Novel Highly Active Recombinant Glutaredoxin from Chlorella

Dec 30, 2013 - Agricultural Biotechnology Center, National Chung Hsing University, ... of Biological Science and Technology, I-Shou University, Kaohsi...
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Novel Highly Active Recombinant Glutaredoxin from Chlorella sorokiniana T‑89 Hsu-Han Chuang,†,‡ Chu-Ying Cheng,†,‡ Yu-Ting Chen,*,‡,§ and Jei-Fu Shaw*,†,‡,∥ †

Department of Food Science and Biotechnology, National Chung Hsing University, Taichung, Taiwan Agricultural Biotechnology Center, National Chung Hsing University, Taichung, Taiwan § Institute of Genomics and Bioinformatics, National Chung Hsing University, Taichung, Taiwan ∥ Department of Biological Science and Technology, I-Shou University, Kaohsiung, Taiwan ‡

S Supporting Information *

ABSTRACT: Glutaredoxin (Grx) is a thiol/disulfide oxidoreductase that maintains the cellular thiol/disulfide ratio. A 321 bp cDNA fragment encoding a putative Grx (named CsT-89Grx) was cloned from heat-tolerant Chlorella sorokiniana T-89 and expressed in an Escherichia coli system. The sequence analysis of CsT-89Grx and site-directed mutations showed that the putative active site within the CPYC motif belonged to the dithiol superfamily. The biochemical property analyses showed that the optimal pH and temperature of CsT-89Grx are pH 8.5 and 50 °C, respectively. The activity of CsT-89Grx showed high thermal stability (retained 70% activity at 80 °C for 30 min) and broad pH stability (retained over 70% activity for 1 h) ranging from pH 3 to 11. The kinetic parameter kcat/Km was 20 982 min−1 mM−1, which suggested that CsT-89Grx exhibited the highest catalytic efficiency in reducing the disulfide bond among all the Grx reported in the related literature and is therefore potentially useful for industrial applications. KEYWORDS: Chlorella sorokiniana T-89, glutaredoxin, β-hydroxyethylene disulfide (HED), reactive oxygen species (ROS), glutathione (GSH)

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INTRODUCTION Chlorella spp. are edible green algae that are among the eukaryotic unicellular microalgae.1 These algae have been widely used as functional (healthcare) food and bioactive chemical sources because they are a rich source of nutrients, including high-quality proteins, vitamins, minerals, dietary fiber, nucleic acid, chlorophyll, lipid, carbohydrates, and bioactive substances.2,3 Previous studies reported that Chlorella spp. possess diverse biological functions including antioxidant, antiinflammatory, and immunomodulatory functions, as well as the mitigation of hyperlipidemia, wound healing, suppression of hypertension and elevated serum glucose level, and detoxification.3−9 Chlorella spp. are also used in environmental and industrial applications, such as wastewater treatment, degradation of toxic materials, and heavy metal removal.1,10,11 Oxygen is necessary to sustain the life of organisms. However, reactive oxygen species (ROS) are formed when oxygen is incompletely reduced.12 ROS containing free radicals, such as superoxide radicals (O2−), peroxyl radicals (ROO−), nitric oxide radicals (NO−), and hydroxyl radicals (OH−) can attack cells, thus damaging proteins, nucleic acids, and cell membranes; such damage is associated with numerous diseases.12−16 In oxidative stress, ROS might react with the cysteine residues of protein to form sulfenic acid,12,13 which can disturb protein function. Several antioxidant defense mechanisms were thus developed to prevent thiol oxidation and to avoid the destruction of cellular functions.12,17,18 Among antioxidative defense mechanisms, the glutaredoxin (Grx) system serves an important function in maintaining and regulating cellular redox balance.19 © 2013 American Chemical Society

