Projects

Our primary research focus is on enzymes involved in carbohydrate metabolism. Carbohydrates comprise an almost limitless variety of molecular species due to differences in constituent monosaccharides, linkage types, degrees of polymerization, and chemical modifications. These structurally diverse molecules perform distinct and non-substitutable roles, including energy storage, structural roles, and biological signaling. Living organisms utilize enzymes (proteins) to accurately distinguish among these diverse carbohydrate structures and to employ them in a highly regulated manner. Understanding the structure and function of carbohydrate-metabolizing enzymes is therefore important not only from a fundamental perspective—aimed at elucidating carbohydrate-related biological phenomena—but also for practical applications such as drug development and the production of functional carbohydrates.

To understand enzyme function, we place particular emphasis on quantitative evaluation. Qualitative assessments—such as whether a reaction proceeds or not—are insufficient for comparing enzyme functions. Because enzymes act as catalysts that accelerate reaction rates by lowering activation energy, their function can be quantitatively described by measuring reaction rates (e.g., the amount of product formed per unit time). By analyzing the relationship between reaction rates and various conditions—such as substrate type and concentration, pH, and temperature—we can gain detailed insights into enzyme function. Based on such kinetic analyses, we aim to elucidate the relationship between enzyme structure and function by combining protein engineering approaches (e.g., analysis of mutant enzymes) with structural biology techniques, including X-ray crystallography and single-particle cryo-electron microscopy.

Below, we introduce our ongoing research projects along with the experimental approaches employed.

L-glucoside-metabolizing enzymes

Glucose, the constituent monosaccharide of starch, glycogen, and cellulose, has long been believed to exist exclusively in the D-form in nature. We questioned this “homochirality hypothesis of glucose” and set out to identify enzymes capable of hydrolyzing glycosides of its enantiomer, the L-form. By mining genome databases for genes encoding proteins homologous to enzymes that hydrolyze α-L-fucosides—naturally abundant L-sugars—we identified candidate genes and produced the corresponding recombinant enzymes for functional analysis. As a result, we discovered a novel enzyme, α-L-glucosidase, encoded in the genome of the bacterium Cecembia lonarensis (ACS Omega, 2022). Through X-ray crystallographic analysis, we elucidated the molecular mechanism by which this enzyme recognizes L-glucose. Furthermore, by exploiting its transglycosylation activity, we achieved the enzymatic synthesis of α-L-glucooligosaccharides. We also demonstrated that this enzyme hydrolyzes 6-deoxy-α-L-glucosides (α-L-quinovosides) (J. Appl. Glycosci., 2024). In the course of these studies, we also carried out the organic synthesis of candidate substrates, including α-L-glucosides and α-L-quinovosides.

The discovery of α-L-glucosidase suggests that α-L-glucosides may exist in nature. We are currently pursuing collaborative studies to identify the biosynthetic enzymes of α-L-glucosides, as well as enzymes involved in the transport and catabolism of α-L-glucose. In addition, we aim to elucidate the functional properties of α-L-gluco-oligosaccharides and to develop simple methods for the detection and quantification of α-L-glucose (and its glycosides).

Starch-metabolizing enzymes

Starch is a carbohydrate produced exclusively by plants through photosynthesis; however, it serves as an energy source for nearly all living organisms. Over the course of evolution, organisms have developed diverse pathways for starch metabolism, involving a wide variety of enzymes and transport proteins. While the hydrolytic pathway—where starch is degraded into glucose by enzymes such as α-amylases and α-glucosidases—is widely distributed, some microorganisms have acquired unique metabolic routes that convert starch into structurally distinct oligosaccharides using transglycosylation enzymes (transglycosidases) (Biosci. Biotechnol. Biochem., 2024).

In the human small intestine, α-glucosidase is known as maltase–glucoamylase (MGAM), and its inhibition can suppress the rapid postprandial increase in blood glucose levels (glycemic spikes). We demonstrated that MGAM can be readily prepared in large quantities from porcine blood and determined its three-dimensional structure by cryo-electron microscopy single-particle analysis (J. Enzym. Inhib. Med. Chem., 2026). Furthermore, through kinetic analysis of inhibition, we revealed that because MGAM possesses two active sites, competitive inhibitors can exhibit apparent behavior resembling mixed inhibition. These findings clearly indicate that careful interpretation is required when determining the mode of inhibition in MGAM inhibitor studies. We are currently conducting research to identify inhibitors of MGAM that could serve as lead compounds for therapeutics or functional foods aimed at the treatment and prevention of type 2 diabetes.

In addition, we are working on the rational engineering of transglycosylation enzymes to enable the efficient production of functional oligosaccharides from starch and maltooligosaccharides.

Elucidation of Structural Factors Determining Catalytic Mechanisms

Glycoside hydrolases are broadly classified into two types based on the stereochemical outcome at the anomeric center: inverting enzymes, which invert the anomeric configuration between substrate and product, and retaining enzymes, which preserve it. Their catalytic mechanisms are generally explained by a single-displacement mechanism and a double-displacement mechanism, respectively. In addition to hydrolysis, retaining enzymes can catalyze transglycosylation reactions and are therefore widely utilized for oligosaccharide synthesis.

Although the amino acid sequences of these two enzyme types are typically quite distinct, our group has demonstrated the existence of enzyme families—such as GH97 and GH15—that exhibit different catalytic mechanisms despite having similar amino acid sequences (Biosci. Biotechnol. Biochem., 2011; J. Biol. Chem., 2025). Even among retaining enzymes, the relative catalytic efficiencies of hydrolysis and transglycosylation vary depending on the enzyme. Moreover, some inverting enzymes are known to produce and accumulate oligosaccharides from monosaccharides via the reverse reaction of hydrolysis (i.e., condensation). We investigate these enzyme systems from multiple perspectives, ranging from fundamental studies on catalytic mechanisms of retaining and inverting enzymes in the context of molecular evolution, to applied research aimed at efficient monosaccharide production and oligosaccharide synthesis.

Others

In addition to glycoside hydrolases, we also study a variety of carbohydrate-related proteins, including solute-binding proteins (SBPs) that specifically recognize and bind carbohydrates for transport into cells, as well as carbohydrate oxidoreductases and isomerases.

Furthermore, leveraging our expertise in recombinant protein production and protein engineering, we are involved in collaborative research aimed at developing protein-based therapeutics to improve fertility in dairy cattle.

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