Inhibitory Efficacy of Cycloartenyl Ferulate against α-Glucosidase and α-Amylase and Its Increased Concentration in Gamma-Irradiated Rice (Germinated Rice).

Cycloartenyl ferulate is a derivative of γ-oryzanol with varied biological activity, including diabetes mellitus treatment. This research focused on improving the cycloartenyl ferulate accumulation in germinated rice by gamma irradiation under saline conditions. Moreover, the inhibitory potential of cycloartenyl ferulate against carbohydrate hydrolysis enzymes (α-glucosidase and α-amylase) was investigated through in vitro and in silico techniques. The results revealed that cycloartenyl ferulate increased in germinated rice under saline conditions upon gamma irradiation. A suitable condition for stimulating the highest cycloartenyl ferulate concentration (852.20±20.59 µg/g) in germinated rice was obtained from the gamma dose at 100 Gy and under 40 mM salt concentration. The inhibitory potential of cycloartenyl ferulate against α-glucosidase (31.31±1.43%) was higher than against α-amylase (12.72±1.11%). The inhibition mode of cycloartenyl ferulate against α-glucosidase was demonstrated as a mixed-type inhibition. A fluorescence study confirmed that the cycloartenyl ferulate interacted with the α-glucosidase's active site. A docking study revealed that cycloartenyl ferulate bound to seven amino acids of α-glucosidase with a binding energy of -8.8 kcal/mol and a higher binding potential than α-amylase (-8.2 kcal/mol). The results suggested that the gamma irradiation technique under saline conditions is suitable for stimulating γ-oryzanol, especially cycloartenyl ferulate. Furthermore, cycloartenyl ferulate demonstrated its potential as a candidate compound for blood glucose management in diabetes mellitus treatment.

Nanozymes paper-based analytical device for the detection of organophosphate pesticides in fruits and vegetables.

In this work, copper oxide nanoparticles (CuONPs) nanozymes paper-based analytical device was designed for the rapid detection of organophosphate pesticides in fruits and vegetables. The paper-based analytical device was modified with silica oxide nanoparticles to enhance the assay sensitivity. CuO nanozymes displayed peroxidase-like activity and catalyzed the oxidation of o-dianisidine in the presence of H2O2 from the hydrolysis of acetylthiocholine. This results in the formation of a brown-colored product. In the presence of organophosphate pesticides such as malathion, acetylcholinesterase activity was inhibited, resulting in reduced color intensity production, and which was measured with a smartphone. The proposed nanozymes paper-based analytical device exhibited a good linear detection range (0.1-5 mg L-1), a low detection limit of 0.08 mg L-1, and the analysis time was only about 10 min for malathion detection under optimal conditions. Moreover, the CuONPs had excellent catalytic activity and higher stability than peroxidase. Finally, this device can be applied to detect organophosphate pesticides in fruits and vegetables with rapidity, accuracy, portability, and ease of handling in the field.

A portable device as a paper test strip platform with smartphone application for detection of branched-chain amino acids in edible insects.

A novel strategy is presented for simple, sensitive and selective branched-chain amino acids (BCAAs) detection in edible insects on a paper test strip device readout with a smartphone. Silver nanoparticles (AgNPs) were applied, interacting with dye, which provided 1.5 times higher color intensity than the one without AgNPs. The proposed paper test strip exhibiting the detection limit of leucine, isoleucine and valine were 6.0, 6.2 and 7.2 mg/L, respectively. The linear dynamic ranges were 20-120 mg/L for leucine and 20-110 mg/L for isoleucine and valine detections. The proposed paper test strip was successfully applied for the detection of BCAAs in edible insects. The analytical results obtained using paper test strips were in good agreement with those obtained via a commercial test kit. This study shows the successful integration of the paper test strip and the smartphone to afford an easy-to-use, inexpensive and portable device alternative for BCAAs analysis.

