Background:The origin of specificity of plant ␣-glucosidases for long malto-oligosaccharides remains uncertain. Results: The crystal structure and mutational analyses of sugar beet ␣-glucosidase revealed its substrate binding properties. Conclusion:The long-substrate specificity was described as two structural elements, the N-loop and subdomain b2. Significance: A slight structural difference leads to significant differences in specificity for varying chain lengths of substrate.

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.

Endo-β-1,4-mannanases from Streptomyces thermolilacinus (StMan) and Thermobifida fusca (TfMan) demonstrated different substrate specificities. StMan hydrolyzed galactosylmannooligosaccharide (GGM5; 6(III) ,6(IV) -α-d-galactosyl mannopentaose) to GGM3 and M2, whereas TfMan hydrolyzed GGM5 to GGM4 and M1. To determine the region involved in the substrate specificity, we constructed chimeric enzymes of StMan and TfMan and evaluated their substrate specificities. Moreover, the crystal structure of the catalytic domain of StMan (StMandC) and the complex structure of the inactive mutant StE273AdC with M6 were solved at resolutions of 1.60 and 1.50 Å, respectively. Structural comparisons of StMandC and the catalytic domain of TfMan lead to the identification of a subsite around -1 in StMandC that could accommodate a galactose branch. These findings demonstrate that the two loops (loop7 and loop8) are responsible for substrate recognition in GH5 actinomycete mannanases. In particular, Trp281 in loop7 of StMan, which is located in a narrow and deep cleft, plays an important role in its affinity toward linear substrates. Asp310 in loop8 of StMan specifically bound to the galactosyl unit in the -1 subsite.

α-Glucosidase from Aspergillus niger (AgdA; typical α-1,4-glucosidase) is known to industrially produce α-(1→ 6)-glucooligosaccharides. This fungus also has another α-glucosidase-like protein, AgdB. To learn its function, wild-type AgdB was expressed in Pichia pastoris. However, the enzyme displayed two electrophoretic forms due to heterogeneity of Nglycosylation at Asn354. The deglycosylation mutant N354D shared the same properties with wild-type AgdB. N354D demonstrated hydrolytic specificity toward α-(1→3)and α-(1→4)-glucosidic linkages, indicating that AgdB is an α-1,3-/α-1,4glucosidase. N354D-catalyzed transglucosylation from maltose was analyzed in short-and long-term reactions, enabling us to learn the transglucosylation specificity and product accumulation, respectively. A short-term reaction (<15 min) synthesized 3 II -O-α-glucosyl-maltose and maltotriose, indicating α-1,3-/α-1,4-transferring specificity. A long-term reaction (<24 h) accumulated kojibiose and nigerose using formed glucose as an acceptor substrate. AgdA and AgdB are distinct α-glucosidases. At a high concentration of glucose added exogenously, AgdB largely generated the rare sugars kojibiose and nigerose (exhibiting beneficial physiological functions) with 19% and 24% yields from maltose, respectively.

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