Glycoside hydrolase (GH) enzymes are classified into 190 families, each defined by sequence similarity, and are crucial in carbohydrate metabolism by catalyzing the hydrolysis of glycosidic bonds in diverse organisms [1], [2], [3], [4], [5]. Traditionally, α-glucoside hydrolases have been divided into two groups, α-glucosidases (EC 3.2.1.20) and glucoamylases (EC 3.2.1.3), which utilize different catalytic mechanisms, with α-glucosidases catalyzing the hydrolysis of α-glycosidic linkages through a retaining mechanism, whereas glucoamylases hydrolyze these linkages using an inverting mechanism [6], [7], [8]. However, investigation of α-glycoside hydrolases in the GH97 family from Bacteroides thetaiotaomicron (B. thetaiotaomicron) caused the classic classification of α-glucoside hydrolases to change and extended our knowledge of structure-related catalytic mechanisms [9]. For example, BtGH97a (also known as SusB) liberates the β-anomer of glucose through an inverting mechanism and has significant similarities in the catalytic domain with GH27, GH36, and GH31 α-glucoside hydrolases, all of which employ a retaining catalytic mechanism [9], [10], [11]. The GH97 family is of interest not only because few of its enzymes have been characterized, but also because this family contains both retaining and inverting enzymes [12]. According to the Carbohydrate-Active enZymes (CAZy) database (http://www.cazy.org/), few bacterial genera possess GH97 family enzymes; to date, only 12 members of the GH97 family have been characterized: BtGH97a, BtGH97b, BT0683, BT2620, BT3294, BT3661, BT3664 from B. thetaiotaomicron [10], [13], [14], [15], [16], Aglu (BFO_2272) from Tannerella forsythensis [17], Ag97A from Pseudoalteromonas sp. K8 [18], and FjGH97A (Fjoh_4429) from Flavobacterium johnsoniae [19].

The GH97 family of enzymes can be divided into three subgroups based on sequence alignment [20]. In Fig. 1, the amino acid sequence of BtGH97a in Group 1 is used as the reference, with the sequences of the other enzymes aligned to their actual positions within their full-length sequences, corresponding to the same functional regions. Group 1 enzymes, including BtGH97a and Aglu, contain three critical glutamic acid residues (Glu439, Glu508, and Glu532), which are essential for the inverting catalytic mechanism typical of α-glucosidases, allowing these enzymes to efficiently hydrolyze α-glycosidic bonds. In Group 2, enzymes possess aspartic acid and glutamic acid residues corresponding to Glu508 and Glu532 in Group 1, indicating partial sequence similarity and likely similar catalytic roles, but with subtle functional differences due to the substitution of Asp for Glu508. Group 3 enzymes, such as BT2620 and BtGH97b, retain only the Glu532 residue, lacking equivalents to Glu439 and Glu508. This difference leads Group 3 enzymes to adopt a retaining mechanism, commonly associated with alpha-galactosidases, highlighting a distinct catalytic process compared to Groups 1 and 2.

B. thetaiotaomicron is a Gram-negative anaerobic organism found in human colons [21], [22], [23]. It possesses a 4,776-member proteome that enables the uptake and hydrolysis of polysaccharides not digested by the host [24], [25]. B. thetaiotaomicron possesses an extensive starch utilization system with multiple sus genes facilitating starch binding and utilization, and among sequenced prokaryotes, it is one of the richest in glycosyl hydrolases, enabling it to cleave most glycosidic bonds found in nature [26], [27]. Interestingly, the CAZy database (http://www.cazy.org/) shows that B. thetaiotaomicron contains at least 10 enzymes in the GH97 family, whereas this GH family is very rare in other human gut bacteria.

Among its diverse glycoside hydrolases, α-glucosidases play a pivotal role in liberating glucose from dietary and endogenous α-glucans. These enzymes enable B. thetaiotaomicron to efficiently access carbon sources within the competitive and nutrient-variable environment of the human colon. Despite their metabolic importance, many GH97 α-glucosidases in B. thetaiotaomicron remain uncharacterized. Understanding the functional diversity of these enzymes is essential to reveal how this organism coordinates glycan foraging and adapts to complex glycan landscapes. In this study, we characterized three novel glycoside hydrolase (GH97) enzymes cloned from B. thetaiotaomicron. By expressing and purifying these enzymes, we sought to investigate their substrate specificity, kinetic properties, and dependency on metal ions. These enzymes were selected based on their unique sequence alignment within the GH97 family, representing different subgroups with potential catalytic diversity. This investigation provides insights into the structural and functional diversity within the GH97 family, contributing to a broader understanding of carbohydrate-active enzymes.