Structure-function relationship of substrate length specificity of dextran glucosidase from Streptococcus mutans

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.

An enzyme family reunion — similarities, differences and eccentricities in actions on α-glucans

“…The structures of the free and oligosaccharide binding form were solved (Hondoh et al 2008) representing a very significant advancement of knowledge as the closest relative, oligo-1,6-glucosidase of GH13, was structure determined only in its free form. Substitution of the catalytic aspartate nucleophile of the dextran glucosidase to a cysteine and subsequent oxidation to sulphinic acid improved the transglycosylation capacity of this enzyme (Saburi et al 2007).…”

Structural Comparison of <i>Streptococcus mutans</i> Dextran Glucosidase with Glucoside Hydrolases in GH13

“…The β α loop 4 (residue number 199 213 in DGase) of DGase is much shorter than those of the other two glucoside hydrolases, and is thought to be related with the preference in the substrate chain length. 22) The β α loop 6 of GSJ (residue number 281 297 in GSJ), in which a short disordered structure is found, is shorter than those of other α 1,6 hydrolyzing glucoside hydrolases, and the direction of this loop is different. The β α loops 6 of O16G and DGase (residue numbers 280 300 in O16G and 261 283 in DGase) make turn toward the active site, and the side chain of Lys residue (Lys275 of DGase and Lys293 of O16G; see Fig.…”