Purification and Characterization of a Novel Redox-Regulated Isoform

Article pubs.acs.org/JAFC

Purification and Characterization of a Novel Redox-Regulated Isoform of Myrosinase (β-Thioglucoside Glucohydrolase) from Lepidium latifolium L. Rohini Bhat,†,§ Tarandeep Kaur,†,§ Manu Khajuria,† Ruchika Vyas,‡ and Dhiraj Vyas*,†,§ †

Biodiversity and Applied Botany Division, ‡Formulation and Drug Development Division, and §Academy of Scientific and Innovative Research, Indian Institute of Integrative Medicine (CSIR), Canal Road, Jammu, Jammu and Kashmir 180001, India S Supporting Information *

ABSTRACT: Myrosinase (ExPASy entry EC 3.2.1.147) is involved in the hydrolysis of glucosinolates to isothiocyanates, nitriles, and thiocyanates that are responsible for various ecological and health benefits. Myrosinase was purified from the leaves of Lepidium latifolium, a high-altitude plant, to homogeneity in a three-step purification process. Purified enzyme exists as dimer in native form (∼160 kDa) with a subunit size of ∼70 kDa. The enzyme exhibited maximum activity at pH 6.0 and 50 °C. With sinigrin as substrate, the enzyme showed Km and Vmax values of 171 ± 23 μM and 0.302 μmol min−1 mg−1, respectively. The enzyme was found to be redox-regulated, with an increase in Vmax and Kcat in the presence of GSH. Reduced forms of the enzyme were found to be more active. This thiol-regulated kinetic behavior of myrosinase signifies enzyme’s strategy to fine-tune its activity in different redox environments, thus regulating its biological effects. KEYWORDS: myrosinase, enzyme purification, enzyme kinetics, redox regulation, glutathione

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INTRODUCTION Glucosinolates are nitrogen- and sulfur-containing plant secondary metabolites present in the family Brassicaceae, of which more than 120 different glucosinolates have been identified.1 Because of their occurrence in important crops such as cabbage, broccoli, oilseed rape (canola), and the model plant Arabidopsis, they are a well-studied class of plant secondary metabolites. Glucosinolates by themselves have little biological activity, but upon plant damage, they are hydrolyzed by myrosinases (β-thioglucosidase glucohydrolase, ExPASy entry EC 3.2.1.147) to form a variety of hydrolysis products, including isothiocyanates, nitriles, epithionitriles, and thiocyanates.2 These hydrolysis products are responsible for “mustard bomb”: the typical taste and smell of cruciferous vegetables. Glucosinolates and their degradation products, mainly isothiocyanates, have been found to give protection against cancer, cardiovascular and central nervous system diseases, diabetic nephropathy and neuropathy, skin integrity, and Helicobacter pylori infection.3 Apart from their health benefits, these degradation products are well-known for their ecological importance in plant−insect and plant−microbe interactions.4,5 Glucosinolate hydrolysis is avoided in situ because glucosinolates and myrosinase are present in different tissues or cellular compartments. Myrosinases are members of the family 1 O-glycoside hydrolase superfamily6 with different degrees of glycosylation. They have been reported to be composed of two identical subunits ranging from 60 to 75 kDa with apparent molecular masses of 135−150 kDa.7 However, different studies have shown that they can form high-molecularweight complexes with myrosinase-binding proteins.7−9 3D structural analysis of various myrosinases on the basis of the crystal structure of Sinapis alba myrosinase has suggested that the protein folds into a (β/α)8-barrel structure.7,10 The active © 2015 American Chemical Society

site has a hydrophobic pocket for binding the variable but generally hydrophobic side chain of glucosinolates and includes specific amino acids that participate in catalysis or interact with the ascorbate cofactor or the glucose or sulfate moieties of glucosinolates. To date several myrosinases from various Brassicaceae members have been purified, including Brassica napus,11,12 Lepidium sativum,13 Sinapis alba,14−16 Raphinus sativus,17,18 Armoracia rusticana,19 Crambe abyssinica,20 and Brassica oleracea.21 Their characterization has led to a deeper understanding of the glucosinolate−myrosinase system in plants, including activation of myrosinase by ascorbic acid and their specificities with various glucosinolates. Lepidium latifolium, also known as perennial pepperweed, is an ecologically important plant22 that has attracted the attention of ecologists after been recognized as noxious weed along the western coast of North America.23 However, the Western Himalayan ecotype of this plant is used as phytofood,24 and several stress-responsive genes have recently been isolated from this ecotype.25,26 It has also been shown to be a rich source of sinigrin24 and possesses a very efficient glutathione-mediated redox mechanism.27 The role of myrosinase in thiol-based signaling in Brassica napus guard cells28 and allyl isothiocyanate in Arabidopsis stomatal closure29 have been recently deciphered. The present study was therefore envisaged to purify and characterize myrosinase from this Himalayan ecotype of L. latifolium. We hypothesize a responsive glucosinolate−myrosinase system in this plant owing to its high glucosinolate content. Received: Revised: Accepted: Published: 10218