Grx, also known as thioltransferase, is a thiol/disulfide oxidoreductase with antioxidative capacity that participates in various cellular functions.20−22 Grx can reduce disulfides by coupling to glutathione (GSH), GSH reductase (GR), and nicotinamide adenine dinucleotide phosphate (NADPH)23,24 to maintain the cellular thiol/disulfide ratio. On the basis of the active site sequence and conserved motifs involved in GSH binding, Grxs are categorized into six classes.22 Grx can also be classified into two groups on the basis of their catalytic reactions, namely, monothiol- and dithiol reaction mechanisms.24−26 The active site of dithiol Grxs contains the conserved CXXC motif, which reduces protein disulfides or protein−S−S−glutathione substrates.26 The active site of monothiol Grxs contains the CXXS motif, which reduces the mixed disulfides formed between proteins and GSH.25,27 In this study, the CsT-89Grx cDNA was amplified from heattolerant Chlorella sorokiniana T-89, and the recombinant enzyme, CsT-89Grx, was successfully expressed in Escherichia coli. The biochemical properties of CsT-89Grx, including the optimal pH and temperature, stability of pH and temperature, effects of various metal ions, and chemical reagents, were thoroughly investigated. Studies showed that the novel recombinant Grx from Chlorella sorokiniana T-89, called CsT89Grx, is the most active enzyme that exhibits the highest catalytic efficiency among all reported glutaredoxins. CsTReceived: Revised: Accepted: Published: 927

August 22, 2013 December 27, 2013 December 30, 2013 December 30, 2013 dx.doi.org/10.1021/jf405213h | J. Agric. Food Chem. 2014, 62, 927−933

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E. coli BL21(DE3) (Novagen, Germany) was used as the expression host. The BL21(DE3) containing pET-20b_CsT89Grx was grown until OD600 0.4 at 37 °C in LB broth with ampicillin (100 μg/mL). Thereafter, the recombinant protein was induced by adding isopropyl thio-β-D-galactoside (IPTG) to a final concentration of 0.4 mM. Protein induction was performed at 28 °C for 4 h before cell harvesting. The cells were lysed in BugBuster Protein Extraction Reagent (Novagen, Germany). The crude protein lysate was obtained after 10 000g centrifugation for 15 min. The rCsT-89Grx was purified using BD TALON Superflow Resin, which was used for the purification of polyhistidine-tagged protein (BD Biosciences Clontech, U.S.A.). The purified enzyme was desalted using a Protein Desalting Spin Column following affinity purification (Pierce, U.S.A.). The recombinant protein was analyzed using 16% Tricine sodium dodecyl sulfate polyacrylamide gel electrophoresis (Tricine SDS-PAGE).28 Protein Quantitation and Enzyme Activity Assay. Protein concentrations were quantified according to Bradford’s 29 method using bovine serum albumin as the standard. Grx activity was assayed using the HED method.30 The reaction mixture consisted of 50 mM of Tris-Cl (pH 7.4), 0.6 μg of GR, 0.3 mM of HED, 0.2 mM of NADPH, 0.8 mM of GSH, and 0.1 μg of rCsT-89Grx in a total volume of 500 μL. Briefly, the disulfide bond was spontaneously formed between HED and GSH within 2 min, and the reaction was subsequently commenced by adding 0.1 μg of rCsT-89Grx. Activity was monitored on the basis of the decrease in NADPH absorbance at 340 nm. One unit of activity was defined as the enzyme necessary to deplete 1 μmol of NADPH per minute. The value was calculated by (ΔA340 × V)/(min × 6.2), where V indicates the cuvette volume in milliliters, and 6.2 is the millimolar extinction coefficient for NADPH.30 Biochemical Characterization of the Recombinant CsT-89Grx. The activities of CsT-89Grx under various conditions were studied using the described HED assay. The optimal pH and temperature, stability of pH and temperature, kinetic analysis as well as the effects of metal ions, chemical reagents, and inhibitors were studied. For the optimal pH of CsT-89Grx, the activity of the enzyme over the pH range of 3.0 to 11.0 was measured using a 50 mM acetate buffer (pH 3.0− 6.0), a 50 mM Tris-HCl buffer pH (pH 7.0−9.0), and a 50 mM glycine-NaOH buffer (pH 10.0−11.0) at 30 °C. Optimal enzyme activities were also tested at various temperatures (15 to 60 °C) at pH 8.5 in 50 mM Tris-HCl buffer. To analyze thermal tolerance, the enzyme was incubated at various temperatures from 4 to 100 °C for 1 h, and residual activity was analyzed in 50 mM Tris-HCl buffer (pH 8.5) at 50 °C. The pH stability of CsT-89Grx was investigated at different pH values (pH 3, 5, 7, 9, and 11) and at optimum pH and temperature. The kinetic analyses of the purified CsT-89Grx with different concentrations of HED (0.06 mM to 0.2 mM) were conducted under the optimum pH and temperature conditions. To study the effects of various metal ions (NaCl, MgCl2, MnCl2, CaCl2, CoCl2, CuSO4, ZnCl2, and FeCl3) on the enzyme, 1 or 10 mM of the metal ions and CsT-89Grx was preincubated in 50 mM Tris-HCl (pH 8.5) at 37 °C for 30 min. The mixture was then analyzed for enzyme activity under optimal conditions. The effects of chemical reagents and inhibitors on enzyme activity were measured. Chemical reagents and inhibitors, including urea, ethylenediaminetetraacetic acid (EDTA), sodium dodecyl disulfate (SDS), dithiotheretiol (DTT), phenylmethanesulfony fluoride

