Wataru Saburi

α-Amylase, ubiquitously distributed across diverse organisms, catalyzes the hydrolysis of the internal α-(1→4)-linkage of α-(1→4)-glucan including starch as an essential energy source. Insect α-amylases, which belong to the glycoside hydrolase family 13 subfamily 15 (GH13_15), are important for optimal larval growth and adult longevity. Honeybee (Apis mellifera) α-amylase (AMA) is expressed in the hypopharyngeal glands of forager bees and secreted into honey. AMA in honey, which is important for food quality control, has been partly characterized. However, its structure–function relationship is poorly understood. Herein, we present biochemical, structural, and mutational analyses of AMA. Kinetic analysis using p-nitrophenyl maltooligosaccharides and their 4,6-benzylidene-modified derivatives revealed a subsite affinity map of AMA. AMA contains high-affinity subsites −3, −2, +1, and +2, similar to those of the mammalian α-amylases of GH13_24. Most AMA substrate-binding residues are conserved in the GH13_24 enzymes. Mutational analysis revealed that Leu175 is crucial at subsites −3/−2 for reactions with oligo- and polysaccharides. Furthermore, Trp77 at subsites −3/−2 and Lys210 at subsite +2 are suggested to be involved in the proper binding of long-chain substrates. AMA shares a surface sugar-binding site with mammalian α-amylases, where Asn281, Trp285, and Trp293 are essential for the binding and degradation of starch granules.

Isomaltosaccharides, composed of α‐(1 → 6)‐ d ‐glucosyl residues, exhibit diverse beneficial properties depending on the degree of polymerization (DP) and attract great interest across multiple industries. The anomer‐retaining transglucosidase, dextran dextrinase (DDase)—which synthesizes the α‐(1 → 6)‐ d ‐glucose polymer dextran from α‐(1 → 4)‐glucan maltooligosaccharides (MOSs)—shows structural similarity to anomer‐inverting α‐glucohydrolases belonging to glycoside hydrolase family 15 (GH15). Here, we show a new GH15 transglucosidase, α‐glucan 4(6)‐α‐glucosyltransferase, from Tepidibacillus decaturensis (Td46GT) and its mechanism for converting MOS into isomaltooligosaccharide (IMO). Td46GT catalyzes DDase‐like d ‐glucosyl‐transfer reactions: α‐(1 → 4)‐transglucosylation to MOS and α‐(1 → 6)‐transglucosylation to short‐chain MOS of DP 2–3 and IMO. Unlike DDase, it does not produce dextran. Kinetic analyses of the two‐substrate reactions revealed that the acceptors determined formed linkages. Relative acceptor‐substrate specificity constants (RASCs) indicated that maltotriose and maltose were the best acceptors for α‐(1 → 4)‐ and α‐(1 → 6)‐transglucosylations, respectively. The subsite affinities calculated from the RASCs were consistent with those obtained from k _cat / K _m values for single‐substrate reactions. Time‐dependent changes in the MOS and IMO concentrations during the reaction were quantitatively simulated using RASCs and k _cat / K _m values. Structure prediction suggested Td46GT possesses a substrate‐binding site similar to DDase, and site‐directed mutagenesis identified Phe418 and Tyr612 as critical residues at subsite +2 for MOS and IMO binding. Our results suggested that Td46GT possesses distinct MOS‐ and IMO‐binding subsites in a single active pocket, which are shared by both acceptor and donor substrates, and that the binding manner of the acceptor determines the product specificity of transglucosylation.

Starch degradation in malted barley produces yeast-fermentable sugars. In this study, we compared the amylolytic enzymes and composition of the malt starch hydrolysates of two barley cultivars, Hokudai 1 (the first cultivar established in Japan) and Kitanohoshi (the currently used cultivar for beer production). Hokudai 1 malt contained lower activity of amylolytic enzymes than Kitanohoshi malt, although these cultivars contained α-amylase AMY2 and β-amylase Bmy1 as the predominant enzymes. Malt starch hydrolysate of Hokudai 1 contained more limit dextrin and less yeast-fermentable sugars than that of Kitanohoshi. In mixed malt saccharification, a high Hokudai 1 malt ratio increased the limit dextrin levels and decreased the maltotriose and maltose levels. Even though Kitanohoshi malt contained more amylolytic enzymes than Hokudai 1 malt, addition of Kitanohoshi extract containing the amylolytic enzymes did not enhance malt starch degradation of Hokudai 1. Hokudai 1 malt starch was less degradable than Kitanohoshi malt starch.