Gamma Irradiation Promotes the Growth Rate of Thai Pigmented Rice As Well As Inducing the Accumulation of Bioactive Compounds and Carbohydrate Hydrolyzing Enzymes Inhibitors (α-Glucosidase and α-Amylase) under Salt Conditions.

Rice contains many bioactive compounds that perform various biological activities. Some of these compounds have been identified as α-glucosidase and α-amylase inhibitors, including guaiacol, vanillin, methyl vanillate, vanillic acid, syringic acid, and 2-pentyl furan. In this study, we assessed the growth rate, photosynthetic pigment content, phenolic content, and flavonoid content of gamma-irradiated Thai pigmented rice. Bioactive components of gamma-irradiated rice that had been subjected to salt treatment were also investigated. The findings showed that production of photosynthetic pigments, which are associated with plant growth, was induced by low gamma exposure. Phenolic and flavonoid content of rice was increased after gamma irradiation at 5 to 1,000 Gy. Both gamma irradiation and the salt conditions changed the quantity of vanillin, methyl vanillate, and vanillic acid in the rice. However, at a salt concentration of 40 mM, the salt stress had more of an effect than the gamma dosage. However, the high concentrations of methyl vanillate and vanillic acid detected in the rice under salt conditions were ameliorated by gamma irradiation. Guaiacol served as the substrate of guaiacol peroxidase for catalyzed reactive oxygen species, as evidenced by the observation that the guaiacol content of rice decreased between increased gamma dosages. A gamma dose of 40 to 1,000 Gy resulted in the production of syringic acid. Under salt stress, syringic acid buildup was also seen to be ameliorated by gamma irradiation. In comparison to salt conditions, particularly for 20 mM salt, gamma irradiation had less of an impact on the 2-pentyl furan in rice.

Inhibitory potential of phenolic compounds of Thai colored rice (Oryza sativa L.) against α-glucosidase and α-amylase through in vitro and in silico studies.

BACKGROUND: This study investigated the inhibitory efficiency of phenolic compounds content methyl vanillate, syringic acid and vanillic acid against α-glucosidase and α-amylase. The phenolic compound contents of 10 Thai colored rice cultivars were also determined, and the relationship between the inhibitory efficiency of colored rice extract with methyl vanillate, syringic acid and vanillic acid was evaluated. RESULTS: The results revealed that the inhibition efficiency of methyl vanillate, syringic acid and vanillic acid was higher against α-glucosidase than against α-amylase. Inhibitory activity of vanillic acid against α-glucosidase and α-amylase was highest, with IC50 of 0.100 ± 0.01 and 0.130 ± 0.02 mmol L-1 , respectively. Docking study showed strong binding by three hydrogen bonds and four hydrogen bonds between vanillic acid with the amino acid in the binding site of α-glucosidase and α-amylase, respectively. Inhibition modes of these phenolic compounds were defined as a mixed type inhibition against α-glucosidase. Highest phenolic compound contents of methyl vanillate, syringic acid and vanillic acid were obtained from methanol extracts of all rice cultivars. The methanol extracts of all colored rice cultivars such as Khao Leum Pua also showed the highest inhibition potential against α-glucosidase and α-amylase. The results indicated that these phenolic compound contents were closely related to the inhibition potential of colored rice extracts against α-glucosidase and α-amylase. CONCLUSION: Our results suggest that rice, especially colored rice cultivars, has the source of phenolic compounds. Moreover, the phenolic compounds had the greatest source of natural inhibitor against α-glucosidase and α-amylase. © 2022 Society of Chemical Industry.

Effect of gamma irradiation on growth, proline content, bioactive compound changes, and biological activity of 5 popular Thai rice cultivars.