May 15, 2015 November 2, 2015 November 3, 2015 November 3, 2015 DOI: 10.1021/acs.jafc.5b04468 J. Agric. Food Chem. 2015, 63, 10218−10226

Article

Journal of Agricultural and Food Chemistry

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Size-exclusion chromatography of glycoprotein fraction was done on a 1260 Infinity series HPLC (Agilent Technologies, Santa Clara, CA, USA) using an SEC-5 column (7.8 × 300 mm2, 5 μm, 300 Å) with autosampler and fraction collector. For elution of proteins, 150 mM sodium phosphate buffer (pH 7.0) was used as solvent with a flow rate of 0.8 mL/min. Detection of various peaks was done at 214 nm using a diode array detector. The peaks were collected and tested for myrosinase activity. The active enzyme peak was further concentrated as described above. PAGE and Western Blotting. Protein samples were separated in a reducing environment on a 10% separating SDS-PAGE gel, and bands were visualized by staining with SimplyBlue Safestain (Life Technologies Ltd., Carlsbad, CA, USA). For activity staining, native gel electrophoresis was conducted without SDS and DTT at 4 °C. Native gel was equilibrated with citrate buffer (pH 5.0) for 10 min and BaCl2 assay mixture (50 mM BaCl2, 1 mM ascorbic acid, and 18 mM sinigrin). The gel was then covered with a glass plate and incubated at 37 °C until white bands of BaSO4 appeared. Protein samples were separated on 10% SDS-PAGE as above and transferred to a PVDF membrane (Westran Clear Signal, Whatman Inc., Sanford, ME, USA) using a submerged wet transfer buffer (25 mM Tris-HCl (pH 7.6), 192 mM glycine, 20% methanol, and 0.03% SDS) through electroblotting. Complete transfer was evaluated using ponceau S staining, and the membrane was blocked with 3% BSA. Furthermore, it was incubated with 3D7 monoclonal antibodies (a kind gift from Prof. Meijer, Sweden) for 30 min and washed three times with TBS-T buffer for 5 min. The blot was incubated with goat anti-mouse secondary antibody for 30 min and developed using ECL Advance Western blotting detection kit (GE Healthcare, Buckinghamshire, UK). LC-MS/MS Analysis. Following SDS-PAGE, the purified protein band was cut and sent to Ms/- Xcelris Inc., Ahmedabad, India, for protein identification. The sample was analyzed by LC-MS/MS using AB Sciex TripleTOF 6600 System. After the sample is loaded, the peptide is trapped (ChromXP nanoLC Trap column 350 μm i.d. × 0.5 mm, ChromXP C18 3 μm 120 Å) and eluted at a flow rate of 300 nL/ min into a reverse-phase C18 column (ChromXP nano LC column 75 μm i.d. × 15 cm, ChromXP C18 3 μm 120 Å) using a linear gradient of acetonitrile (3−36%) in 0.1% formic acid. Raw data files were converted to Mascot Generic Format (MGF) and mzXML format using OpenMS. The MGF files are searched against UniProt, NCBI, and common MS contaminant databases using Mascot 2.5 (Matrix Science) Software. Protein Characterization. Molecular Weight Determination. The molecular weight of the purified protein was determined using high-performance size-exclusion chromatography.30 The column (SEC-5 column (7.8 × 300 mm2, 5 μm, 300 Å) was equilibrated using 150 mM sodium phosphate buffer (pH 7.0) with a flow rate of 1 mL/min. Different protein standards including catalase (220 kDa), alcohol dehydrogenase (160 kDa), transferrin (81 kDa) albumin bovine (66.7 kDa), β-lactoglobulin (35 kDa), and ribonuclease (13.7 kDa) were calibrated in the column. Purified myrosinase was also run through the column, and its native molecular weight was estimated in comparison to those of the protein standards by plotting log molecular weight versus retention time. pH and Temperature Optima. The effect of pH on enzyme activity was tested in the pH range of 3.0−10.7 using sodium citrate buffer (3.0−5.0), sodium malonate buffer (5.0−7.0), Tris-HCl buffer (7.0− 9.0), and sodium carbonate buffer (9.2−10.7). Purified protein was incubated in different pH solutions for 1 h, and the activity was measured as described above. The effect of temperature on enzyme activity was determined at 4, 10, 20, 30, 40, 50, 60, 70, 80, and 90 °C at optimum pH 6.0. Kinetic Analysis of Myrosinase. Kinetic study of the purified enzyme was done by studying the velocity of catalysis of sinigrin at different sinigrin concentrations (0.02−0.3 mM). The Lineweaver− Burk plot (double-reciprocal plot) method was used to determine the Michaelis−Menten constant (Km), maximum reaction velocity (Vmax), and the turnover rate of enzyme−substrate complex (Kcat) of the purified protein.

