their homologs in prokaryotes are the most extensively studied members of this groupJ Thioredoxin and glutaredoxin, with a molecular mass of about 12 kDa, have at the N terminus the conserved active site sequence CGPC and CPYC, respectively.2 They typically transfer electrons from NADPH to the substrate in reactions coupled with other specific enzymes. Glutaredoxin derives its reducing equivalents from glutathione, which in turn is linked to the glutathione reductase/NADPH system, whereas thioredoxin utilizes the thioredoxin reductase/NADPH pathway for the same purpose. Thioredoxin is involved in maintaining the redox status of sulfhydryl groups inside the cell and participates in catalyzing and/or regulating a variety of cellular functions, including the ability to activate surface receptors and regulate microtubule assembly) In other systems thioredoxin is I H. Loferer and H. Hennecke, TrendsBiochem. Sci. 19, 169 (1994). 2 A. Holmgren and E Aslund, Methods Enzymol. 252, 283 (1995).

METHODSINENZYMOLOGY,VOL.334

Copyright© 2001 by AcademicPress All rightsof reproductionin any formreserved. 0076-6879/00 $35.00

[7]

P. furiosus PDIS" BIOCHEMICALPROPERTIES

63

reduced by a photoreduced ferredoxin via an iron-sulfur ferredoxin-thioredoxin reductase. 4 The endoplasmic reticulum PDI, which catalyzes the reduction, oxidation, and reshuffling of protein disulfides in eukaryotes, 5'6 is a homodimer of two 57 kDa subunits. Each subunit contains two functional domains with significant sequence homology to Escherichia coli thioredoxin. 7 The functional equivalent of PDI in prokaryotes, DsbA, is a periplasmic, monomeric protein characterized by a low similarity to E. coli thioredoxin with the active site sequence motif CPHC. 8'9 Little information is availble on protein disulfide oxidoreductases in Archaea. A glutaredoxin-like protein, isolated from Methanobacterium thermoautotrophicum, was shown to have a molecular mass of 9 kDa, and a low sequence identity (<20%) to known glutaredoxin, and not to function as glutaredoxin-dependent enzyme.l° A highly thermostable protein disulfide oxidoreductase was first isolated from Sulfolobus solfataricus. I I From its ability to catalyze the reduction of insulin disulfides in the presence of dithiothreitol (DTT), the protein was considered a thioredoxin. The protein showed an unusually high molecular mass of 25 kDa and from amino acid composition analysis contained four cysteine residues. A homologous protein was subsequently purified from Pyrococcusfuriosus. 12 From its amino acid sequence, which showed two distinct CXXC motifs, and from its thioltransferase activity the protein was considered to be a glutaredoxin-like protein. More recently the protein was crystallized, and its three-dimensional (3-D) structure at high resolution revealed structural details suggesting it may be related to the multidomain eukaryotic PDI. For this reason the protein was more correctly named protein disulfide oxidoreductase from P furiosus (Pf PDO). 13-t5 In this paper we describe the purification of the P f PDO and the cloning and its gene expression in E. coli.

3 j. H. Wong,K. Kobrehel,and B. B. Buchanan,Methods Enzymol. 252, 228 (1995). 4 p. Schurmann,Methods Enzymol. 252, 274 (1995). 5 j. C. Edman, L. Ellis, R. Blacher, R. A. Roth, and W. J. Rutter,Nature 319, 267 (1985). 6 j. Kemmink,N. J. Darby,Dijkstra, M. Nilges, and T. E. Creighton, Curr. Biol. 7, 239 (1997). 7 j. Bardwelland J. Beckwith, Cell 74, 769 (1993). 8 j. L. Martin,J. C. A. Bardwell, and J. Kurigan,Nature 365, 464 (1993). 9 R. E Golberg,C. J. Epstein,and C. B. Anfinsen,J. Biol. Chem. 238, 628 (1968). 10 S. McFarlan,C. A. Terrell,and H. P. C. Hogenkamp,Z Biol. Chem. 267, 10561 (1992). II A. Guagliardi,V. Nobile,S. Bartolucci,and M. Rossi, Int. J. Biochem. 26, 375 (1994). 12A. Guagliardi,D. de Pascale, R. Cannio,V. Nobile,S. Bartolucci,and M. Rossi,J. Biol. Chem. 270, 5748 (1995). J3 B. Ren, G. Tibellin,D. de Pascale, M. Rossi, S. Bartolucci, and R. Ladenstein,J. Struct. Biol. 119, (1997). 14 B. Ren, G. Tibellin,D. de Pascale, M. Rossi, S. Bartolucci,and R. Ladenstein,Nature Struct. Biol. 5, 602 (1998). 15 B. Ren and R. Ladenstein,Methods in Enzymology 334 [8] (2001) (this volume).