Gamma irradiation is the technique used to induce plant mutation and it has affected both the physiological and biochemical compounds of the plant. Some new rice lines are also created through the gamma-irradiation technique. We investigated the effect of gamma irradiation on plant growth, volatile compounds, and the biological activity of gamma-irradiated rice extract compared to non-gamma-irradiated rice extract. The results reveal that the gamma-irradiated rice growth was related to the proline content, as the low gamma dose induced rice growth and proline accumulation in gamma-irradiated rice. We induced the bioactive compounds, including the flavonoid content and phenolic content of gamma-irradiated rice, through the low gamma irradiation dose at 60-100 Gy. Interestingly, bioactive compounds were stimulated by a gamma dose similar to that of the biological activity (antioxidant activity and enzyme inhibition) of gamma-irradiated rice. The results suggest that gamma-irradiated rice extract's biological activity was closely related to the flavonoid and phenolic content of rice. We also identified the variety of volatile compounds in gamma-irradiated rice and they were also reported for the biological activity. Our results can generate a new rice line that exhibits high plant growth and is rich with bioactive compounds such as flavonoid and phenolic compounds which are related to the improvement of human health.

Combination effects of rice extract and five aromatic compounds against α-glucosidase, α-amylase and tyrosinase.

Rice is a source of bioactive compounds related to human health and has been used for both consumption and traditional medicine. The authors investigated the synergistic and additive effect of rice extract (RE) combined with five aromatic compounds against three enzymes: α-glucosidase, α-amylase and tyrosinase. RE was purified by thin-layer chromatography (TLC) and preparative TLC (PTLC) with different solvent systems. RE had higher α-glucosidase and α-amylase inhibitory activity than the five aromatic compounds, while the five aromatic compounds had higher tyrosinase inhibitory activity than RE. The combination of RE/acarbose produced synergic inhibition of α-glucosidase and α-amylase, whereas RE showed additive inhibition of both enzymes when combined with aromatic compounds. The five aromatic compounds showed additive inhibition of tyrosinase when combined with RE. The combination of 2-methoxy-4-vinylphenol/vanillin/guaiacol produced synergistic inhibition of α-amylase while showing antagonism of α-glucosidase and tyrosinase. Interestingly, the RE produced additive inhibition of α-glucosidase, α-amylase and tyrosinase when combined with the 2-methoxy-4-vinylphenol/vanillin/guaiacol combination. RE had rich bioactive compounds related to α-glucosidase, α-amylase and tyrosinase inhibitory activity. Volatile compounds, including 2-methoxy-4-vinylphenol, vanillin and guaiacol, enhanced the inhibitory activity of RE against α-glucosidase, α-amylase and tyrosinase activities.

Evaluation of Cyanide Content, Volatile Compounds Profile, and Biological Properties of Fresh and Boiled Sliced Thai Bamboo Shoot (Dendrocalamus asper Back.).

This study evaluated the cyanide content, bio-active compounds profile, volatile compounds profile, and biological activity of fresh and boiled sliced bamboo. Cyanide was only detected in fresh bamboo shoots, at a content of 140.40±5.34 mg/kg. Furthermore, the fresh bamboo shoots extracts had free radical scavenging properties, as demonstrated by ABTSï½¥+ and DPPHï½¥ assays, and contained phytochemical compounds, such as flavonoid, terpenoid, and reducing sugar. Indeed, the total phenolic and flavonoid contents were 12.12±0.12 mg gallic acid equivalent/dw and 1.60±0.11 mg quercetin equivalent/dw, respectively. In addition, these extracts demonstrated inhibitory activity against α-glucosidase (61.30±0.45%), α-amylase (37.00±1.82%), and tyrosinase (26.57±0.57%). Some volatile compounds, such as 2-methoxyphenol and 2-pentylfuran, show α-glucosidase inhibitory activity, and these compounds exerted α-amylase inhibitory activity in the fresh sliced bamboo shoots. The major volatile compound 4-methylphenol (68.15%), which exerts tyrosinase inhibitory activity, was also detected in fresh sliced bamboo shoots. The boiled sliced bamboo shoots extracts also contained bio-active compounds and exhibited biological activity similar to those in the fresh sliced bamboo shoots extracts. However, the boiling process and sliced technique reduced the bio-active compounds and biological properties as well as some of volatile compounds.