89Grx is therefore potentially useful for biotechnological applications.

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MATERIALS AND METHODS Materials. C. sorokiniana T-89 (isolated from the paddy field of Chiayi, Taiwan) was provided by Dr. Tan-Chi Huang. The restriction enzymes, NdeI and XhoI, were purchased from NEB (New England BioLabs, U.S.A.). β-Hydroxyethylene disulfide [HED, (HOCH2CH2)2S2], as a low molecular weight substrate, was purchased from Sigma-Aldrich-Fluka (St. Louis, MO). Glutathione reductase (GR), NADPH, and glutathione (GSH) were obtained from Sigma (St. Louis, MO). The Highspeed Plasmid Mini Kit and Gel/PCR DNA Fragments Extraction Kit were purchased from Geneaid (Geneaid Biotech, Bade City, Taiwan). Protein molecular mass markers were obtained from Fermentas (Canada). RNA Extraction and cDNA Synthesis. Total RNA of C. sorokiniana T-89 was prepared using RNAzol RT (Molecular Research Center). The mRNA was then purified using Oligotex mRNA Mini kit (Qiagen, U.S.A.). The cDNA was carried out using the 5′/3′ RACE kit (Roche, Germany). Cloning of C. sorokiniana T-89 Grx Gene and Its Mutant Forms. After total RNA was extracted from C. sorokiniana T-89, the cDNA was synthesized for sequencing analysis. Gene-specific primers of CsT-89Grx were designed on the basis of the RNA sequencing analysis information of C. sorokiniana T-89. The full length Grx gene from C. sorokiniana T-89 was amplified by polymerase chain reaction (PCR) using gene-specific primers, and the CsT-89 Grx gene was obtained using C. sorokiniana T-89 cDNA as a template. The forward primer Grx-F (5′-GGAATTCCATATGAGCGCGGCCAAGCAGCTGG-3′) and the reverse primer Grx-R (5′CCGCTCGAGGAGCACGCCGGCATTGCGCAGCAT-3′) were incorporated with the cloning sites of NdeI and XhoI (in italics), respectively. After digestion with NdeI and XhoI, the CsT-89Grx DNA fragment was ligated into a pET-20b(+) expression vector (Novagen, Germany), which was previously treated with the same restriction enzymes. Consequently, the recombinant pET-20b_CsT-89Grx plasmid was transformed into the cloning host E. coli DH5α (GeneMark, Taiwan). The site-directed mutagenesis of CsT-89Grx active sites (C25A and C28A) were constructed using the QuikChange Site-Directed Mutagenesis Kit (Agilent Technologies, U.S.A.). The mutant primers were designed to replace cysteine by alaline (in italics) as follows: CsT-89GrxC25A-F (5′- AGCAAGACCTACGCGCCTTACTGCGTG-3′), CsT-89GrxC25A-R (5′CACGCAGTAAGGCGCGTAGGTCTTGCT-3′); CsT-89Grx C28A-F (5′-TACTGCCCTTACGCGGTGAAGGGCAAG-3′), CsT-89GrxC28A-R (5′-CTTGCCCTTCACCGCGTAAGGGCAGTA-3′); and CsT-89GrxC25,28A-F (5′-TTTAGCAAGACCTCGCGCCTTACGCGGTGAAGGGCAAGCGG-3′), CsT-89Grx C25,28A-R (5′-CCGCTTGCCCTTCACCGCGTAAGGCGCGTAGGTCTTGCTAA A-3′). Bioinformatic Analysis of CsT-89Grx. The protein sequence of CsT-89Grx was analyzed using protein−protein BLAST in NCBI (http://www.ncbi.nlm.nih.gov/). The molecular mass and pI of the encoded protein were computed using ExPASy (http://www.expasy.ch/tools/protparam.html). The multiple amino acid sequence alignment of CsT-89Grx was compared using ClustalW2 (http://www.ebi.ac.uk/Tools/ clustalw2/index.html). Expression and Purification of Recombinant CsT89Grx. For recombinant CsT-89Grx (rCsT-89Grx) expression, 928

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with AfGrx, 43% with TcGrx, 36% with HsGrx and ScGrx, and 27% with EcGrx), the active site (CPYC motif) and GSHbinding sites were highly conserved (Figure 2).

