Bacterial β-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 β-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

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

Identification of Rice β-Glucosidase with High Hydrolytic Activity towards Salicylic Acid β-D-Glucoside

β-Glucosidases (EC 3.2.1.21) split β-glucosidic linkages at the non-reducing end of glucosides and oligosaccharides to release β-D-glucose. One of the important functions of plant β-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 β-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 β-D-glucoside, an inactive storage-form of salicylic acid, than towards tuberonic acid β-D-glucoside (TAG), although OsTAGG2 was originally isolated from rice based on TAG-hydrolytic activity

Expression, purification, crystallization and preliminary X-ray analysis of rice (Oryza sativa L.) Os4BGlu12 β-glucosidase

Recombinant rice Os4BGlu12 β-glucosidase purified from E. coli was crystallized with and without 2,4-dinitrophenyl-2-deoxy-2-fluoro-β-d-glucopyranoside