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[7]

P u r i f i c a t i o n o f Native P r o t e i n

Pf PDO is purified to homogeneity by two chromatography steps involving anion exchange and gel filtration. SDS-PAGE analysis performed according to Laemmli 16 of the active sample from the gel filtration column shows one protein band with molecular mass of about 26 kDa. The reductase activity was found to be associated with this protein throughout the purification steps. As it is difficult to quantify the assay of the activity, based on insulin precipitation, no yield and purification factors are calculated. In general 0.5 mg of homogeneous PfPDO is obtained from 10 g of cells (wet weight). Preparation of Crude Extract P.furiosus is grown at 95 ° and pH 7.5 in a medium containing the following components (per liter): Bacto-peptone (5 g), yeast extract (1 g), NaCI (19.4 g), MgSO4 (12.6 g), NazSO4 (3.2 g), CaCI2 (2.4 g), KCI (0.5 g), NaHCO3 (0.16 g), KBr (0.08 g), SrC12 (0.057 g), H3PO3 (0.022 g), Na2SO3 (0.004 g), NaF (0.0024 g), KNO3 (0.0016 g), NazHPO4 (0.01 g), and sulfur (25 g). The culture is bubbled with N2 (5 liter/min). The cells are harvested after 8 h, and immediately frozen in liquid N2 and stored at - 8 0 ° until required. For enzyme purification, 10 g (wet weight) of cells is freeze-thawed and resuspended in 40 ml of 20 mM Tris buffer, pH 8.4, 2 mM EDTA, 1 M NaC1, and homogenized in a French press for 20 rain. The homogenate is centrifuged at 160,000g for 90 min at 4 °; the supernatant (30 ml) represents the crude extract, and this catalyzes the reduction of insulin in the presence of DTT at 30 ° as described below. Anion-Exchange Chromatography The crude extract (1.0 g of protein in 30 ml) is extensively dialyzed against buffer A (20 mM Tris buffer, pH 8.4, 2 mM EDTA) and loaded onto a DEAESepharose Fast Flow column (Pharmacia, Uppsala, Sweden, 2.2 × 18 cm) equilibrated in the same buffer. Bound proteins are eluted at a flow rate of 50 ml/hr by a linear gradient from 0 to 0.3 M NaC1 in buffer A, and 5-ml fractions are collected. The active fractions are pooled, concentrated in a Savant centrifuge (Savant Speed Vac SC 110), and dialyzed against buffer A.

Gel-Filtration Chromatography The active fractions from the DEAE-Sepharose Fast Flow column (0.1 g of protein in 6 ml) are loaded in three separate runs onto a HiLoad Superdex 75 column (Pharmacia, 2.6 x 60 cm) connected to an FPLC system (Pharmacia) eluted with buffer B (20 mM riffs buffer, pH 8.4, 2 mM EDTA, 1 M NaC1) at a 16U. K. Laemmli,Nature227, 680 (1970).

[7]

P furiosus PDIS: BIOCHEMICALPROPERTIES

65

flow rate of 2 ml/min. The active fractions (3 ml each) are pooled and concentrated in a Savant centrifuge. The yield was 0.5 mg of homogeneous protein (95% of homogeneity). The protein concentration is determined by the Bio-Rad (Hercules, CA) dye-binding assay, 17 using bovine serum albumin (BSA) as the standard.

