SUMMARYBecause structural modifications of flavonoids are closely related to their properties, such as stability, solubility, flavor and coloration, characterizing the enzymes that catalyze the modification reactions can be useful for engineering agriculturally beneficial traits of flavonoids. In this work, we examined the enzymes involved in the modification pathway of highly glycosylated and acylated anthocyanins that accumulate in Lobelia erinus. Cultivar Aqua Blue (AB) of L. erinus is blue-flowered and accumulates delphinidin 3-O-p-coumaroylrutinoside-5-O-malonylglucoside-3 0 5 0 -O-dihydroxycinnamoylglucoside (lobelinins) in its petals. Cultivar Aqua Lavender (AL) is mauve-flowered, and LC-MS analyses showed that AL accumulated delphinidin 3-O-glucoside (Dp3G), which was not further modified toward lobelinins. A crude protein assay showed that modification processes of lobelinin were carried out in a specific order, and there was no difference between AB and AL in modification reactions after rhamnosylation of Dp3G, indicating that the lack of highly modified anthocyanins in AL resulted from a single mutation of rhamnosyltransferase catalyzing the rhamnosylation of Dp3G. We cloned rhamnosyltransferase genes (RTs) from AB and confirmed their UDP-rhamnose-dependent rhamnosyltransferase activities on Dp3G using recombinant proteins. In contrast, the RT gene in AL had a 5-bp nucleotide deletion, resulting in a truncated polypeptide without the plant secondary product glycosyltransferase box. In a complementation test, AL that was transformed with the RT gene from AB produced blue flowers. These results suggest that rhamnosylation is an essential process for lobelinin synthesis, and thus the expression of RT has a great impact on the flower color and is necessary for the blue color of Lobelia flowers.

Abbreviations: α-GalF, α-galactopyranosyl fluoride; Gal-Lac, β-lactosyl α-D-galactopyranoside; 20 GH, glycoside hydrolase family; HMBC, heteronuclear multiple bond correlation; 21 high-performance anion exchange chromatography-pulsed amperometric detection. The preparation of a glycosynthase, a catalytic nucleophile mutant of a glycosidase, is a well-5 established strategy for the effective synthesis of glycosidic linkages. However, glycosynthases 6 derived from α-glycosidases can give poor yields of desired products because they require 7 generally unstable β-glycosyl fluoride donors. Here, we investigate a transglycosylation catalyzed 8 by a catalytic nucleophile mutant derived from a glycoside hydrolase family (GH) 97 α-9 galactosidase, using more stable β-galactosyl azide and α-galactosyl fluoride donors. The mutant 10 enzyme catalyzes the glycosynthase reaction using β-galactosyl azide and α-galactosyl transfer 11 from α-galactosyl fluoride with assistance of external anions. Formate was more effective at 12 restoring transfer activity than azide. Kinetic analysis suggests that poor transglycosylation in the 13 presence of the azide is because of low activity of the ternary complex between enzyme, β-14 galactosyl azide, and acceptor. A three-dimensional structure of the mutant enzyme in complex 15 with the transglycosylation product, β-lactosyl α-D-galactoside, was solved to elucidate the 16 ligand-binding aspects of the α-galactosidase. Subtle differences at the β→α loops 1, 2, and 3 of 17 the catalytic TIM barrel of the α-galactosidase from those of a homologous GH97 α-glucoside 18 hydrolase seem to be involved in substrate recognitions. In particular, the Trp residues in β→α 19 loop 1 have separate roles. Trp312 of the α-galactosidase appears to exclude the equatorial 20 hydroxy group at C4 of glucosides, whereas the corresponding Trp residue in the α-glucoside 21 hydrolase makes a hydrogen bond with this hydroxy group. The mechanism of α-galactoside-22 recognition is conserved among GH27, 31, 36, and 97 α-galactosidases. 23 24

We investigated the antidepressant-like effect of lactoferrin (Lf) in a repeated forced-swim test (FST) stress mouse model. FST was performed on days 1, 2, 7 and 14. Bovine Lf (bLf) or bovine serum albumin (BSA) was supplemented at 1% to the commercial diet after the first FST throughout the experimental period. The FST-control and FST+BSA group showed a marked increase in immobility time on day 2, which remained increased up to the 14th day, while the FST+bLf group showed a significant lower immobility time. Brain-derived neurotrophic factor (BDNF) content in the hippocampus significantly decreased in all of FST treated groups. These results suggest that bLf may improve the depressive-like symptoms induced by repeated FST.

Glycoside hydrolase family 97 (GH97) is one of the most interesting glycosidase families, which contains inverting and retaining glycosidases. Currently, only two enzyme types, α-glucoside hydrolase and α-galactosidase, are registered in the carbohydrate active enzyme database as GH97 function-known proteins. To explore new specificities, BT3661 and BT3664, which have distinct amino acid sequences when compared with that of GH97 α-glucoside hydrolase and α-galactosidase, were characterized in this study. BT3664 was identified to be an α-galactosidase, whereas BT3661 exhibits hydrolytic activity toward both β-l-arabinopyranoside and α-d-galactopyranoside, and thus we designate BT3661 as a β-l-arabinopyranosidase/α-d-galactosidase. Since this is the first dual substrate specificity enzyme in GH97, we investigated the substrate recognition mechanism of BT3661 by determining its three-dimensional structure and based on this structural data generated a number of mutants to probe the enzymatic mechanism. Structural comparison shows that the active-site pocket of BT3661 is similar to GH97 α-galactosidase BT1871, but the environment around the hydroxymethyl group of the galactopyranoside is different. While BT1871 bears Glu361 to stabilize the hydroxy group of C6 through a hydrogen bond with its carboxy group, BT3661 has Asn338 at the equivalent position. Amino acid mutation analysis indicates that the length of the side chain at Asn338 is important for defining specificity of BT3661. The k/K value for the hydrolysis of p-nitrophenyl α-galactoside decreases when Asn338 is substituted with Glu, whereas an increase is observed when the mutation is Ala. Interestingly, mutation of Asn338 to Ala reduces the k/K value for hydrolysis of p-nitrophenyl β-l-arabinopyranoside.

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