MATERIALS AND METHODS

Plant Material. Authenticated seeds of L. latifolium were collected from Leh and grown at the experimental farm of Indian Institute of Integrative Medicine (IIIM), Jammu (32°43′ N, 74°54′ E; 305 m above sea level). For purification, mature leaves were collected during full vegetative growth phase (during May) and immediately stored in liquid nitrogen. Leaves from plants growing at Leh (34°10′ N, 77°40′ E; 3505 m above sea level) were also collected during the same phenological stage and stored in liquid nitrogen until further analysis. Comparisons were made between leaves from the top (more than 50 cm from the base) and bottom (0−25 cm from the base). Myrosinase Activity Assay. Myrosinase activity was determined by evaluating the rate of glucose production. Sinigrin (5 mM in 25 mM sodium malonate buffer, pH 6.0) was incubated for 30 min with the requisite amount of enzyme at 37 °C. The enzyme reaction was stopped by heating at 95 °C for 10 min, and the released glucose was measured by the peroxidase−glucose oxidase coupled reaction using a glucose assay kit (Sigma-Aldrich Life Science, St. Louis, MO, USA). One unit of enzyme was defined as 1 μmol of glucose produced per minute using sinigrin as substrate. Alternatively, myrosinase activity was also determined by evaluating the rate of sinigrin hydrolysis as described earlier19 with some modifications. Briefly, sinigrin (0.2 mM) was mixed with 300 μL of phosphate buffer (25 mM), and the mixture was allowed to equilibrate for 5 min in a microtiter plate. The reaction was initiated by addition of requisite amount of crude/purified enzyme. Hydrolysis of sinigrin was continuously monitored at 229 nm in a microplate reader (Synergy H1 hybrid reader, Biotek US) over a period of 30 min at room temperature. One unit of enzyme was defined as the amount of myrosinase that catalyzes the hydrolysis of 1 μmol of sinigrin per minute. Protein content at every step was measured using Bradford reagent (Sigma Life Science, St. Louis, MO, USA), and the specific activity was calculated as units of enzyme per milligram of protein. Glutathione Estimation. Glutathione reduced (GSH) was measured on the basis of preferential acid−alkali destruction of reduced−oxidized forms using a coupled recycling assay as described previously.27 Experimental rates were derived from standard curve made for 0−500 pmol of GSH using an initial rate of change. Enzyme Purification. Protein Extraction. All the steps for purification of enzyme were carried out at 4 °C. At different steps in purification protocol, samples were assayed for myrosinase activity, and the protein content was estimated as described below. Leaf material (50 g) from plants grown at Jammu was crushed in a prechilled pestle and mortar using liquid nitrogen. The leaf powder was thoroughly mixed with 500 mL of 25 mM potassium phosphate extraction buffer (pH 7.0) containing 1 mM EDTA, 2 mM DTT, 1 mM PMSF, 0.05% Triton-X, and 1% glycerol. The homogenate was vortexed for 30 min at 4 °C and filtered through four folds of muslin cloth. The filtrate was centrifuged at 10 000 g for 30 min. The supernatant was transferred to another tube and centrifuged again for 10 min. The supernatant fraction thus obtained was used for further purification of the enzyme. Ammonium Sulfate Precipitation. Ammonium sulfate was added to crude enzyme fraction at 50% saturation followed by vortexing for 4 h. The homogenate was centrifuged at 10 000 g for 30 min. Resultant supernatant was then brought to 80% saturation with (NH4)2 SO4, vortexed for 4 h, and then centrifuged at 10 000 g for 30 min. The pellet thus obtained was dissolved in 25 mM phosphate buffer (pH 7.0) containing protease inhibitor cocktail (Pierce Thermo Scientific, Bonn, Germany). The dissolved protein was desalted using a 10 kDa Amicon Ultra-4 filter (Merck Millipore Ltd., Co. Cork, IRL) and 25 mM phosphate buffer (pH 7.0). Affinity Chromatography and HPLC. Glycoprotein-rich fraction was purified using a concanavalin-A agarose column (FOCUS glycoprotein, G-Biosciences, St. Louis, MO, USA) as per the manufacturer’s protocol. The eluted protein sample was concentrated using a 10 kDa Amicon Ultra-4 filter (Merck Millipore Ltd., Co. Cork, IRL) and used in HPLC. 10219