(PMSF), and iodoactamide, were incubated with the enzyme for 30 min at 37 °C in 50 mM Tris-HCl (pH 8.5). Residual activity was determined at 50 °C in 50 mM Tris-HCl buffer (pH 8.5). Enzyme activity without treatment was defined as 100%.

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RESULTS AND DISCUSSION Cloning of Grx Gene from C. sorokiniana T-89 and Sequence Analysis. C. sorokiniana T-89 isolated from a paddy field in Chiayi, Taiwan, belonged to the heat-tolerant Chlorella species because it could grow well up to 38 °C.31 Cultures of C. sorokiniana T-89 at different growth stages were used for RNA extraction, and mRNA was converted to cDNA for sequence analysis. The contig data of sequences were obtained using the Illuina sequencing system. Contigs were predicted for their function via sequence alignment using NCBI BLAST. Among the sequence data, Grx was identified. The forward and reverse specific primers of CsT-89Grx were designed on the basis of sequence alignment using NCBI BLAST. Using C. sorokiniana T-89 cDNA as template, the 321 bp DNA fragment encoding the Grx protein was amplified by PCR. CsT-89Grx was further constructed into pET-20b(+) and was confirmed to have the correct sequence by DNA sequencing. Using the ExPASy Compute pI/MW program, the theoretical molecular mass and pI of CsT-89Grx were computed as 11.4 and 8.97 kDa, respectively. The bioinformatics of CsT-89Grx was analyzed using the NCBI protein-BLAST program. According to the data analysis, the predicted active sites of CsT-89Grx were Cys25 and Cys28, and the conserved redox active site CPYC motif suggested that CsT-89Grx belonged to the dithiol superfamily.26,32,33 The residues of Lys22, Cys25, Tyr27, Arg69∼Pro72, and Gly83∼Asp86 were predicted to be the putative GSH binding sites (Figure 1).

Figure 2. Multiple amino acid sequence alignments of CsT-89Grx with diverse organisms using the ClustalW2 program. Accession numbers and abbreviations of the sequences: Chlorella sorokiniana T89 (CsT-89Grx, this study), Chlorella variabilis (CvGrx, EFN57573.1), Aspergillus fumigates Af293 (AfGrx, XP_750359.1), Chlamydomonas reinhardtii (CrGrx, XP_001694001.1), Escherichia coli (EcGrx, AAA23936.1), Homo sapiens (HsGrx, NP_002055.1), Saccharomyces cerevisiae S288c (ScGrx, NP_009895.1), and Taiwanofungus camphorates (TcGrx, ABY58974.1). The conserved residual amino acids are displayed in the black box.

Expression and Purification of Recombinant CsT89Grx. The DNA fragment of 321 bp of CsT-89Grx was amplified by PCR using the specific primers (Grx-F/Grx-R) and cloned into the pET-20b(+) expression vector, as described in the Materials and Methods. The result was analyzed by 16% Tricine SDS-PAGE (Figure 3). After induction by IPTG, the rCsT-89Grx containing the 6-His tag was overexpressed in E.

Figure 1. Deduced nucleotide and amino acid sequence of C. sorokiniana T-89 glutaredoxin (CsT-89Grx). The stop codon is shown in asterisks. The predicted active sites of CsT-89Grx, Cys25, and Cys28 are shown as black triangles, and the putative conserved CPYC motif is indicated by a gray block. The putative GSH-binding sites are composed of Lys22, Cys25, Tyr27, Arg69∼Pro72, and Gly83∼Asp86, as presented by bold letters. Figure 3. Expression and purification of the recombinant CsT-89Grx in the E. coli expression system. The total protein and purified enzyme were analyzed with 16% Tricine SDS-PAGE followed by Coomassie blue staining. Lane 1, crude soluble extract from Bl21(DE3) with pET20b(+); lane 2, crude soluble extract from Bl21(DE3) expressing CsT89Grx containing the 6-His tag; lane 3, pass-through protein from TALON Co2+ column; lane 4, the purified CsT-89Grx by TALON Co2+ column; and lane M is a PageRuler Prestained protein marker (Fermentas).

