title: Molecular basis for the recognition of long-chain substrates by plant α-glucosidases.
abstract: Sugar beet α-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher kcat/Km values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific α-glucosidases. To gain structural insights into the long-chain substrate recognition of SBG, a crystal structure complex with the pseudotetrasaccharide acarbose was determined at 1.7 Å resolution. The active site pocket of SBG is formed by a (β/α)8 barrel domain and a long loop (N-loop) bulging from the N-terminal domain similar to other related enzymes. Two residues (Phe-236 and Asn-237) in the N-loop are important for the long-chain specificity. Kinetic analysis of an Asn-237 mutant enzyme and a previous study of a Phe-236 mutant enzyme demonstrated that these residues create subsites +2 and +3. The structure also indicates that Phe-236 and Asn-237 guide the reducing end of long substrates to subdomain b2, which is an additional element inserted into the (β/α)8 barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.
authors.affiliation: Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan.
authors.forename: Takayoshi
authors.initials: T
authors.lastname: Tagami
authors.forename: Keitaro
authors.initials: K
authors.lastname: Yamashita
authors.forename: Masayuki
authors.initials: M
authors.lastname: Okuyama
authors.forename: Haruhide
authors.initials: H
authors.lastname: Mori
authors.forename: Min
authors.initials: M
authors.lastname: Yao
authors.forename: Atsuo
authors.initials: A
authors.lastname: Kimura
mesh_terms: D020909:Acarbose / Q000737:chemistry; D000595:Amino Acid Sequence; D027461:Beta vulgaris / Q000201:enzymology*; D004252:DNA Mutational Analysis; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010940:Plant Proteins / Q000737:chemistry*; D011485:Protein Binding; D017434:Protein Structure, Tertiary; D011994:Recombinant Proteins / Q000737:chemistry; D013379:Substrate Specificity; D000520:alpha-Glucosidases / Q000737:chemistry*
chemical_list: D010940:Plant Proteins; D011994:Recombinant Proteins; D000520:alpha-Glucosidases; D020909:Acarbose
keywords: Acarbose Recognition; Crystal Structure; Enzyme Catalysis; Enzyme Kinetics; Enzyme Structure; Glycoside Hydrolase Family 31; Long-chain Specificity; Plant; α-Glucosidase