DOI: 10.1021/acs.jafc.5b04468 J. Agric. Food Chem. 2015, 63, 10218−10226

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Figure 1. (A) Myrosinase activity (bars) and corresponding GSH concentrations (black line and points) in top and bottom leaves of Lepidium latifolium growing at Leh and Jammu. LT, Leh top leaves; JT, Jammu top leaves; LB, Leh bottom leaves and JB, Jammu bottom leaves. (B) Myrosinase activity after treatment with different concentrations of oxidizing and reducing agents for 1 h: open bars, Jammu; solid bars, Leh. Each value is a mean of three independent readings (n = 3) ± SD. *, statistically significant values over control using Student’s t test at p ≤ 0.05. Effect of Ions and Other Compounds. The effect of different ions and compounds, including Ba2+, Ca2+, Mg2+, Mn2+, Zn2+, EDTA, glucose, SDS and urea was investigated on the purified enzyme at 5 mM concentration. Effect of different concentrations of ascorbic acid (0.02−2 mM) on purified enzyme activity was also investigated. Redox Treatment. Crude extract and purified enzyme were treated with different concentrations of reducing agent DTT (0.5, 5, and 10 mM) and oxidizing agent CuCl2 (0.5, 5, and 10 mM). Reversible redox behavior was checked by first oxidizing the purified enzyme with CuCl2 (10 mM), followed by its reduction with DTT (5, 10 mM). The effect of reduced (GSH; 1 and 10 mM) and oxidized (GSSG; 1 and 10 mM) glutathione was also tested to understand the oligomerization status of the purified myrosinase. Kinetics analysis of the purified enzyme was also conducted in the presence of 0.1, 0.5, and 1 mM GSH. In a separate experiment, 0.25 mM sinigrin was incubated in the presence of purified enzyme (0.8 μg), purified enzyme + 0.5 mM GSH, or purified enzyme + 1 mM GSH for 1 h at 37 °C. Degradation of sinigrin was quantified on HPLC using an Eclipse XDB-C18 column (7.8 × 300 mm2, 5 μm, 300 Å) in isocratic flow (A: 10% methanol, B: 90% water) at a rate of 1 mL/min. Detection of the sinigrin peak was done at 229 nm using a diode array detector. Statistical Analysis. All the data were produced in triplicate (n ≥ 3) and represented as the mean ± SD. Student’s t test was carried out

using online GraphPad Software, USA (http://www.graphpad.com/ quickcalcs/) to identify the significant differences (p ≤ 0.05).

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RESULTS AND DISCUSSION Glucosinolates are hydrolyzed by myrosinase into isothiocyanates, thiocyanates, nitriles, and epithioalkanes, among others, depending on the pH, metal ions, epithiospecifier proteins, and other cofactors. This is important in nutraceutical and ecological contexts because the hydrolysis products are biologically active. In our study, the myrosinase activity from the leaves of L. latifolium plants growing at Jammu was higher (∼1.5-fold) as compared to that of the plants growing at Leh (Figure 1A) in both the top and bottom leaves. In an earlier study using the same genotype, we have found that GSHinduced changes in redox potential at these locations27 may have resulted from differences in various abiotic conditions including temperature, light intensity, and atmospheric pressure. Positive correlation (r = 0.81) was found in the metabolic content of GSH (reduced glutathione) with the activity of myrosinase at these locations (Figure 1A). Although these metabolites and enzymes are present in different compartments in vivo, they come in contact with each other 10220


