Molecular Determinants of Substrate Recognition in Thermostable -glucosidases Belonging to Glycoside Hydrolase Family 13
| ISSN: | 0021-924X |
|---|---|
| DOI: | 10.1093/jb/mvm110 |
| Přístupová URL adresa: | https://explore.openaire.eu/search/publication?articleId=doi_dedup___::218cac902eecd22f233daafcb68266c6 https://doi.org/10.1093/jb/mvm110 |
| Přírůstkové číslo: | edsair.doi.dedup.....218cac902eecd22f233daafcb68266c6 |
| Autor: | Reiko Takemura, Hiroshi Matsui, Hiroyuki Tanaka, Yoshiyuki Tsujimoto, Shin-ichi Kashiwabara, Yuzuru Suzuki, Atsushi Shimonaka, Tomohiko Yokogawa, Kunihiko Watanabe |
| Rok vydání: | 2007 |
| Předmět: |
Glycoside Hydrolases
Stereochemistry Molecular Sequence Data Biology Biochemistry Substrate Specificity Geobacillus stearothermophilus Structure-Activity Relationship chemistry.chemical_compound Enzyme Stability Structure–activity relationship Glycoside hydrolase Amino Acid Sequence Maltose Bacillaceae Molecular Biology Peptide sequence chemistry.chemical_classification Hydrolysis Mutagenesis Temperature Protein primary structure alpha-Glucosidases General Medicine Isomaltose Recombinant Proteins Kinetics Enzyme chemistry Mutation Mutagenesis Site-Directed Sequence Alignment |
| Zdroj: | Journal of Biochemistry. 142:87-93 |
| ISSN: | 0021-924X |
| DOI: | 10.1093/jb/mvm110 |
| Popis: | Bacillus stearothermophilus alpha-1,4-glucosidase (BS) is highly specific for alpha-1,4-glucosidic bonds of maltose, maltooligosaccharides and alpha-glucans. Bacillus thermoglucosdasius oligo-1,6-glucosidase (BT) can specifically hydrolyse alpha-1,6 bonds of isomaltose, isomaltooligosaccharides and alpha-limit dextrin. The two enzymes have high homology in primary structure and belong to glycoside hydrolase family 13, which contain four conservative regions (I, II, III and IV). The two enzymes are suggested to be very close in structure, even though there are strict differences in their substrate specificities. Molecular determinants of substrate recognition in these two enzymes were analysed by site-directed mutagenesis. Twenty BT-based mutants and three BS-based mutants were constructed and characterized. Double substitutions in BT of Val200 -->Ala in region II and Pro258 -->Asn in region III caused an appearance of maltase activity compared with BS, and a large reduction of isomaltase activity. The values of k(0)/K(m) (s(-1). mM(-1)) of the BT-mutant for maltose and isomaltose were 69.0 and 15.4, respectively. We conclude that the Val/Ala200 and Pro/Asn258 residues in the alpha-glucosidases may be largely responsible for substrate recognition, although the regions I and IV also exert a slight influence. Additionally, BT V200A and V200A/P258N possessed high hydrolase activity towards sucrose. |
| Databáze: | OpenAIRE |
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