High-Performance Liquid Chromatographs For analytical purposes Pf PDO can be further purified by high-performance liquid chromatography (HPLC) with a C4 reversed-phase column (Vydac 0.46 × 25 cm, 0.5 Ixm size particles) with 0.1% trifluoroacetic acid TFA (v/v) in H20 as buffer C, and 0.07% TFA in acetonitrile as buffer D. Starting from buffer C a linear gradient (0-100% D) was performed in 60 min. N-Terminal Sequence Analysis The N-terminal sequence of the homogeneous protein from P. furiosus can be determined by automatic Edman degradation using an Applied Biosystems 473A sequencer (Foster City, CA), according to the manufacturer's instructions. The following 29 residues were identified: GLISDADKKVIKEE FFSKMVNPVKLIVF'v: Cloning, O v e r e x p r e s s i o n , a n d P u r i f i c a t i o n of P f PDO Since the purification procedure using R furiosus required a large amount of cells, Pf PDO was produced as a soluble recombinant protein in E. coli. The recombinant Pf PDO is indistinguishable from the native protein isolated from P. furiosus in all activity assays tested, including insulin reduction.

Isolation of Chromosomal P. furiosus DNA Ten grams of Pfuriosus cells are suspended in 25 ml of 50 mM Tris-HC1 buffer, pH 8.0, 10 mM EDTA, and centrifuged at 3000g for 10 min at 4 °. Cells are resuspended in 20 ml in the same buffer containing 0.2% Triton X- 100 and 1% SDS; the soluble fraction, cleared by centrifugation at 129,500g for 30 min at 4 °, is heated for 10 min at 65 ° and centrifuged again in the same conditions. Cesium chloride (1 g/m1) and ethidium bromide (0.6 mg/ml) are added and the samples are ultracentrifuged at 352,000g for 16 hr at 16-18°C. Chromosomal DNA bands are revealed by irradiation using a 340-nm UV lamp and withdrawn as described by Sambrook et al. 18 Ethidium bromide and cesium chloride are removed, respectively, by 17M. M. Bradford,Anal. Biochem.72, 248 (1976). 18j. Sambrook,E. E Fritsch,and T. Maniatis, "MolecularCloning: A LaboratoryManual,"2nd ed. Cold SpringHarborLaboratory,Cold SpringHarbor,NY, 1989.

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[7]

extraction with isoamyl alcohol and extensive dialysis against 10 mM Tris-HC1 buffer, pH 8.0, 1 mM EDTA. DNA concentration is determined spectrophotometrically at 260 nm, and the molecular weight is estimated by electrophoresis on 0.6% agarose gel in 90 mM riffs borate, 20 mM EDTA (TBE buffer) using suitable DNA molecular size markers (Roche) according to Sambrook et al. 18 Construction of P.furiosus Gene Bank A representative genomic bank in pGEM7Zf(+) (Promega, Madison, WI) was found to be highly efficient for the insertion of one single DNA fragment from P.furiosus genome per vector molecule. 19 To prepare the library, high molecular weight DNA of P.furiosus is partially digested with Sau3AI (1 hr of incubation with 0.15 units per Ixg of genomic DNA), and the fragments 2- 4 kb in size are isolated by electroelution from 3.5% polyacrylamide gel electrophoresis. The fragments are subjected to partial filling in with E. coli DNA polymerase (Klenow fragment), dGTP, and dATP and inserted into the vector pGEM7Zf(+), which previously has been made end-compatible by linearization with XhoI and by partial filling in with the Klenow fragment, dCTP, and dTTP. E. coli BO3310-competent cells are transformed with the ligation mixture. Growth in Luria-Bertani medium (LB medium) (10 g Bacto-tryptone, 5 g Bacto-yeast, 5 g NaC1 in 1 liter) containing 100 Ixg/ml ampicillin for 4 h 18 allows the propagation and amplification of the gene bank. Cloning of Gene The screening of the genomic bank of P.furiosus is performed using colony hybridization experiments carried out under different stringency conditions with 5 × SSC, 0.5% SDS (two changes of 30 min each) at 45 ° and at 55 °.The oligonucleotide mixture for the screening was designed on the N-terminal sequence of the protein from residue 8 to residue 20 and the codon usage in Archaea. 2° Ten pmol of 41mer oligonucleotide mixture (5'-GATAAGAAGGT (G/T)AT(T/A)AAGGAGGAGTrTTTr(TIA)(C/G)(T/G)AAGATGGT-3') is end-labeled with ['y-32p]ATP (Amersham International) by T4 polynucleotide kinase and utilized as probe. Replica filters are used as a control of signal specificity. Clones positive to a first selection are definitively isolated by a second colony hybridization screening performed according to the same protocol. After the sequential screenings, 5 clones OUt of 5 × 10 4 exhibiting positive hybridization signals are isolated; one clone, named pDR7, had a 2.3-kb region encompassing the complete gene sequence. The whole coding sequence is labeled by incorporation of [a-32p]dATP using the random priming method and the specific kit purchased from Roche Molecular Biochemicals (Mannheim, Germany). The hybridization of the whole coding 19 R. Cannio, M. Rossi, and S. Bartolucci, Eur. J. Biochem. 222, 345 (1994). 20 j. Hain, W.-D. Reiter, U. Hudepohl, and W. Zilling, Nucleic Acids Res. 20, 5423 (1992).