Effect of rainfall and altitude on the 2-acetyl-1-pyrroline and volatile compounds profile of black glutinous rice (Thai upland rice).

BACKGROUND: The aroma intensity of rice is mixed of more than 100 volatile compounds. Fragrant rice is famous in Thailand due to specific aroma. One important volatile compound that contributes to aromatic intensity and represents a characteristic compound for fragrant rice is 2-acetyl-1-pyrroline (2AP). The production of volatile compounds and 2AP in rice is affected by various environmental factors, such as cultivation area, water content and temperature. RESULTS: Our research employed 29 samples of fragrant colored rice (Luempua black glutinous rice cultivar) from Phetchabun province, Thailand from three harvested rice seasons (2015-2017). The cultivation areas of rice samples exhibited differences in rainfall (976.6-1260.5 mm y r-1 ) and altitude (26.26-1033.41 m). All volatile compounds and 2AP were identified by gas chromatography-mass spectrometry (GC-MS). In 29 rice samples, 2AP levels decreased with increasing of rainfall. The increase in altitude seems to increase 2AP levels, but some high-altitude cultivation areas resulted in low 2AP content. A variety of volatile compounds was observed in 29 rice samples from different cultivation areas. The results showed that differences in altitude and rainfall affected the production of volatile compounds in fragrant colored rice. Moreover, our results indicated that different environmental conditions in cultivation areas potentially affect volatile compound production during rice growth. CONCLUSION: The increase in 2AP levels under low rainfall conditions and the assortment of volatile compound varieties produced in colored rice grown in different cultivation areas may be useful information for rice cultivation management striving to produce rice with high aromatic intensities. © 2021 Society of Chemical Industry.

Synthesis and inhibitory activity of N-acetylpyrrolidine derivatives on α-glucosidase and α-amylase.

BACKGROUND AND PURPOSE: Carbohydrate hydrolysis enzymes including α-glucosidase and α-amylase are related to type 2 diabetes mellitus. The inhibiting of these enzymes might use for type 2 diabetes mellitus treatment. EXPERIMENTAL APPROACH: N-substituted-acetylpyrrolidine linked with -benzyl- (N-(benzyl)-2-acetylpyrrolidine (4a)) and -tosyl- (N-(tosyl)-2-acetylpyrrolidine (4b)) were synthesized and evaluated for their pharmaceutical properties against α-glucosidase and α-amylase and free radical scavenging activity. The structures of 4a and 4b were determined through spectral studies (1H-NMR). FINDINGS / RESULTS: Both compounds 4a and 4b had highest inhibitory potential on α-glucosidase with the IC50 values of 0.52 ± 0.02 and 1.64 ± 0.08 mM, respectively. The kinetic investigation of 4a and 4b against α-glucosidase and α-amylase were functioned in mixed type inhibition. Moreover, both compounds are more likely to bind with the free enzyme than the enzyme-substrate complex based on the Ki < Ki´ on the α-glucosidase and α-amylase enzymes. Regarding the free radical scavenging, 4a had a higher capacity than 4b with IC50 values of 1.01 ± 0.010 mM for 4a and 1.82 ± 0.048 mM for 4b. CONCLUSION AND IMPLICATIONS: Our results indicated that a derivative of N-substitute-acetylpyrrolidine had high potential to inhibit α-glucosidase and α-amylase, and their free radical scavenging properties might be applied to the therapeutic care of patients with type 2 diabetes mellitus.

α-Glucosidase, α-amylase, and tyrosinase inhibitory potential of capsaicin and dihydrocapsaicin.