Moreover, multiple amino acid sequence alignments of Grx containing C. sorokiniana T-89 (in this study), Aspergillus fumigates Af293 (XP_750359.1), Chlamydomonas reinhardtii (XP_001694001.1), E. coli (AAA23936.1), Homo sapiens (NP_002055.1), Saccharomyces cerevisiae S288c (N P_0 09 895 . 1) , and T a i w an o f u n gu s c a m ph o r a t e s (ABY58974.1) were also conducted. Despite low sequence similarity (CsT-89Grx shared 53% identity with CrGrx, 44% 929

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Figure 4. Effect of pH and temperature on purified CsT-89Grx using HED assay. (A) Optimal pH of CsT-89Grx measured at different pH values (pH 5 to 11). Activity at pH 8.5 corresponds to 100%. (B) Optimal temperature of CsT-89Grx measured from 15 to 60 °C. Activity at 50 °C corresponds to 100%. (C) Effect of pH on the stability of CsT-89Grx. Purified CsT-89Grx was incubated at 37 °C from 50 mM acetate buffer (pH 3, 5), 50 mM Tris-HCl (pH 7, 9), and 50 mM glycine-NaOH (pH 11) for 1 h. (D) Thermal stability of the purified CsT-89Grx. Enzyme was incubated at pH 7.4, 50 mM Tris-HCl buffer at different temperatures (4 to 100 °C) for 1 h. Grx activity was monitored on the basis of the decrease in NADPH absorbance at 340 nm.

Table 1. Kinetic Parameters of CsT-89Grx and the Published Grxs from Other Organisms HED

Km (mM) kcat (min−1) kcat/Km

CsT-89Grx

EcGrx1a

ScGrx1b

CrGrx1c

HsGrx1d

TcGrxe

0.17 ± 0.02 3566.8 ± 269.94 20982 ± 390.26

3.0 6900 2300

0.12 132 1100

0.34 1800 5294

1.07 490 458

0.57 1633.0 2864.9

a

The reaction mixture, 100 mM Tris-Cl, pH 8.0, 6 mg/mL yeast glutathione reductase (GR), 1 mM GSH, 0.7 mM HED, 0.2 mM NADPH, 0.1 mg/ mL bovine serum albumin (BSA), and 2 mM EDTA, was incubated at 25 °C for the enzyme activity measurements.35 b The reaction mixture, 100 mM Tris−HCl, pH 7.4, 6 μg/mL GR, 1 mM GSH, HED (0.03 to 2.2 mM), 0.2 mM NADPH, 0.1 mg/mL BSA, and 2 mM EDTA, was incubated at 30 °C for 3 min for the enzyme activity measurements.36 c The reaction mixture contained: 50 μmol Tris-Cl, pH 8.0, 1 μmol EDTA, 0.5 μmol GSH, 3.8 μg yeast GR, 0.2 μmol NADPH, 50 μg BSA, (0.1−2 mM) HED, in a final volume of 0.5 mL.37 d The reaction mixture contained: 100 mM potassium phosphate, pH 7.0, 1 mM EDTA, 1 mM GSH, 6 μg/mL yeast GR, 240 μM NADPH, 0.1 mg/mL BSA, and 0.1−3 mM HED.38 eThe reaction mixture, 100 mM Tris-Cl, pH 7.4, 0.8 mM GSH, 0.6 μg GR, 0.2 mM NADPH, and 0.3 mM to 2.4 mM HED in a final volume of 100 μL, was incubated at 25 °C for 2 min for the enzyme activity measurements.32

coli BL21(DE3) (Figure 3, lane 2). A molecular mass of approximately 12 kDa of the purified rCsT-89Grx was also shown in the gel and was consistent with the expected size of rCsT-89Grx, including the 6-His tag (Figure 3, lane 4). Effects of pH and Temperature on the Purified CsT89Grx. The effects of pH and temperature on the purified rCsT-89Grx were evaluated using the HED assay. The optimal pH of rCsT-89Grx was determined at pH 8.5. High activity was observed in a broad temperature range from 30 to 55 °C (>80% relative activity of maximum activity), and the optimum activity was observed at 50 °C (Figure 4A,B). Meanwhile, the stability of pH and temperature on the rCsT-89Grx was