P. f u r i o s u s PDIS" BIOCHEMICAL PROPERTIES

[7] 1

67 M

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FIG. 1. The deduced amino acid sequence of Pyrococcusfuriosus protein disulfide oxidoreductase (Pf PDO). The amino acids in bold-face type represent the two putative active sites.

sequence as a probe to the P. furiosus chromosomal DNA produces results consistent with the restriction maps of the isolated clone, thus confirming that the recombinant clone underwent no rearrangements during the cloning procedures. The isolated clone is sequenced completely. Suitable restriction fragments are subcloned into pUC18 plasmid and sequenced using the universal M13 forward and reverse primers or specific synthetic oligonucleotides. DNA sequencing is carried out by the dideoxy chain termination method 2l with [35S]dATP, using Sequenase version 2.0 sequencing kit (Amersham) on alkali-denatured double stranded templates. A 675-bp open reading frame (ORF) starting from ATG and ending at TAA as well as extended flanking regions were found. Figure 1 shows the deduced primary structure of the P. furiosus protein. The 226 amino acid residues derived from the ORF matched the calculated size of the native protein whose N-terminal sequence lacks the initial methionine. The residues from Phe-141 to Cys-149 identify the FXXXXCXXC motif, which is a part of the active site of the enzymes that catalyze dithiol/disulfide interchange reactions. When the primary structure of the P. furiosus protein was compared with GenBank using the Fasta program, similarity with the active site sequence of the glutaredoxin family was detected, el F. Sanger, S. Nicklen, and A. R. Coulson, Proc. Natl. Acad. Sci. U.S.A. 74, 5463 (1977).

68

REDOX AND THIOL-DEPENDENT PROTEINS

[7]

with the sequence CPYC at residues 146-149 typical of this family. The sequence CQYC at resides 35-38 is not conserved in the active site of any of the known protein disulfide oxidoreductases. Construction of Expression Vector The complete coding sequence of the gene is obtained by gene amplification performed according to previously described procedure22 for 25 cycles at 55 ° annealing temperature, on a Perkin-Elmer (Norwalk, CT) cycler temp. The oligonucleotides used as primers were 5'-GgccATGGGATTGATTAGTGAC-3' (PfN) and 5'-gagAGTCGAGAGTCGACTAGATCTGCG-Y (PfC), where the underlined letters indicate the initiation and termination codons and small letters indicate point mutations inserted to generate the NcoI and XbaI sites. The pDR7 plasmid, containing the whole gene, is linearized with BamHI and used as template for Pfu (Stratagene, La Jolla, CA, Cloning Systems) polymerase. The amplified fragment is digested with NcoI and XbaI and inserted into pTRC99A plasmid (Pharmacia), previously made end-compatible with the same restriction endonucleases. Restriction analysis and subsequent sequencing confirmed the correct sequence of the recombinant clone, which is designated as pPfPDO. The newly constructed plasmid pPfPDO is used as expression vector: in fact, the transcription of the recombinant gene is controlled by the strong hybrid regulatory sequence promoter ptac, derived from the fusion of the lac and trp promoters, and can still be inducible by isopropyl-13-D-thiogalactopyranoside (IPTG).