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activity in the purification table. With the exception of myrosinase purified from broccoli that showed 88% yield and 1318-fold purification,21 other studies have reported 34−40fold purification.13,19 SDS-PAGE analysis of the crude extract suggested a mixture of various molecular weight proteins (Figure 2A) in leaf homogenate of L. latifolium. Molecular weight of band obtained by CBB staining of the purified peak from HPLC was found to be ∼70 kDa (Figure 2A). Higher load (2 μg) of protein also resulted in a single band (Figure S1). A protein band at a similar position was also found in SDS-PAGE analysis of other purification steps. This suggested enrichment of specific myrosinase protein with each purification step. Previously, myrosinases from different members of family Brassicaceae have been reported to be a homodimer of subunit sizes ranging from 62 to 75 kDa.11,19,32,33 The presence of additional bands, either high or low molecular weight, have been credited to other myrosinase-associated proteins.7−9 However, we found a single band, suggesting that it consists of the same subunit and follows the general trend of myrosinase enzyme family from different plants. The protein was confirmed to be myrosinase, showing its reactivity with anti-myrosinase monoclonal antibody 3D7 at similar molecular weight (Figure 2B). This antibody has been previously used for identification and characterization of different myrosinase gene families.15,34,35 The appearance of white bands of BaSO4 in native gel staining confirmed myrosinase activity (Figure 2C). Native molecular weight of the purified protein as determined by size-exclusion chromatography was found to be ∼160 kDa (Figure S2). This suggests that the purified enzyme exists as a homodimer with a subunit of about 70 kDa. The existence of the dimeric form of myrosinase has been widely reported in different plant species.18,19,31 Thus, purified myrosinase from L. latifolium was found to be identical to group III (140−200 kDa) myrosinase from Brassica species.9 The mass spectrometry based peptide sequence BLAST search of known protein databases showed homology with Armoracia rusticana myrosinase (GKYPDIMR and GYATGTDAPGR) and TGG2 of Arabidopsis thaliana (IGPVMITR and DLDVMEELGVKGYR) (Table 2). Both these enzymes also show dimeric composition with subunit sizes of ∼65 kDa19,31 and are phylogenetically very close.36 Thus, purified enzyme from L. latifolium is also thought to be encoded by the MYR I gene subfamily. Characterization of the Purified Enzyme. Figure 3A shows that the optimum pH of the purified myrosinase is slightly acidic (pH 6.0). Loss of enzyme activity was observed in the basic range (20%) except for pH 9.2 where enzyme retained nearly 40% activity. Variation in activity was also observed with different buffers, which suggests the effect of various salts and ions in enzyme activity. Even in the optimum range, malonate buffer showed higher activity as compared to that of the phosphate buffer (data not shown). The pH characteristics of the purified enzyme are in agreement with the pH optima of myrosinase from horseradish roots (5.7),19 Lepidium sativum seedlings (5.5),13 and TGG1 from Arabidopsis leaves (6.0).31 The temperature optimum was found to be in the range of 50 °C as observed for other myrosinases (Figure 3B).19,21,31,37 Most of the myrosinases have been found to work well in a broad range of temperature (37−75 °C).7,33 The rate of sinigrin hydrolysis at pH 6.0 (sodium malonate) and 50 °C was found to be linear for at least 60 min. As already reported for other myrosinases, ascorbate activated the purified

during herbivory. To investigate the redox response of myrosinase in this plant, the enzyme activity of both the Jammu and Leh crude leaf extract was analyzed after incubation with different oxidizing and reducing agents (Figure 1B). A significant increase (p ≤ 0.05) in the myrosinase activity in the presence of GSH (10 mM) was observed, whereas no change was observed in the presence of GSSG (oxidized form). Similar enhancement of enzyme activity was observed with DTT (reducing agent) treatment, whereas a decrease in the enzyme activity was observed in the presence of CuCl2 (oxidizing agent). This result with crude enzyme extract suggested a possible role of redox-mediated regulation in the glucosinolate−myrosinase system in this important food plant of high altitudes. Because higher myrosinase activity was observed in plants growing at Jammu, the enzyme purification process was therefore initiated from L. latifolium growing at Jammu. Both product formation coupling assay and sinigrin degradation assay were used for determination of myrosinase activity, with both the assays showing similar results. However, the product estimation assay showed interference (no color formation) in the presence of DTT and GSH (data not shown). Also, this assay could not be used in some experiments where glucose is used. Hence, all the results for myrosinase activity assay, except for that of enzyme purification, were shown using sinigrin degradation assay. Homogeneously pure enzyme (see below) and proper controls were used for the assays to avoid nonspecific degradation of the substrate. Purification. A summary of different purification steps such as ammonium sulfate precipitation, concanavalin A affinity chromatography, and size-exclusion HPLC that were used for myrosinase purification have been provided in Table 1. The Table 1. Purification Table of Myrosinase from 50 g of Leaves from Lepidium latifolium L.a purification step crude extract (NH4)2SO4 precipitation concanavalin affinity size-exclusion HPLC

