Yanling Hua

Rice Os9BGlu31 Is a Transglucosidase with the Capacity to Equilibrate Phenylpropanoid, Flavonoid, and Phytohormone Glycoconjugates

Background: Glycosylation regulates the activities of plant metabolites and is mediated by glycosyltransferases (GT), glycoside hydrolases (GH), and transglycosidases (TG). Results: The vacuolar TG Os9BGlu31 transfers glucose between phenolic acid esters and other compounds. Conclusion: Os9BGlu31 equilibrates phenolic acids, phytohormones, and their glucosyl conjugates. Significance: Os9BGlu31 and similar TG can broaden glycoconjugate diversity in planta. Glycosylation is an important mechanism of controlling the reactivities and bioactivities of plant secondary metabolites and phytohormones. Rice (Oryza sativa) Os9BGlu31 is a glycoside hydrolase family GH1 transglycosidase that acts to transfer glucose between phenolic acids, phytohormones, and flavonoids. The highest activity was observed with the donors feruloyl-glucose, 4-coumaroyl-glucose, and sinapoyl-glucose, which are known to serve as donors in acyl and glucosyl transfer reactions in the vacuole, where Os9BGlu31 is localized. The free acids of these compounds also served as the best acceptors, suggesting that Os9BGlu31 may equilibrate the levels of phenolic acids and carboxylated phytohormones and their glucoconjugates. The Os9BGlu31 gene is most highly expressed in senescing flag leaf and developing seed and is induced in rice seedlings in response to drought stress and treatment with phytohormones, including abscisic acid, ethephon, methyljasmonate, 2,4-dichlorophenoxyacetic acid, and kinetin. Although site-directed mutagenesis of Os9BGlu31 indicated a function for the putative catalytic acid/base (Glu169), catalytic nucleophile residues (Glu387), and His386, the wild type enzyme displays an unusual lack of inhibition by mechanism-based inhibitors of GH1 β-glucosidases that utilize a double displacement retaining mechanism.

Expression and enzymatic properties of rice (Oryza sativa L.) monolignol β-glucosidases

Monolignol glucosides and their β-glucosidases are found in monocots, but their biological roles are unclear. Phylogenetic analysis of rice (Oryza sativa L.) glycoside hydrolase family GH1 β-glucosidases indicated that Os4BGlu14, Os4BGlu16, and Os4BGlu18 are closely related to known monolignol β-glucosidases. An optimized Os4BGlu16 cDNA and cloned Os4BGlu18 cDNA were used to express fusion proteins with His6 tags in Pichia pastoris and Escherichia coli, respectively. The secreted Os4BGlu16 fusion protein was purified from media by immobilized metal affinity chromatography (IMAC), while Os4BGlu18 was extracted from E. coli cells and purified by anion exchange chromatography, hydrophobic interaction chromatography and IMAC. Os4BGlu16 and Os4BGlu18 hydrolyzed the monolignol glucosides coniferin (kcat/KM, 21.6mM(-1)s(-1) for Os4BGlu16 and for Os4BGlu18) and syringin (kcat/KM, 22.8mM(-1)s(-1) for Os4BGlu16 and 24.0mM(-1)s(-1) for Os4BGlu18) with much higher catalytic efficiencies than other substrates. In quantitative RT-PCR, highest Os4BGlu14 mRNA levels were detected in endosperm, embryo, lemma, panicle and pollen. Os4BGlu16 was detected highest in leaf from 4 to 10 weeks, endosperm and lemma, while Os4BGlu18 mRNA was most abundant in vegetative stage from 1 week to 4 weeks, pollen and lemma. These data suggest a role for Os4BGlu16 and Os4BGlu18 monolignol β-glucosidases in both vegetative and reproductive rice tissues.

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.

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

In order to identify a rice gibberellin ester β-D-glucosidase, gibberellin A4 β-D-glucosyl ester (GA4-GE) was synthesized and used to screen rice β-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 β-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 β-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 β-glucosidase uses the same retaining catalytic mechanism to hydrolyze 1-O-acyl glucose ester and glucoside.

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.

Structure of a plant β-galactosidase C-terminal domain.

Most plant β-galactosidases, which belong to glycoside hydrolase family 35, have a C-terminal domain homologous to animal galactose and rhamnose-binding lectins. To investigate the structure and function of this domain, the C-terminal domain of the rice (Oryza sativa L.) β-galactosidase 1 (OsBGal1 Cter) was expressed in Escherichia coli and purified to homogeneity. The free OsBGal1 Cter is monomeric with a native molecular weight of 15kDa. NMR spectroscopy indicated that OsBGal1 Cter comprises five β-strands and one α-helix. The structure of this domain is similar to lectin domains from animals, but loops A and C of OsBGal1 Cter are longer than the corresponding loops from related animal lectins with known structures. In addition, loop A of OsBGal1 Cter was not well defined, suggesting it is flexible. Although OsBGal1 Cter was predicted to be a galactose/rhamnose-binding domain, binding with rhamnose, galactose, glucose, β-1,4-d-galactobiose and raffinose could not be observed in NMR experiments.

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.