H i g h Level E x p r e s s i o n a n d P u r i f i c a t i o n o f R e c o m b i n a n t P r o t e i n E. coli Rb791-competent cells are transformed with the newly constructed pPfPDO expression vector and grown at 37 ° in 1 liter of Luria-Bertani medium. E. coli strain Rb791 cells transformed with pTRC99A (Pharmacia) represents a negative control. The expression of the P.furiosus protein is induced by the addition of 1 mM IPTG (final concentration) to the culture medium, when the growth was at Ar00 of 1. The induction time was 18 hr. Cells are harvested by centrifugation at 3000g for 10 min at 4 ~, washed with 30 ml of ice-cold 50 mM sodium phosphate buffer, pH 8.0, 0.1 M NaC1, 1 mM EDTA, and resuspended in 48 ml of the same buffer supplemented with 0.7 mM phenylmethanesulfonyl fluoride (PMSF). Ceils are sonicated three times with a frequency of 20 kHz (Sonicator Ultrasonic liquid processor; Heat System Ultrasonic Inc., Farmingdale, NY) and ultracentrifuged at 160,000g for 90 rain at 4 °. The supernatant of the ultracentrifugation constitutes the crude extract. 22 R. K. Saiki, "PCR Protocols: A Guide to Methods and Applications" (M. A. Innis, D. A. Gelfand, J. J. Sninski, and T. J. White, eds.). Academic Press, San Diego, 1990.

[7]

e. furiosus PDIS: BIOCHEMICALPROPERTIES

69

Heat Treatments The crude extract undergoes two successive thermal precipitation steps. Most of the E. coli proteins become insoluble and are removed by centrifugation, whereas the recombinant protein is recovered in an active form in the supernatant. The crude extract (85 mg of protein in 40 ml) is heated at 70 ° for 10 min and centrifuged at 5000g at 4 ° for 10 min; the supernatant is concentrated by ultrafiltration in an Amicon (Danvers, MA) cell (membrane cutoff 2000 Da), subjected to a second heating at 75 ° for 10 min, and centrifuged as above. Following the thermoprecipitation steps, about 80% of the E. coli proteins are removed.

Gel-Filtration Chromatography The sample from heat treatment (28 mg of protein in 2 ml) is loaded onto a HiLoad Superdex 75 column (Pharmacia, 2.6 × 60 cm) connected to an FPLC (fast protein liquid chromatography) system (Pharmacia) eluted with buffer B (20 mM Tris buffer, pH 8.4, 2 mM EDTA, 1 M NaC1) at a flow rate of 2 ml/min. The active protein (18 mg) is stored at - 2 0 °. This protein is generally >90% homogeneous, as judged by (12%, w/v) SDS-PAGE. The molecular mass of the recombinant protein is measured on a Bio-Q triple quadruple mass spectrometer (Micromass, Manchester, UK). Samples are dissolved in 1% (v/v) acetic acid/50% (v/v) acetonitrile and injected into the ion source at a flow rate of 10 ml/min using a Phoenix syringe pump. Spectra are collected and elaborated using the MASSLYNX software provided by the manufacturer. Assay Methods

Insulin Activity Assays Insulin reductase activity is assayed according to Holmgren 23 with a few modifications by measuring the catalytic reduction of insulin disulfide bonds at 30 ° . P f P D O (approximately 20 ixg) is added in 1 ml of 100 mM sodium phosphate buffer, pH 7.0, containing 2 mM EDTA and 1 mg of bovine insulin. A control cuvette contains only buffer and insulin. The reaction is started by addition of 1 mM dithiothreitol (DTT) to both cuvettes. Increasing turbidity from precipitation of insulin B chain is recorded at 650 nm. The stock solution of insulin (10 mg/ml) is prepared according to the Holmgren23 protocol.