investigated. After preincubation under different pH conditions for 1 h, over 70% residual activity of CsT-89Grx was detected in a broad pH range from 3 to 11, which suggested stability at a broad pH range (Figure 4C). Analysis of the stability of CsT89Grx at the temperature range of 4 to 100 °C showed relative stability below 70 °C (>50% of residual activity) and even at 80 °C for 1 h, with approximately 30% of CsT-89Grx activity retained (Figure 4D). Ken et al. 32 reported that the residual activity of TcGrx remained at approximately 50% at 100 °C for 8.5 min. The Grx from Ipomoea batatas maintained 60% activity after heating at 80 °C for 16 min.34 Compared with TcGrx and Ipomoea batatas glutaredoxin, CsT-89Grx retained 70% activity 930

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under 100 °C for 8.5 min and 80 °C for 30 min (data not shown). These results suggested that the recombinant CsT89Grx is the most heat-stable Grx reported. Kinetic Parameters of the Purified CsT-89Grx. The kinetic analysis of rCsT-89Grx was illustrated with the Lineweaver−Burk plot. The kinetic parameters Km (0.17 mM), kcat (3566.9 min−1), and kcat/Km (20 982 min−1 mM−1) were calculated. The kinetic parameters of CsT-89Grx were compared with those of Grx from other organisms, including E. coli,35 Saccharomyces cerevisiae,36 Chlamydomonas reinhardtii,37 Taiwanofungus camphorates,32 and Homo sapiens.38 The rCsT89Grx showed that a relatively lower Km corresponds to a higher kcat value, and the highest kcat/Km value compared with Grx from other organisms. These results suggested that rCsT89Grx had the highest catalytic efficiency in reducing the disulfide bond (Table 1). Effects of Metal Ions and Chemical Reagents on the Purified CsT-89Grx. The effects of metal ions and chemical reagents on the activities of rCsT-89Grx are presented in Table 2. Metal ions, such as Na+, Mg2+, Mn2+, and Ca2+, displayed no

Table 3. Effects of Chemicals on the Activities of the Purified CsT-89Grx chemicals

SDS Urea iodoacetamide PMSF DTT removing DTTa

none Na+ Mg2+ Mn2+ Ca2+ Co2+ Cu2+ Zn2+ Fe3+

concentration

relative activity (%)

1 mM 10 mM 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM 1 mM 10 mM

100 93.2 ± 3.3 104.6 ± 6.2 84.0 ± 3.6 99.3 ± 4.5 92.4 ± 8.0 109.1 ± 4.7 81.2 ± 6.2 109.5 ± 4.0 89.1 ± 14.2 34.6 ± 2.7 9.4 ± 9.0 7.7 ± 3.9 5.0 ± 3.9 2.4 ± 1.6 21.0 ± 7.0 1.5 ± 1.7

1 5 1 5 1 4 1 5 1 5 1 5 1 5

mM mM mM mM M M mM mM mM mM mM mM mM mM

relative activity (%) 100 103.1 ± 7.0 100 ± 6.3 37.1 ± 4.2 23.8 ± 11.8 76.7 ± 7.6 40.0 ± 5.2 68.1 ± 0.9 49.3 ± 4.1 96.4 ± 6.7 89.8 ± 5.7 94.0 ± 12.3 68.9 ± 3.0 101.1 ± 4.4 98.7 ± 4.8

a

The purified CsT-89Grx was preincubated with DTT for 30 min at 37 °C in 50 mM Tris-HCl (pH 8.5). DTT was then removed from the reaction solution by a protein desalting spin column. The residual activity was determined at 50 °C in 50 mM Tris-HCl buffer (pH 8.5).

