Chi-Tsai Lin

Subunit interaction enhances enzyme activity and stability of sweet potato cytosolic Cu/Zn-superoxide dismutase purified by a His-tagged recombinant protein method

The coding region of copper/zinc-superoxide dismutase (Cu/Zn-SOD) cDNA from sweet potato, Ipomoea batatas (L.) Lam. cv. Tainong 57, was introduced into an expression vector, pET-20b(+). The Cu/Zn-SOD purified by His-tagged technique showed two active forms (dimer and monomer). The amount of proteins of dimer and monomer appeared to be equal, but the activity of dimeric form was seven times higher than that of monomeric form. The enzyme was dissociated into monomer by imidazole buffer above 1.0 M, acidic pH (below 3.0), or SDS (above 1%). The enzyme is quite stable. The enzyme activity is not affected at 85 °C for 20 min, in alkali pH 11.2, or in 0.1 M EDTA and also quite resistant to proteolytic attack. Dimer is more stable than monomer. The thermal inactivation rate constant kdcalculated for the monomer at 85 °C was 0.029 min-1 and the half-life for inactivation was about 28 min. In contrast, there is no significant change of dimer activity after 40 min at 85 °C. The enzyme dimer and monomer retained 83% and 58% of original activity, respectively, after 3 h incubation with trypsin at 37 °C, while those retained 100% and 31% of original activity with chymotrypsin under the same condition. These results suggest subunit interaction might change the enzyme conformation and greatly improve the catalytic activity and stability of the enzyme. It is also possible that the intersubunit contacts stabilize a particular optimal conformation of the protein or the dimeric structure enhances catalytic activity by increasing the electrostatic steering of substrate into the active site.

Characterization of fish Cu/Zn-superoxide dismutase and its protection from oxidative stress.

Copper/zinc superoxide dismutase was cloned from the zebrafish (Danio rerio). The full coding region of the zebrafish superoxide dismutase (ZSOD) complementary DNA was ligated with pET-20b(+) and successfully expressed in Escherichia coli strain AD494(DE3)pLysS. The active enzyme was purified by His tagging. The ZSOD yield was 6 mg from 0.2 L of E. coli culture, and the specific activity was 2000 U/mg as assayed using a RANSOD kit. The enzyme stability was characterized by reaction to temperature, pH, and detergent treatment. The results showed enzyme activity was still active after heat treatment at 70°C for 10 minutes, resistant to pH treatment from 2.3 to 12, and resistant to treatment with sodium dodecyl sulfate (SDS) under 4%. In addition, the recombinant ZSOD was used to protect fish from 100 ppm of paraquat-induced oxidative injury by soaking fish larva in 55 µg/ml SOD enzyme. The results were significant.

Characterization, molecular modelling and developmental expression of zebrafish manganese superoxide dismutase.

A 977 bp cDNA containing an open reading frame encoding 224 amino acid residues of manganese superoxide dismutase was cloned from zebrafish (zMn-SOD). The deduced amino acid sequence showed high identity with the sequences of Mn-SODs from human (85.1%) to nematode (61.6%). The 3-D structure model was superimposed on the relative domains of human Mn-SOD with the root mean square (rms) deviation of 0.0919 A. The recombinant mature zMn-SOD with enzyme activity was purified using His-tag technique. The half-life of the enzyme is approximately 48 min and its thermal inactivation rate constant k(d) is 0.0154 min(-1)at 70 degrees C. The enzyme was active under a broad pH (2.2-11.2) and in the presence of up to 4% SDS. Real-time RT-PCR assay was used to detect the zMn-SOD mRNA expression during the developmental stages following a challenge with paraquat. A high level expression of Mn-SOD mRNA was detected at the cleavage stage, but decreased significantly under paraquat treatment. The results indicated that Mn-SOD plays an important role during embryonic development.

The gene structure of starch phosphorylase from sweet potato.

