期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 288, 期 26, 页码 19296-19303出版社
ELSEVIER
DOI: 10.1074/jbc.M113.465211
关键词
-
资金
- Grants-in-Aid for Scientific Research [24658089] Funding Source: KAKEN
Sugar beet alpha-glucosidase (SBG), a member of glycoside hydrolase family 31, shows exceptional long-chain specificity, exhibiting higher k(cat)/K-m values for longer malto-oligosaccharides. However, its amino acid sequence is similar to those of other short chain-specific alpha-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 angstrom resolution. The active site pocket of SBG is formed by a (beta/alpha)(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 (beta/alpha)(8) barrel domain. Subdomain b2 of SBG includes Ser-497, which was identified as the residue at subsite +4 by site-directed mutagenesis.
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