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SUMMARY: Purple carrot accumulates anthocyanins modified with galactose, xylose, glucose, and sinapic acid. Most of the genes associated with anthocyanin biosynthesis have been identified, except for the glucosyltransferase genes involved in the step before the acylation in purple carrot. Anthocyanins are commonly glycosylated in reactions catalyzed by UDP‐sugar‐dependent glycosyltransferases (UGTs). Although many studies have been conducted on UGTs, the glucosylation of carrot anthocyanins remains unknown. Acyl‐glucose‐dependent glucosyltransferase activity modifying cyanidin 3‐xylosylgalactoside was detected in the crude protein extract prepared from purple carrot cultured cells. In addition, the corresponding enzyme was purified. The cDNA encoding this glucosyltransferase was isolated based on the partial amino acid sequence of the purified protein. The recombinant protein produced in Nicotiana benthamiana leaves via agroinfiltration exhibited anthocyanin glucosyltransferase activity. This glucosyltransferase belongs to the glycoside hydrolase family 3 (GH3). The expression pattern of the gene encoding this GH3‐type anthocyanin glucosyltransferase was consistent with anthocyanin accumulation in carrot tissues and cultured cells. Significance Statement: Glucosylation of the purple carrot anthocyanin cyanidin 3‐O‐(2″‐O‐xylosyl)‐(6″‐O‐sinapoylglucosyl)‐galactoside is mediated by a glycoside hydrolase family 3 vacuolar enzyme that catalyzes a reaction that uses acyl‐glucose as the glucosyl donor. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae isoprimeverose‐producing oligoxyloglucan hydrolase (IpeA) releases isoprimeverose units (α‐d‐xylopyranosyl‐(1→6)‐d‐glucose) from the non‐reducing end of xyloglucan oligosaccharides and belongs to glycoside hydrolase family 3. In this paper, we report the X‐ray crystal structure of the IpeA complexed with a xyloglucan oligosaccharide, (XXXG: Glc4Xyl3). Trp515 of IpeA plays a critical role in XXXG recognition at positive subsites. In addition, docking simulation of IpeA‐XXXG suggested that two Tyr residues (Tyr268 and Tyr445) are involved in the catalytic reaction mechanism of IpeA. Tyr268 plays an important role in product turnover, whereas Tyr445 stabilizes the acid/base Glu524 residue, which serves as a proton donor. Our findings indicate that the substrate recognition machinery of IpeA is specifically adapted to xyloglucan oligosaccharides. [ABSTRACT FROM AUTHOR]

The glycoside hydrolase family 3 (GH3) β‐glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β‐Glucosidases hydrolyse the β‐1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N‐glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β‐glucosidase from Rasamsonia emersonii. NcCel3A is a three‐domain protein with a number of extended loops that deepen the active‐site cleft of the enzyme. These structures characterize this subfamily of GH3 β‐glucosidases and account for the high cellobiose specificity of this subfamily. The glycoside hydrolase family 3 β‐glucosidase Cel3A from Neurospora crassa (NcCel3A) is highly specific for cellobiose. The crystal structure of NcCel3A has been solved to 2.25 Å resolution. The structure is a dimer and exhibits a high degree of N‐glycosylation. [ABSTRACT FROM AUTHOR]

β-Glucosidases belong to families 1, 3 and 9 of the glycoside hydrolases and act on cello-oligosaccharides. Family 1 and 3 enzymes are retaining and are reported to have transglycosylation activity, which can be used to produce oligosaccharides and glycoconjugates. Family 3 enzymes are less well characterized than their family 1 homologues and to date only two crystal structures have been solved. Here, the expression, purification, crystallization and X-ray diffraction data of a family 3 β-glucosidase from the hyperthermophilic bacterium Thermotoga neapolitana are reported. Crystals of selenomethionine-substituted protein have also been grown. The crystals belong to space group C2221, with unit-cell parameters a = 74.9, b = 127.0, c = 175.2 Å. Native data have been collected to 2.4 Å resolution and the structure has been solved to 2.7 Å using the selenomethionine MAD method. Model building and refinement of the structure are under way. [ABSTRACT FROM AUTHOR]

The intracellular β-glucosidase from Kluyveromyces marxianus NBRC1777 ( KmBglI) belongs to glycoside hydrolase family 3 and has a unique domain architecture. Selenomethionine-labelled KmBglI was purified and crystallized by the hanging-drop vapour-diffusion method using the purified enzyme at 30 mg ml−1, 0.04 M potassium dihydrogen phosphate pH 5.1, 16%( w/ v) PEG 8000 and 20%( v/ v) glycerol. The crystal belonged to space group C2, with unit-cell parameters a = 245.8, b = 148.7, c = 119.9 Å, β = 112.9°. Multiple-wavelength anomalous dispersion data were collected at 2.4 and 2.5 Å resolution. A tetramer was assumed to be present in the asymmetric unit, which gave a Matthews coefficient of 2.6 Å3 Da−1. [ABSTRACT FROM AUTHOR]