Chili is a spicy plant and is widely used in traditional medicine. Capsaicin and dihydrocapsaicin belong to the capsaicinoid group, which is produced from chili. This study aims to investigate the antidiabetic properties and anti-melanin synthesis of capsaicinoids by studying the inhibitory activity of capsaicin and dihydrocapsaicin with α-glucosidase, α-amylase, and tyrosinase. The results revealed that dihydrocapsaicin with IC50 had 4.13-fold and 3.00-fold for α-glucosidase and α-amylase, respectively, which are lower than capsaicin. Moreover, the IC50 of capsaicin with tyrosinase had 1.73 times less than dihydrocapsaicin. The inhibition constant (Ki ) also supported that the dihydrocapsaicin had higher inhibitory activity than capsaicin against α-glucosidase and α-amylase, but lower inhibitory activity than capsaicin on tyrosinase. Capsaicin and dihydrocapsaicin functioned in mixed-type inhibition on each enzyme, except that capsaicin functioned in competitive inhibition of tyrosinase. The results indicated that capsaicin and dihydrocapsaicin had more potent anti-melanin synthesis than antidiabetic properties. PRACTICAL APPLICATIONS: This study presents the inhibition potential of capsaicin and dihydrocapsaicin on antidiabetes and anti-melanin properties by standard methods for inhibitory activity against α-glucosidase, α-amylase, and tyrosinase. We suggest the application of these results in the development of antidiabetes and anti-melanin drugs for pharmaceutical and cosmetic industries.

The Correlation between 2-Acetyl-1-pyrroline Content, Biological Compounds and Molecular Characterization to the Aroma Intensities of Thai Local Rice.

Aroma intensities of rice are correlated with the mixture of aroma compounds it contains. 2-acetyl-1-pyrroline (2AP) has been reported as a major aroma compound and as a characteristic compound in fragrant rice. In this study, Thai local cultivars were classified into fragrant and non-fragrant rice based on the 2AP content and molecular characterization. Local rice cultivars were also examined for their proline content and volatile compounds profile, which are important factors in determining aroma. The results suggested that 43 Thai local rice cultivars were classified into 25 fragrant rice cultivars and 18 non-fragrant cultivars. The type of fragrant rice cultivars included 16 non-colored and 9 colored rice cultivars, while the type of non-fragrant rice cultivars included 14 non-colored and 4 colored rice cultivars. The proline content of local rice cultivars was determined and showed no correlation with the 2AP content; however, the proline level appears to be associated with the environmental stress in the rice cultivation area. One hundred and forty volatile compounds were identified from local rice cultivars. Among the detected compounds, 18 volatile compounds, including hexanal 1-pentanol octanal (E)-2-heptenal 6-methyl-5-hepten-2-one 1-hexanol nonanal 2-butoxy-ethanol (E)-2-octenal 1-tetradecene 1-octen-3-ol decanal benzaldehyde (E)-2-nonenal 1-nonanol benzyl alcohol isovanillin and vanillin contributed to the aroma intensities of both fragrant and non-fragrant rice. Aroma compounds were more abundant in fragrant than in non-fragrant rice. Moreover, the levels of aroma compounds recorded in non-colored cultivars were higher than those in colored rice cultivars. In contrast, the 2AP content of colored rice cultivars was higher than that in non-colored rice cultivars. Our findings may assist rice breeding programs in producing a new aromatic genotype rice with high potential aroma intensities.

Effect of Gamma Irradiation on 2-Acetyl-1-pyrroline Content, GABA Content and Volatile Compounds of Germinated Rice (Thai Upland Rice).