β1-3/1-6 Glucans, known for their diverse structures, comprise a β1-3-linked main chain and β1-6-linked short branches. Laminarin, a β1-3/1-6 glucan extracted from brown seaweed, for instance, includes β1-6 linkages even in the main chain. The diverse structures provide various beneficial functions of the glucan. To investigate the relationship between structure and functionality, and to enable the characterization of β1-3/1-6 glucan-metabolizing enzymes, oligosaccharides containing exact structures of β1-3/1-6 glucans are required. We synthesized the monomeric units for the synthesis of β1-3/1-6 mixed-linked glucooligosaccharides. 2-(Trimethylsilyl)ethyl 2-O-benzoyl-4,6-O-benzylidene-β-d-glucopyranoside served as an acceptor in the formation of β1-3 linkages. Phenyl 2-O-benzoyl-4,6-O-benzylidene-3-O-(tert-butyldiphenylsilyl)-1-thio-β-d-glucopyranoside and phenyl 2,3-di-O-benzoyl-4,6-di-O-levulinyl-1-thio-β-d-glucopyranoside acted as donors, synthesizing acceptors suitable for the formation of β1-3- and β1-6-linkages, respectively. These were used to synthesize a derivative of Glcβ1-6Glcβ1-3Glcβ1-3Glc, demonstrating that the proposed route can be applied to synthesize the main chain of β-glucan, with the inclusion of both β1-3 and β1-6 linkages.

Mannose 2-epimerase (ME), a member of the acylglucosamine 2-epimerase (AGE) superfamily that catalyzes epimerization of D-mannose and D-glucose, has recently been characterized to have potential for D-mannose production. However, the substrate-recognition and catalytic mechanism of ME remains unknown. In this study, structures of Runella slithyformis ME (RsME) and its D254A mutant [RsME(D254A)] were determined in their apo forms and as intermediate-analog complexes [RsME–D-glucitol and RsME(D254A)–D-glucitol]. RsME possesses the (α/α)₆-barrel of the AGE superfamily members but has a unique pocket-covering long loop (loop_α7–α8). The RsME–D-glucitol structure showed that loop_α7–α8 moves towards D-glucitol and closes the active pocket. Trp251 and Asp254 in loop_α7–α8 are only conserved in MEs and interact with D-glucitol. Kinetic analyses of the mutants confirmed the importance of these residues for RsME activity. Moreover, the structures of RsME(D254A) and RsME(D254A)–D-glucitol revealed that Asp254 is vital for binding the ligand in a correct conformation and for active-pocket closure. Docking calculations and structural comparison with other 2-epimerases show that the longer loop_α7–α8 in RsME causes steric hindrance upon binding to disaccharides. A detailed substrate-recognition and catalytic mechanism for monosaccharide-specific epimerization in RsME has been proposed.

β-Galactosidase (EC 3.2.1.23) hydrolyzes β-D-galactosidic linkages at the nonreducing end of substrates to produce β-D-galactose. Lacticaseibacillus casei is one of the most widely utilized probiotic species of lactobacilli. It possesses a putative β-galactosidase belonging to glycoside hydrolase family 35 (GH35). This enzyme is encoded by the gene included in the gene cluster for utilization of lacto-N-biose I (LNB; Galβ1-3GlcNAc) and galacto-N-biose (GNB; Galβ1-3GalNAc) via the phosphoenolpyruvate: sugar phosphotransferase system. The GH35 protein (GnbG) from L. casei BL23 is predicted to be 6-phospho-β-galactosidase (EC 3.2.1.85). However, its 6-phospho-β-galactosidase activity has not yet been examined, whereas its hydrolytic activity against LNB and GNB has been demonstrated. In this study, L. casei JCM1134 LBCZ_0230, homologous to GnbG, was characterized enzymatically and structurally. A recombinant LBCZ_0230, produced in Escherichia coli, exhibited high hydrolytic activity toward o-nitrophenyl β-D-galactopyranoside, p-nitrophenyl β-D-galactopyranoside, LNB, and GNB, but not toward o-nitrophenyl 6-phospho-β-D-galactopyranoside. Crystal structure analysis indicates that the structure of subsite −1 of LBCZ_0230 is very similar to that of Streptococcus pneumoniae β-galactosidase BgaC and not suitable for binding to 6-phospho-β-D-galactopyranoside. These biochemical and structural analyses indicate that LBCZ_0230 is a β-galactosidase. According to the prediction of LNB’s binding mode, aromatic residues, Trp190, Trp240, Trp243, Phe244, and Tyr458, form hydrophobic interactions with N-acetyl-D-glucosamine residues of LNB at subsite +1.