Thioltranferase Activity Pf PDO has a glutathione (GSH)-dependent disulfide reductase activity (named the thioltransferase activity) that can be assayed by coupling to saturating 23A. Holmgren,J. Biol. Chem.254,9627 (1979).

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REDOX AND THIOL-DEPENDENTPROTEINS

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concentrations of glutathione reductase, which reduces glutathione, oxidized form GSSG in the presence of NADPH, 24 by using L-cystine as the disulfide substrate. The standard assay mixture consists of a 0.1 M sodium phosphate buffer, pH 8.0, 1 mM EDTA, 10 mM GSH, 2.5 mM L-cystine, 0.2 units of glutathione reductase (Sigma Aldrich, Milwaukee, WI), 0.3 mM NADPH in the absence or presence of 5 p.g PfPDO (final volume, 1 ml); enzyme activity is monitored at 340 nm and 30 °. Activity dependence on pH is determined by the standard assay method except that 0.1 M sodium phosphate buffer is used in the pH range 6.0 to 8.0 and 0.1 M Tris-HC1 buffer 8.0 to 9.0. The enzyme shows a sharp optimum at pH 8.0.

Oxidation Activity The disulfide bond-forming activity of PfPDO is monitored using the synthetic decapeptide NRCSQGSCWN containing two cysteine residues at position 3 and 8 designed by Ruddock et al. 25 The peptide contains a fluorescent group (tryptophan) on one side of one cysteine residue and a protonated group (arginine) on the other side of the second cysteine residue, and the two cysteine residues are separated by a flexible linker region. The linker is long enough to permit the formation of an unstrained disulfide bond, and the peptide is small and water soluble. Oxidation of this dithiol peptide to the disulfide state is accompanied by a change in tryptophan fluorescence emission intensity. In fact, on oxidation, the fluorescent group and the protonated group are brought close together and quenching of the fluorophore occurs where arginine is the charged quencher. Fluorescence quenching was used as the basis for monitoring the disulfide bond-forming activity of the eukaryotic enzyme protein disulfide-isomerase (PDI) and the bacterial enzyme DsbA at pH 7.0, 25 and with this assay it is possible to detect the disulfide bond activity of Pf PDO. The catalyzed oxidation of the peptide was also observed with HPLC analysis 26 by incubating the peptide in the presence of GSH, GSSG, and PfPDO.

Spectroscopic Determination of Oxidation of Substrate Peptide The substrate peptide NRCSQGSCWN is synthesized from Primm s.r.1. Italia using a Shimadzu PSSM8 automated peptide synthesizer. The purified peptide is eluted in a single peak by HPLC analysis and stored at - 2 0 ° in the elution buffer (30% acetonitrile in 0.1% TFA). The peptide concentration is determined spectrophotometrically using an adsorption coefficient of 5600 M -1 cm-1 at 278 nm. 24 K. Axelsson, S. Eriksson, and B. Mannervik,Biochemistry17, 2978 (1978). 25 L. W. Ruddock,R. Hirst, and R. B. Freedman,Biochem.J. 315, 1001 (1996). 26 A. Toscoand L. Birolo,personal communication, 1999.

P.furiosus PDIS: BIOCHEMICALPROPERTIES

[71

71

The assay is performed in McIlvaine buffer (0.2 M disodium hydrogen phosphate/().l M citric acid; pH 7.0) with 2mM GSH (stock solution 60.1 mg/ml), 0.5 mM GSSG (stock solution 30.7 mg/ml), and 5~M Pf PDO (stock solution, 1.0 mg/ml in Tris-C1 pH 8.4). It is placed in a fluorescence cuvette with a final assay volume of 1 ml. 25 After mixing, the cuvette is placed in a thermostatically controlled Perkin-Elmer LS50B spectrofluorimeter for 1 min to allow thermal equilibration of the solution to 50°C. Next, 5 p~M substrate peptide (1.05 mM, in 30% acetonitrile/0.1% TFA) is added, mixed, and the change in fluorescence intensity (excitation 280 nm, emission 350 nm, slits 5/5 nm) is monitored over an appropriate time (15 min); 900 data points are collected. As a control, the same experiment is carried out in the absence of Pf PDO and the decrease in fluorescence intensity is not observed (Fig. 2). At pH 8.0 the spontaneous oxidation of the peptide substrate is observed, presumably due to air oxidation, but at pH 7.0 only the catalyzed oxidation of the substrate is measured.

g O

150

B

100

300

500 Time (sec)

700

FIG.2. Timecourseof the peptidein the oxidizedform(A), and in the reducedform(B).