Aroma intensity in rice is related to the level of 2-acetyl-1-pyrroline (2AP). The accumulation of 2AP in rice has been synthesized via l-proline metabolism by inactive betaine aldehyde dehydrogenase enzyme (BADH2), which activates 2AP accumulation. Meanwhile, active BADH2 inhibits 2AP accumulation but activates γ-aminobutyric acid (GABA) accumulation. The improvement of 2AP content in rice has been reported under certain conditions, such as high salinity, water treatment, and reduction of high intensity solar exposure. In this study, we conducted the effects of gamma irradiation on 2AP content, GABA content and volatile compounds of germinated rice (Thai upland rice). Our results showed that the GABA content was highest when rice seeds germinated within a 24-h. The 2AP content of irradiated rice (germinated within a 24-h duration) was higher than non-irradiated rice for all gamma doses, particularly at 20 Gy, which showed a 23-fold higher level of 2AP than non-irradiated rice. On the other hand, the reduction of the GABA content of irradiated rice was caused by an increase in the gamma dose. At 300 Gy, irradiated rice had a GABA content approximately 2.6-fold lower than non-irradiated rice. Moreover, we observed that a reduction of volatile compounds occurred when increasing gamma dose. However, some volatile compounds appeared in the irradiated rice at gamma doses of 60 Gy, 80 Gy, 100 Gy and 300 Gy. Furthermore, we observed that the level of Octanal, which is the compound most related to aroma intensity, of irradiated rice was stronger than that of non-irradiated rice. Our results demonstrate for the first time that 2AP and GABA contents are sensitive to gamma irradiation conditions. Moreover, the results indicate that the gamma irradiation technique can be used to improve the aroma intensity of rice.

Bacterial ß-Glucosidase Reveals the Structural and Functional Basis of Genetic Defects in Human Glucocerebrosidase 2 (GBA2).

Human glucosylcerebrosidase 2 (GBA2) of the CAZy family GH116 is responsible for the breakdown of glycosphingolipids on the cytoplasmic face of the endoplasmic reticulum and Golgi apparatus. Genetic defects in GBA2 result in spastic paraplegia and cerebellar ataxia, while cross-talk between GBA2 and GBA1 glucosylceramidases may affect Gaucher disease. Here, we report the first three-dimensional structure for any GH116 enzyme, Thermoanaerobacterium xylanolyticum TxGH116 ß-glucosidase, alone and in complex with diverse ligands. These structures allow identification of the glucoside binding and active site residues, which are shown to be conserved with GBA2. Mutagenic analysis of TxGH116 and structural modeling of GBA2 provide a detailed structural and functional rationale for pathogenic missense mutations of GBA2.

Identification of rice Os4BGlu13 as a ß-glucosidase which hydrolyzes gibberellin A4 1-O-ß-d-glucosyl ester, in addition to tuberonic acid glucoside and salicylic acid derivative glucosides.

Gibberellin 1-O-ß-d-glucose ester hydrolysis activity has been detected in rice seedling extracts, but no enzyme responsible for this activity has ever been purified and identified. Therefore, gibberellin A4 glucosyl ester (GA4-GE) ß-d-glucosidase activity was purified from ten-day rice seedling stems and leaves. The family 1 glycoside hydrolase Os4BGlu13 was identified in the final purification fraction. The Os4BGlu13 cDNA was amplified from rice seedlings and expressed as an N-terminal thioredoxin-tagged fusion protein in Escherichia coli. The purified recombinant Os4BGlu13 protein (rOs4BGlu13) had an optimum pH of 4.5, for hydrolysis of p-nitrophenyl ß-d-glucopyranoside (pNPGlc), which was the best substrate identified, with a kcat/Km of 637 mM(-1) s(-1). rOs4BGlu13 hydrolyzed helicin best among natural glycosides tested (kcat/Km of 74.4 mM(-1) s(-1)). Os4BGlu13 was previously designated tuberonic acid glucoside (TAG) ß-glucosidase (TAGG), and here the kcat/Km of rOsBGlu13 for TAG was 6.68 mM(-1) s(-1), while that for GA4-GE was 3.63 mM(-1) s(-1) and for salicylic acid glucoside (SAG) is 0.88 mM(-1) s(-1). rOs4BGlu13 also hydrolyzed oligosaccharides, with preference for short ß-(1 â†’ 3)-linked over ß-(1 â†’ 4)-linked glucooligosaccharides. The enzymatic data suggests that Os4BGlu13 may contribute to TAG, SAG, oligosaccharide and GA4-GE hydrolysis in the rice plant, although helicin or a similar compound may be its primary target.