total protein (mg)

total units (units)

specific activity (units/mg)

purification fold

yield (%)

604 227

27.3 14.2

0.045 0.062

1 1.4

100 52.2

2.5

3.1

1.19

26.4

11.2

0.57

1.8

3.12

69.3

6.5

a

Enzyme activity was measured using product estimation assay using 5 mM sinigrin as substrate.

first purification step led to approximately 1.4-fold purification of myrosinase with 52.2% yield (specific activity of 0.062 μmol min−1 mg−1 protein). Lectin-based affinity chromatography further increases purification to 26.4-fold and specific activity to 1.19 μmol min−1 mg−1 protein with a yield of around 11.2%. The final purification step resulted in 6.5% yield of myrosinase enzyme with 69.3-fold purification (specific activity of 3.12 μmol min−1 mg−1 protein) and showed a single band in reducing SDS-PAGE analysis. Our results match with 4% yield and 30−40-fold purification for Crambe seed myrosinase in a three-step process.20 Similarly, 51.9-fold TGG1 purification was achieved when the leaves of Arabidopsis mutants were used as a source.31 We have also used the leaves as a source for myrosinase in this study. Because the myrosinase activity assay was carried out without ascorbate to avoid any redox-related effects, this might be a reason for fewer units and lower specific 10221


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Figure 2. Coomassie-stained SDS-PAGE gel (A), Western blot using 3D7 antibody (B), and activity staining using BaSO4 (C) of various protein fractions at different purification steps. CE, crude extract; AS, 50−80% ammonium sulfate precipitate fraction; CV, concanavalin-eluted fraction; PU, size-exclusion HPLC-eluted fraction; MK, protein marker. Panel D shows the chromatogram of myrosinase purification using size-exclusion chromatography. UV spectra show concanavalin fraction (solid line) and purified enzyme (dashed line).

Table 2. LC-MS/MS Protein Identification and Percentage Identity Matching of Obtained Peptides % identity peptide sequence GKYPDIMR GYATGTDAPGR IGPVMITR DLDVMEELGVKGYR IGPVMIT

m/z (z) 497.24 532.24 451.76 409.69 372.70

(2) (2) (2) (4) (2)

Mascot score

Armoracia rusticana (MY1) (Q5PXK2_ARMRU)

Arabidopsis thaliana (TGG2) (BGL37_ARATH)

58 58 41 41 37

100 100 100 78.6 100

87.5 91 100 100 100

The purified myrosinase exhibited a Km value of 171 ± 23 μM and a maximum velocity of 0.302 μmoles min−1 mg−1 at pH 6.0 and 37 °C. For kinetic studies, 37 °C was chosen to mimic the temperature being faced by the plant in its natural environment during its growth stage. Also, the enzyme retained more than 80% of its activity at this temperature. Giving a Kcat value of 0.33 s−1 toward sinigrin, the enzyme possesses a comparable/higher glucohydrolase activity than enzymes purified from Arabidopsis.31 The lineage similarity of L. latifolium with Arabidopsis has already been reported with both species belonging to the lineage I clade of the Brassicaceae family.27 However, higher Kcat values were obtained in recombinant cloned myrosinase from Arabidopsis.33 When enzyme activity was assessed in the presence of different substrates, it was observed that this isozyme had the highest affinity toward sinigrin at optimum temperature and pH (Figure 6C). This was followed by gluconapin, whereas the least activity was observed with glucoraphanin. This has direct corroboration with the glucosinolate types present in this plant, with very high content of sinigrin.24 A similar observation was found for the Crambe abyssinica myrosinase where the highest affinity was found for epiprogoitrin, which is the major glucosinolate present in Crambe seeds.20 It can therefore be postulated that the purified isozyme plays a major role in degradation of glucosinolates from this plant, and hence in their ecological effects. Redox Regulation of Purified Enzyme and Its Significance. The presence of reducing agent DTT showed an increase in the activity of the purified enzyme, with lower