a-Glucan phosphorylases are of key importance among the enzymes controlling the entry of storage polysaccharides into the glycolytic pathway in microbes, animals, and plants. The enzymes catalyze the reversible phosphorolysis of cu-l,4-glucosidic linkages in glucan substrates. Among SP from higher plants, those from potato are best studied. They are classified into L and H types depending on their low and high affinities toward branched glucans, respectively (Nakano et al., 1989). The L type is larger than the H type in having a 78-amino acid residue insertion at the central position, causing the molecule to show a low homology toward phosphorylases from other sources (Nakano and Fukui, 1986; Nakano et al., 1989; Lin et al., 1991; Mori et al., 1991). The presence of the extra peptide was suggested to affect the substrate affinity (Nakano and Fukui, 1986), and this was proven to be true by a protein engineering approach (Mori et al., 1993). Animal glycogen phosphorylases are endowed with both covalent and allosteric regulatory mechanisms (Fukui et al., 1982; Hwang and Fletterick, 1986) and their structurefunction relationship has been established, but neither such functions nor the corresponding structures are known for either Lor H-type plant phosphorylases. Previously, we postulated that SP in the growing sweet potato (Ipomoea batatus L.) root could be involved in a starch synthesis pathway. Gel electrophoresis analysis of sweet potato SP always gave multiple bands, which indicated the presence of subunits with different proteolytic susceptibility at the midchain 78-residue segment. Determination of amino acid sequence was hindered by blocking of the N terminus and the difficulty of obtaining intact pure peptide. We have cloned and sequenced a cDNA encoding this L-type isozyme (Lin et al., 1991). The cDNA encodes a 955-residue polypeptide that has 81 % homology toward the L-type potato enzyme (916 residues plus a 50-residue putative transit peptide). Higher divergence of the two enzymes was found in about 70 residues of the N termini, including a putative transit peptide, and in the midchain 78-residue insert. Very high similarity between the potato

Dehydroascorbate Reductase cDNA from Sweet Potato (Ipomoea batatas [L.] Lam): Expression, Enzyme Properties, and Kinetic Studies

A cDNA encoding a putative dehydroascorbate reductase (DHAR) was cloned from sweet potato. The deduced protein showed a high level of sequence homology with DHARs from other plants (67 to approximately 81%). Functional sweet potato DHAR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 12% native PAGE. The proteins half-life of deactivation at 50 degrees C was 10.1 min, and its thermal inactivation rate constant K(d) was 6.4 x 10(-2) min(-1). The enzyme was stable in a broad pH range from 6.0-11.0 and in the presence of 0.8 M imidazole. The K(m) values for DHA and GSH were 0.19 and 2.38 mM, respectively.

Cloning, expression, and characterization of an enzyme possessing both glutaredoxin and dehydroascorbate reductase activity from Taiwanofungus camphorata.

Glutaredoxins (Grxs) play important roles in the reduction of disulfides via reduced glutathione as a reductant. A cDNA (503 bp, EU193660) encoding a putative Grx was cloned from Taiwanofugus camphorata (Tc). The deduced amino acid sequence is conserved among the reported dithiol Grxs. A 3D homology structure was created for this TcGrx. To characterize the TcGrx enzyme, the coding region was subcloned into an expression vector pET-20b(+) and transformed into Escherichia coli . Functional TcGrx was expressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed bands of approximately 15 kDa on 15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The TcGrx encodes a protein possessing both Grx and dehydroascorbate reductase (DHAR) activity. The Michaelis constant (K(m)) values for beta-hydroxyethyl disulfide (HED) and dehydroascorbate (DHA) were 0.57 and 1.85 mM, respectively. The half-life of deactivation of the protein at 100 degrees C was 8.5 min, and its thermal inactivation rate constant K(d) was 6.52 x 10(-2) min(-1). The enzyme was active under a broad pH range from 6.0 to 10.0 and in the presence of imidazole up to 0.4 M. The enzyme was susceptible to SDS denaturation and protease degradation/inactivation.

The Gene Structure of Cu/Zn-Superoxide Dismutase from Sweet Potato

SODs (superoxide:superoxide oxidoreductase, EC 1.15.1.1) catalyze the dismutation of superoxide to dioxygen and hydrogen peroxide to protect organisms from oxidative damage (Hassan, 1984). SODs are metalloproteins that are classified into three types (Mn-, Fe-, and Cu/Zn-SOD) depending on the metal found in the active site. In plants, the most prominent SODs are Cu/Zn isozymes. It has been shown that transgenic plants that overexpress chloroplastic Cu/Zn-SOD increase resistance to oxidative stress (Gupta et al., 1993) and the activity of plant SOD increases in response to a variety of environmental and chemical stimuli (Fridovich, 1986; Perl-Treves and Galun, 1991). Recently, increased levels of SOD activities resulting from differential regulation of individual SOD genes at the transcriptional leve1 were also reported (Perl-Treves and Galun, 1991). Many plant SOD cDNAs from leaf or seedling have been studied, but information concerning the Cu/Zn-SOD gene from root tissue is limited. Previously, we cloned and sequenced a full-length cytosolic Cu/Zn-SOD cDNA from sweet potato root (Lin et al., 1993). In this paper, we report the structural features of a Cu/Zn-SOD gene from the same tissue (Table I). Positive clones derived from the cytosolic Cu/Zn-SOD gene were characterized. The total sequence is 3950 bp long. Structural alignment showed perfect agreement between the cDNA sequence and the open reading frame of genomic DNA constructed from eight exons. The coding sequence, comprising 456 bp, begins in the second and ends in the last exon. The genomic sequence was compared with SOD gene structures derived from eukayotes: human (2.3 kb, five exons, four introns, Levanon et al., 1985), Drosophila melanogaster (1.8 kb, two exons, one intron, Seto et al., 1987), Neurospora crassa (1.0 kb, four exons, three introns, Chary et al., 1990), and rice (2.0 kb, eight exons, seven introns,

Cloning, expression, and purification of a functional glutathione reductase from sweet potato (Ipomoea batatas [L.] Lam): kinetic studies and characterization.

