Structure-function relationship of substrate length specificity of dextran glucosidase from <Emphasis Type="Italic">Streptococcus mutans</Emphasis> |
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Authors: | Wataru Saburi Hironori Hondoh Young-Min Kim Haruhide Mori Masayuki Okuyama Atsuo Kimura |
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Institution: | (1) Research Faculty of Agriculture, Hokkaido University, Sapporo 060-8589, Japan;(2) Nihon Shokuhin Kako Co., LTD., Shizuoka 417-8530, Japan |
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Abstract: | Dextran glucosidase from Streptococcus mutans (SMDG), an exo-type glucosidase of glycoside hydrolase (GH) family 13, specifically hydrolyzes an α-1,6-glucosidic linkage at the non-reducing ends of isomaltooligosaccharides and dextran. SMDG shows the highest sequence
similarity to oligo-1,6-glucosidases (O16Gs) among GH family 13 enzymes, but these enzymes are obviously different in terms
of substrate chain length specificity. SMDG efficiently hydrolyzes both short-and long-chain substrates, while O16G acts on
only short-chain substrates. We focused on this difference in substrate specificity between SMDG and O16G, and elucidated
the structure-function relationship of substrate chain length specificity in SMDG. Crystal structure analysis revealed that
SMDG consists of three domains, A, B, and C, which are commonly found in other GH family 13 enzymes. The structural comparison
between SMDG and O16G from Bacillus cereus indicated that Trp238, spanning subsites +1 and +2, and short β → α loop 4, are characteristic of SMDG, and these structural elements are predicted to be important for high activity toward
long-chain substrates. The substrate size preference of SMDG was kinetically analyzed using two mutants: (i) Trp238 was replaced
by a smaller amino acid, alanine, asparagine or proline; and (ii) short β → α loop 4 was exchanged with the corresponding loop of O16G. Mutant enzymes showed lower preference for long-chain substrates
than wild-type enzyme, indicating that these structural elements are essential for the high activity toward long-chain substrates,
as implied by structural analysis. |
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Keywords: | dextran glucosidase oligo-1 6-glucosidase substrate specificity site-directed mutagenesis crystal structure |
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