Expression and crystallization of a bacterial glycoside hydrolase family 116 ß-glucosidase from Thermoanaerobacterium xylanolyticum.

The Thermoanaerobacterium xylanolyticum gene product TxGH116, a glycoside hydrolase family 116 protein of 806 amino-acid residues sharing 37% amino-acid sequence identity over 783 residues with human glucosylceramidase 2 (GBA2), was expressed in Escherichia coli. Purification by heating, immobilized metal-affinity and size-exclusion chromatography produced >90% pure TxGH116 protein with an apparent molecular mass of 90 kDa on SDS-PAGE. The purified TxGH116 enzyme hydrolyzed the p-nitrophenyl (pNP) glycosides pNP-ß-D-glucoside, pNP-ß-D-galactoside and pNP-N-acetyl-ß-D-glucopyranoside, as well as cellobiose and cellotriose. The TxGH116 protein was crystallized using a precipitant consisting of 0.6 M sodium citrate tribasic, 0.1 M Tris-HCl pH 7.0 by vapour diffusion with micro-seeding to form crystals with maximum dimensions of 120×25×5 µm. The TxGH116 crystals diffracted X-rays to 3.15 Šresolution and belonged to the monoclinic space group P2(1). Structure solution will allow a structural explanation of the effects of human GBA2 mutations.

Enzymatic and structural characterization of hydrolysis of gibberellin A4 glucosyl ester by a rice ß-D-glucosidase.

In order to identify a rice gibberellin ester ß-D-glucosidase, gibberellin A4 ß-D-glucosyl ester (GA4-GE) was synthesized and used to screen rice ß-glucosidases. Os3BGlu6 was found to have the highest hydrolysis activity to GA4-GE among five recombinantly expressed rice glycoside hydrolase family GH1 enzymes from different phylogenic clusters. The kinetic parameters of Os3BGlu6 and its mutants E178Q, E178A, E394D, E394Q and M251N for hydrolysis of p-nitrophenyl ß-D-glucopyranoside (pNPGlc) and GA4-GE confirmed the roles of the catalytic acid/base and nucleophile for hydrolysis of both substrates and suggested M251 contributes to binding hydrophobic aglycones. The activities of the Os3BGlu6 E178Q and E178A acid/base mutants were rescued by azide, which they transglucosylate to produce ß-D-glucopyranosyl azide, in a pH-dependent manner, while acetate also rescued Os3BGlu6 E178A at low pH. High concentrations of sodium azide (200-400 mM) inhibited Os3BGlu6 E178Q but not Os3BGlu6 E178A. The structures of Os3BGlu6 E178Q crystallized with either GA4-GE or pNPGlc had a native α-D-glucosyl moiety covalently linked to the catalytic nucleophile, E394, which showed the hydrogen bonding to the 2-hydroxyl in the covalent intermediate. These data suggest that a GH1 ß-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside.

Identification of rice ß-glucosidase with high hydrolytic activity towards salicylic acid ß-D-glucoside.

