Molecular mechanism for the substrate specificity of Arthrobacter globiformis M6 α-glucosidase CmmB, belonging to glycoside hydrolase family 13 subfamily 30

α-Glucosidase hydrolyzes α-d-glucosides to produce α-d-glucose. Glycoside hydrolase family 13 (GH13) contains α-glucosidases together with various amylolytic enzymes. GH13 α-glucosidases fall into several distinct subfamilies, and subfamily 30 (GH13_30) includes numerous Actinomycetes α-glucosidases. GH13_30 α-glucosidase CmmB from Arthrobacter globiformis, specific to maltooligosaccharides, is involved in the intracellular metabolism of cyclobis-(1 → 6)-α-maltosyl. Herein, the function and structure of CmmB were investigated to advance understanding of the structure–function relationship. CmmB showed the highest kcat/Km for maltose of maltooligosaccharides, and kcat/Km drastically decreased with increasing substrate chain-length. The crystal structures of CmmB in complex with a pseudodisaccharide (acarviosin) and pseudotetrasaccharide (acarbose) were determined at resolutions of 1.60 and 1.70 Å, respectively. The overall structure of CmmB was typical of a GH13 α-glucosidase. Most of the structure of the β→α loop 7 of the catalytic (β/α)8-barrel domain was not determined in the acarbose complex, but the C-terminal side of this loop was modeled in the acarviosin complex. For binding to acarviosin, this loop took on a closed conformation, and I360 and R364 on this loop formed subsite +1 together with H224 and W280. Alanine substitution of these residues indicated that R364 was essential for the catalysis through a hydrogen bond with the O6 of the d-glucose residue in subsite +1. The β→α loop 7 is flexible upon binding to substrates, but I360 and R364 cannot participate in binding to maltooligosaccharides longer than maltose. The loss of interactions with these residues was concluded to result in the low preference for maltotriose and longer maltooligosaccharides.