A cDNA encoding a putative glutathione reductase (GR) was cloned from sweet potato (Ib). The deduced protein showed high level of sequence homology with GRs from other plants (79-38%). A three-dimensional (3-D) homology structure was created. The active site Cys residues are conserved in all reported GR. Functional IbGR was overexpressed and purified. The purified enzyme showed an active monomeric form on a 10% native polyacrylamide gel electrophoresis (PAGE). The monomeric nature of the enzyme was confirmed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and molecular mass determination of the native enzyme. The Michaelis constant (K(m)) values for GSSG (glutathione disulfide) and NADPH (β-nicotinamide adenine dinucleotide phosphate, reduced form) were 0.114 and 0.056 mM, respectively. The enzyme activity was inhibited by Cu(2+) and Zn(2+), but not by Ca(2+). The proteins half-life of deactivation at 70 °C was 3.3 min, and its thermal inactivation rate constant K(d) was 3.48 × 10(-1) min(-1). The enzyme was active in a broad pH range from 6.0 to 11.0 and in the presence of imidazole up to 0.8 M. The native enzyme appeared to be resistant to digestion by trypsin or chymotrypsin.

Production of GST–SOD fusion protein by recombinant E coli XL1 Blue

A recombinant plasmid was constructed by inserting a DNA fragment with the coding region of Cu/Zn–superoxide dismutase (Cu/Zn–SOD) cDNA from sweet potato, Ipomoea batatas (l) Lam cv Tainong 57, into the 3′ end of the open reading frame of the glutathione S-transferase (GST) gene in an expression vector, pGEX-2T. The constructed plasmid was transformed into E coli XL1 Blue. Fusion proteins of Cu/Zn–SOD and GST (GST–SOD) were produced from the recombinant E coli. About 6 mg of GST–SOD fusion proteins could be obtained from 1 dm3 of cultural broth after induction with 0.075 mmol dm−3 Isopropyl-β-D-thiogalactoside (IPTG). Lactose was not an efficient inducer. High cell density culture was performed by fed-batch fermentation using a glucose analyzer to control glucose concentration at 1 g dm−3. The cell density of the fed-batch culture reached an OD600 of 30, the total amount of GST–SOD fusion protein was 100 mg dm−3 which is about 14 times more than that of the batch culture. Most of the fusion proteins were shown to be in an active monomeric form, and the molecular weight was estimated to be 45 kDa by SDS–PAGE and 47 kDa by gel filtration. The specific activity of the purified fusion proteins was about 1200 mg−1 and equal to 3200 unit per mg of SOD domain only. © 2000 Society of Chemical Industry

Monothiol Glutaredoxin cDNA from Taiwanofungus camphorata: A Novel CGFS-type Glutaredoxin Possessing Glutathione Reductase Activity

Glutaredoxins (Grxs) play important roles in the redox system via reduced glutathione as a reductant. A TcmonoGrx cDNA (1039 bp, EU158772) encoding a putative monothiol Grx was cloned from Taiwanofungus camphorata (formerly named Antrodia camphorata). The deduced amino acid sequence is conserved among the reported monothiol Grxs. Two 3-D homology structures of the TcmonoGrx based on known structures of human Grx3 (pdb: 2DIY_A) and Mus musculus Grx3 (pdb: 1WIK_A) have been created. To characterize the TcmonoGrx protein, the coding region was subcloned into an expression vector pET-20b(+) and transformed into E. coli C41(DE3). The recombinant His6-tagged TcmonoGrx was overexpressed and purified by Ni(2+)-nitrilotriacetic acid Sepharose. The purified enzyme showed a predominant band on 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme exhibited glutathione reductase (GR) activity via dithionitrobenzoate (DTNB) assay. The Michaelis constant (K(M)) values for GSSG and NADPH were 0.064 and 0.041 mM, respectively. The enzymes half-life of deactivation at 60 °C was 10.5 min, and its thermal inactivation rate constant (k(d)) was 5.37 × 10(-2) min(-1). The enzyme was active under a broad pH range from 6 to 8. The enzyme retained 50% activity after trypsin digestion at 37 °C for 40 min. Both mutants C(40)→S(40) and C(165)→S(165) lost 40-50% GR activity, whereas the mutant S(168)→C(168) showed a 20% increase in its GR activity.