Table 2. Effects of Metal Ions on the Activities of the Purified CsT-89Grx metal ions

concentration

none EDTA

influence the activity of rCsT-89Grx after being removed from the reaction. PMSF, an irreversible inhibitor, slightly affected the activity of CsT-89Grx at 5 mM. The result suggested that PMSF might influence serine, which is a GSHbinding site, thus causing part of the charge change for serine. Active Site Identification by Site-Directed Mutagenesis. According to the sequence analyses, only two cysteine (putative active sites, Cys25 and Cys28) appeared in CsT-89Grx. Moreover, CsT-89Grx belonged to the dithiol superfamily, and its reaction mechanism is presumably catalyzed by dithiol−disulfide exchange via the coupling of −SH groups to GR and NADPH.20,42 This finding suggested that no disulfide bond appeared in the active site of CsT-89Grx. On the basis of the bioinformatics analysis, the active site of CsT-89Grx was predicted to be the Cys25 and Cys28. As expected, the CsT-89Grx activity was destroyed by iodoacetamide (Table 3). The CsT-89Grx retained 68.1 and 49.3% activity in 1 and 5 mM iodoacetamide, respectively. To confirm the active sites, site-directed mutagenesis of the putative active sites Cys25 and Cys28 was performed (Supplemental Data). The single mutation C25A and C28A and the double mutation C25,28A retained 1.63, 12.87, and 1.5% activity, respectively (Table 4). The above analyses suggested that the Cys25 and Cys28 of CsT-89Grx acted as active sites and served an important function in the antioxidant activity of rCsT-89Grx. In conclusion, we obtained a novel, highly active recombinant Grx enzyme with strong antioxidation capacity

significant effect on enzyme activity. The activity was slightly decreased by 1 mM Co2+ (retaining 89% relative activity) and was significantly inhibited by 10 mM Co2+ (loss of 65.4% relative activity). The activity of rCsT-89Grx was sharply reduced by treatment with Cu2+, Zn2+, and Fe3+. Meanwhile, the activity of rCsT-89Grx was unaffected by 1 or 5 mM of EDTA, which suggested that rCsT-89Grx was not a metalloenzyme (Table 3). Recombinant CsT-89Grx was incubated with different chemical reagents to investigate their effects on its activity. Residual activity was measured following the HED assay. As shown in Table 3, the activity of rCsT-89Grx was significantly reduced by 1 or 5 mM SDS treatment. After treatment with 1 and 4 M urea, enzyme activity was reduced to approximately 23.3 and 60%, respectively. DTT is a strong reducing agent frequently used to reduce the disulfide bonds of proteins.39 Unexpectedly, CsT-89Grx activity was slightly decreased following preincubation with 5 mM DTT. This result suggested that DTT might affect either the activity of GR or GSSG (glutathione, oxidized form).40,41 Indeed, DTT did not

Table 4. Specific Activity of rCsT-89Grx and Mutant Forms protein rCsT-89Grx rCsT-89Grx rCsT-89Grx rCsT-89Grx

C25A C28A C25,28A

specific activitya (unit/mg)

relative activity (%)

259.3 ± 6.35 4.23 ± 7.75 33.37 ± 3.48 3.88 ± 2.81

100 1.63 ± 2.99 12.87 ± 1.34 1.50 ± 1.08

a

Since the enzyme activities of mutant forms were not detected under the same condition of wild type, the specific activity analyses were carried out with 100 ng rCsT-89Grx and 1 μg mutant forms, respectively.

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from C. sorokiniana T-89. The thiol/disulfide oxidoreductase, Grx, from C. sorokiniana T-89 was successfully expressed in the E. coli expression system. On the basis of the biochemical analyses, CsT-89Grx exhibited the highest activity at 50 °C and pH 8.5. CsT-89Grx displayed high heat stability and can adapt to a broader pH range for catalytic reactions. These biochemical properties suggest that the recombinant CsT89Grx is potentially useful for biotechnological applications such as natural foods and pharmaceuticals. CsT-89Grx presented the highest catalytic efficiency (kcat/Km) in reducing the disulfide bond compared with Grx from other organisms. The above conclusions are possible reasons why C. sorokiniana T-89 can grow well at temperatures up to 38 °C, thus aiding in the scavenging of oxidative stress. Moreover, C. sorokiniana T89 can be useful as a functional food.

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ASSOCIATED CONTENT

S Supporting Information *

Site-directed mutagenesis of the putative active sites Cys25 and Cys28. This material is available free of charge via the Internet at http://pubs.acs.org.

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. Fax:886-7-657-7051. Tel.:886-7657-7001 (J.-F.S.). *E-mail: [email protected]. Fax: 886-4-2285-9329. Tel.: 886-4-2284-0338, ext. 7021 (Y.-T.C.). Funding

This research was partially supported by the Ministry of Education, Taiwan, R.O.C. Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors thank Dr. Tan-Chi Huang for providing C. sorokiniana T-89. REFERENCES

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