72

REDOX AND THIOL-DEPENDENT PROTEINS

[71

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FIG.3. HPLCanalysisof the peptide in the oxidizedform (A) and in the reduced form (B). HPLC Analysis High-performance liquid chromatography (Beckman Gold System, Palo Alto, CA) analysis is performed with a reversed-phase C18 column (Spherisorb $5ODS2) equilibrated in buffer E (0.1% TFA in H20). The assay mixture is prepared with 5 t~M reduced peptide, 25 p~MPf PDO, 100 mM GSH, and 25 mM GSSG in McIIvaine buffer pH 7.0, and incubated for 30 rain at 50 °. After incubation the mixture is loaded onto the HPLC column. Chromatography is carried out with a linear gradient 0-100% buffer P (acetonitrile 95%, 0.1% TPA) in Buffer E at flow rate of 1 ml/min in 35 min. The reduced and oxidized form of the peptide have different retention times and are eluted separately, thus demonstrating the capability of Pf PDO to catalyze the formation of the disulfide bond. Indeed, when the same experiment was carried out in the absence of Pf PDO, only one peak of the reduced peptide was observed (Fig. 3). Using these assay conditions the disulfide bond-forming activity of P f P D O shows a linear dependence on the protein concentration up to 50 p~M.

Properties of

Pf

PDO

Homogeneous P f P D O can be stored at - 2 0 ° for at least 3 months without any loss of activity; the presence of a reducing agent does not seem to be essential for its stability. Isoelectric focusing gel of the homogeneous protein reveals one band with isoelectric point of 4.9.

P.furiosus PDIS: BIOCHEMICALPROPERTIES

[7]

73

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lt2

Time (min) FIG. 4. The DTT-dependent reduction of bovine insulin disulfide as carded out in the absence (o) and in the presence at different concentration of pure Pf PDO: (A) 1.2 ~M, (11) 0.6 ixM, (O) 0.2 ~M.

The protein purified from P. furiosus is very thermostable and loses no activity after a 3-hr incubation at 90 °. The molecular mass of the protein was determined by electrospray mass spectrometry; a value of 25,649 Da was calculated, which corresponds to the mass deduced from the amino acid sequence. P f P D O has the ability to act as a reductant at 30 ° and catalyzes the reduction of insulin disulfide in the presence of dithiothreitol. Figure 4 shows the DTTdependent reduction of insulin disulfides at 30 ° in the presence of increasing concentrations of pure P. furiosus protein and in its absence (the spontaneous precipitation reaction). In this case the progress curve obtained for 5 IxM of protein (not shown) was identical to that for 1.2 ~M, indicating that the assay was saturated. Because of substrate instability it is impossible to perform the assay at temperatures higher than 30 °. The protein can also utilize glutathione, glutathione reductase, and NADPH to reduce disulfide substrates, and this demonstrates the thioltransferase activity of the enzyme. The maximal rate of activity with different concentrations of GSH and L-cystine is obtained with 10 mM GSH and 2.5 mM L-cystine; higher concentrations led to inhibition. The thioltransferase activity of the P. furiosus protein shows a linear dependence on the protein concentration up to 0.12 ~M, and inhibition is detected at higher protein concentrations. The functional data demonstrate that the archaeal protein is an oxidant and reductant; in fact, P f P D O is able to catalyze the oxidation of dithiol as well as the reduction of disulfides. The studies of the disulfide bond rearrangement were not successful at the low temperature used (30 °) testing as substrate scrambled RNAse. Acknowledgment This research was supported by grants from EU and MURT.