myrosinase activity from L. latifolium until 0.7 mM concentration (6.5-fold) (Figure 3C). Mutants of AsA-biosynthetic enzyme GDP-mannose pyrophosphorylase have significantly lower myrosinase activity than wild-type Arabidopsis.38 When ascorbic acid binds with the enzyme, it activates the water molecule by acting as a base and abstracting a proton, thus enhancing its nucleophilic attack at the anomeric center.39 Higher concentrations of ascorbic acid have been shown to inhibit the enzyme because the ascorbic acid site overlaps with the aglycon binding site. The effect of various ions/inhibitors/compounds on the activity of purified enzyme has been shown in Figure 3D. All the studied ions at 5 mM concentration inhibited the purified enzyme activity, with Zn2+ causing maximum inhibition. Zn2+ has been previously reported to stabilize the dimeric form of Sinapis alba myrosinase.10 However, it is also reported to be inhibitory even at nanomolar concentrations mostly for enzymes that contain a catalytic cysteine residue.40 It remains to be seen whether the active site of L. latifolium myrosinase contains any cysteine residues. Previous studies report both positive41 and negative effects37 of Zn2+ on myrosinase activity. Chelating agents EDTA and urea showed positive effects on enzyme activity, whereas SDS and glucose had inhibitory effects. Chelation of interfering ions by EDTA could result in activation of enzyme activity. Unlike in Raphanus sativus seedlings,18 our study showed glucose (5 mM) inhibition probably resulting in mixed inhibition by product of myrosinase reaction (Figure S3). 10222


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Figure 3. Effect of pH (A), temperature (B), ascorbic acid (C), and ions/compounds (D) on activity of purified enzyme. A 200 ng amount of purified protein was used for each activity assay. Each value is a mean of three independent readings (n = 3) ± SD. *, statistically significant values over control using Student’s t test at p ≤ 0.05.

intermolecular disulfide bonds between subunits and resulting in multimeric forms.43 Reduced glutathione (GSH) treatment also resulted in an increase in the enzyme activity and changes in the oligomeric forms (Figure 5A,B), whereas GSSG did not show much change. It is therefore postulated that a ratio of different forms is present in different redox environments of the cell, where a specific reduction state favors higher enzyme activity. Enzyme activity in terms of sinigrin degradation was found to be higher in the presence of 0.5 mM GSH as compared to that in the presence of 0 (control) and 1 mM GSH (Figure 5C) suggesting favoring of a specific reduction state. A similar observation was made for kinetic parameters in the presence of GSH (Figure 6A,B), wherein the presence of 0.5 mM GSH showed highest values of Km and Vmax. Similar results on redox regulation were obtained in crude enzyme activity, suggesting that the purified enzyme was key for showing redox regulation in vivo. Endogenous presence of other glutathione-metabolizing enzymes in crude extract such as glutathione reductase, glutathione peroxidase, and class III peroxidases might be the reason for any differential response from purified enzyme. Recently, myrosinase has been identified as a thiol-based redox protein in Brassica napus guard cells, which suggests that the degradation of glucosinolates also has a physiological role for the plant under differential redox environments.28 Myrosinase purified in this study perfectly fits in this role because this ecotype of L. latifolium grows in a high-altitude cold arid zone of the Western Himalayas and possesses a dynamic glutathione-mediated redox regulation.27 This may not show significance under normal conditions

concentrations of DTT (0.5 and 5 mM) showing higher activity (Figure 4A). In contrast, oxidizing agent CuCl2 (10 mM) showed a decrease (∼25−30%) in the enzyme activity. This hints toward the redox regulation of the purified enzyme. To investigate whether the loss of activity under oxidizing conditions was reversible, the CuCl2-treated protein (having decreased activity) was again incubated with DTT (5 and 10 mM). The protein regained its original activity, thus confirming this mechanism to be redox-regulated and not a result of unspecific oxidation (Figure 4C). Reversibility of the activity during different redox regimens is considered to be a key feature in redox-regulating enzymes.42,43 A similar increase in activity in the presence of 5 mM DTT was reported in myrosinase purified from L. sativum seedlings.13 In non-reducing SDS-PAGE, different forms were observed with DTT and CuCl2 treatment (Figure 4B). These were generated with a molecular mass corresponding to homodimer (∼160 kDa) and monomer (∼70 kDa). Another partially reduced form (∼78 kDa) was also visible. Reduction with DTT results in the monomerization and disappearance of the multimeric forms in purified protein, whereas oxidation with CuCl2 results in multimers and a change in monomeric form. Similarly, monomeric form was gradually recovered in the reversibility experiment (Figure 4D). As observed in aldehyde dehydrogenase from A. thaliana, this can be attributed to stepwise reduction/oxidation of catalytic/noncatalytic cysteine residues.42 It has been suggested that oxidation triggers conformational changes that alter the surface charges and the hydrophobicity thereby exposing the thiol groups to form 10223