ß-Glucosidases (EC 3.2.1.21) split ß-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release ß-D-glucose. One of the important functions of plant ß-glucosidase is deglucosylation of inactive glucosides of phytohormones to regulate levels of active hormones. Tuberonic acid is a jasmonate-related compound that shows tuber-inducing activity in the potato. We have identified two enzymes, OsTAGG1 and OsTAGG2, that have hydrolytic activity towards tuberonic acid ß-D-glucoside in rice (Oryza sativa L.). The expression of OsTAGG2 is upregulated by wounding and by methyl jasmonate, suggesting that this isozyme is involved in responses to biotic stresses and wounding, but the physiological substrate of OsTAGG2 remains ambiguous. In this study, we produced recombinant OsTAGG2 in Pichia pastoris (rOsTAGG2P), and investigated its substrate specificity in detail. From 1 L of culture medium, 2.1 mg of purified recombinant enzyme was obtained by ammonium sulfate precipitation and Ni-chelating column chromatography. The specific activity of rOsTAGG2P (182 U/mg) was close to that of the native enzyme (171 U/mg), unlike recombinant OsTAGG2 produced in Escherichia coli, which had approximately 3-fold lower specific activity than the native enzyme. The optimum pH and temperature for rOsTAGG2P were pH 3.4 and 60 °C. After pH and heat treatments, the enzyme retained its original activity in a pH range of 3.4-9.8 and below 55 °C. Native OsTAGG2 and rOsTAGG2P showed 4.5-4.7-fold higher activities towards salicylic acid ß-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid ß-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity.

Exchanging a single amino acid residue generates or weakens a +2 cellooligosaccharide binding subsite in rice ß-glucosidases.

Os3BGlu6, Os3BGlu7, and Os4BGlu12 are rice glycoside hydrolase family 1 ß-glucosidases, the structures of which have been solved by X-ray crystallography. In complex structures, Os3BGlu7 residue Asn245 hydrogen bonds to the second sugar in the +1 subsite for laminaribiose and the third sugar in the +2 subsite for cellotetraose and cellopentaose. The corresponding Os3BGlu6 residue, Met251, appears to block the binding of cellooligosaccharides at the +2 subsite, whereas His252 in this position in Os4BGlu12 could hydrogen bond to oligosaccharides. Mutation of Os3BGlu6 Met251 to Asn resulted in a 15-fold increased k(cat)/K(m) value for hydrolysis of laminaribiose compared to wild type Os3BGlu6 and 9 to 24-fold increases for cellooligosaccharides with degrees of polymerization (DP) of 2-5. On the other hand, mutation of Os3BGlu7 Asn245 to Met decreased the k(cat)/K(m) of hydrolysis by 6.5-fold for laminaribiose and 17 to 30-fold for cellooligosaccharides with DP >2, while mutation of Os4BGlu12 His252 to Met decreased the corresponding k(cat)/K(m) values 2 to 6-fold.

The crystal structure of rice (Oryza sativa L.) Os4BGlu12, an oligosaccharide and tuberonic acid glucoside-hydrolyzing ß-glucosidase with significant thioglucohydrolase activity.

Rice Os4BGlu12, a glycoside hydrolase family 1 (GH1) ß-glucosidase, hydrolyzes ß-(1,4)-linked oligosaccharides of 3-6 glucosyl residues and the ß-(1,3)-linked disaccharide laminaribiose, as well as certain glycosides. The crystal structures of apo Os4BGlu12, and its complexes with 2,4-dinitrophenyl-2-deoxyl-2-fluoroglucoside (DNP2FG) and 2-deoxy-2-fluoroglucose (G2F) were solved at 2.50, 2.45 and 2.40Å resolution, respectively. The overall structure of rice Os4BGlu12 is typical of GH1 enzymes, but it contains an extra disulfide bridge in the loop B region. The glucose ring of the G2F in the covalent intermediate was found in a (4)C(1) chair conformation, while that of the noncovalently bound DNP2FG had a (1)S(3) skew boat, consistent with hydrolysis via a (4)H(3) half-chair transition state. The position of the catalytic nucleophile (Glu393) in the G2F structure was more similar to that of the Sinapsis alba myrosinase G2F complex than to that in covalent intermediates of other O-glucosidases, such as rice Os3BGlu6 and Os3BGlu7 ß-glucosidases. This correlated with a significant thioglucosidase activity for Os4BGlu12, although with 200- to 1200-fold lower k(cat)/K(m) values for S-glucosides than the comparable O-glucosides, while hydrolysis of S-glucosides was undetectable for Os3BGlu6 and Os3BGlu7.