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Figure 4. Effect of different concentrations of DTT and CuCl2 on enzyme activity (A) and various forms on non-reducing SDS-PAGE (B) of the purified myrosinase enzyme. Confirmation of redox regulation of purified enzyme was done by first oxidizing the enzyme with CuCl2 (10 mM) followed by its reduction with DTT (5 and 10 mM). Enzyme activity (C) and various forms on non-reducing SDS-PAGE (D) have been shown: thick black arrows, monomeric form; open arrows, partially reduced monomeric form; thin black arrows, dimeric and other multimeric forms. Each value is a mean of three independent readings (n = 3) ± SD. *, statistically significant values over control using Student’s t test at p ≤ 0.05.

Figure 5. Effect of different concentrations of GSH and GSSG on enzyme activity (A) and various forms on non-reducing SDS-PAGE (B) of the purified myrosinase enzyme. Panel C represents HPLC chromatogram showing sinigrin degradation with purified enzyme (0.8 μg) in absence and presence of GSH (0.5 and 1 mM) for 1 h. All treatments were given for 1 h at pH 6.0 and 37 °C. Thick black arrows, monomeric form; open arrows, partially reduced monomeric form; thin black arrows, dimeric and other multimeric forms. Each value is a mean of three independent readings (n = 3) ± SD. *, statistically significant values over control using Student’s t test at p ≤ 0.05.

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Figure 6. Double reciprocal Lineweaver−Burk plot (A) and kinetic constants (B) of the purified enzyme with different concentrations of GSH (0, 100, 500, and 1000 μM). Sinigrin (0.02−0.3 mM) was used as substrate at pH 6.0 and 37 °C. Panel C represents substrate specificity of purified enzyme toward six glucosinolates, namely, sinigrin (SIN), glucoiberin (GB), glucoerucin (GE), gluconapin (GN), glucoraphinin (GR), and glucotropoelin (GT) at 0.1 mM concentration. Each value is a mean of three independent readings (n = 3) ± SD. *, statistically significant values with respect to sinigrin using Student’s t test at p ≤ 0.05.

Notes

because of their presence in different compartments; however, it might play an important role during biotic interactions and herbivory when they come in contact with the cellular environment. Biological roles of glucosinolate such as plant− insect, plant−microbe, and other health-benefiting properties depend upon the quantitative and qualitative production of hydrolysis products that will be influenced by these redox properties.

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The authors declare no competing financial interest.

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ACKNOWLEDGMENTS We gratefully thank Prof. Dr. Johan Meijer, Department of Plant Biology, Swedish University of Agricultural Sciences, Uppsala, Sweden, for critically reading the manuscript and providing 3D7 antibodies. We are thankful to the Director, IIIM Jammu, for providing the necessary facilities. R.B. and T.K. thank CSIR for providing Senior Research Fellowships, and M.K. thanks DST, GOI, for providing an INSPIRE fellowship.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jafc.5b04468. Higher load (2 μg) of the purified protein in SDSPAGE;molecular weight determination by size-exclusion chromatography; inhibition kinetics in the presence of glucose; and mass spectrum analysis of the identified peptides. (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Author

*E-mail: dhirajvyas@rediffmail.com. Telephone: +91-1912585006-13 (350). Fax: +91-191-2586333. Author Contributions

D.V. conceived the study and designed the experiments. R.B., T.K., M.K., and R.V. carried out the experiments. R.B. and D.V. interpreted the data and wrote the paper. Funding

The authors acknowledge financial support to this study by Council of Scientific and Industrial Research (CSIR), Government of India, under CSIR- networking project (BSC-0109) on ‘Plant Diversity: Studying adaptation biology and understanding/exploiting medicinally important plants for useful bioactives (SIMPLE)’. 10225


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