Structural and Functional Analysis of a Glycoside Hydrolase Family 97 Enzyme from Bacteroides thetaiotaomicron

Abstract

SusB, an 84-kDa ␣-glucoside hydrolase involved in the starch utilization system (sus) of Bacteroides thetaiotaomicron, belongs to glycoside hydrolase (GH) family 97. We have determined the enzymatic characteristics and the crystal structures in free and acarbose-bound form at 1.6 Å resolution. SusB hydrolyzes the ␣-glucosidic linkage, with inversion of anomeric configuration liberating the ␤-anomer of glucose as the reaction product. The substrate specificity of SusB, hydrolyzing not only ␣-1,4-glucosidic linkages but also ␣-1,6-, ␣-1,3-, and ␣-1,2-glucosidic linkages, is clearly different from other well known glucoamylases belonging to GH15. The structure of SusB was solved by the single-wavelength anomalous diffraction method with sulfur atoms as anomalous scatterers using an in-house x-ray source. SusB includes three domains as follows: the N-terminal, catalytic, and C-terminal domains. The structure of the SusB-acarbose complex shows a constellation of carboxyl groups at the catalytic center; Glu 532 is positioned to provide protonic assistance to leaving group departure, with Glu 439 and Glu 508 both positioned to provide base-catalyzed assistance for inverting nucleophilic attack by water. A structural comparison with other glycoside hydrolases revealed significant similarity between the catalytic domain of SusB and those of ␣-retaining glycoside hydrolases belonging to GH27, -36, and -31 despite the differences in catalytic mechanism. SusB and the other retaining enzymes appear to have diverged from a common ancestor and individually acquired the functional carboxyl groups during the process of evolution. Furthermore, sequence comparison of the active site based on the structure of SusB indicated that GH97 included both retaining and inverting enzymes.

Efficient synthesis of α‐galactosyl oligosaccharides using a mutant Bacteroides thetaiotaomicron retaining α‐galactosidase (BtGH97b)

“…7 The initial velocity of the formate ion-mediated reaction was also measured with alternative 8 acceptor substrates, including galactose, glucose, xylose, maltose, and cellobiose. These acceptors 9 followed equation 2 with the exception of galactose, with no increase in the velocity of fluoride 10 release observed for galactose. The constants α, β, and γ for the glycosyl acceptors are 11 summarized in Table 3.…”

“…The NMR spectra indicated that the trisaccharide 6 was β-lactosyl α-D-galactopyranoside [α-D-Galp-(1↔1)-β-D-Glcp-(4←1)β-D-Galp; Gal-Lac], 7 which respective occupancies of β-glucosyl and β-galactosyl moieties at subsites +1 and +2 8 formed. The α-(1↔1)-β linkage was determined by the HMBC-correlation between H-1 (J = 8.2 9 Hz) of the glucose residue and C-1 of the galactose residue, and that between H-1 (J = 3.8 Hz) of 10 the galactose residue and C-1 of the glucose residue (Fig. 4C).…”

“…5C), suggesting that the resistant 8 oligosaccharide was α-Gal-(1→6)-β-Glc-(1→4)-Glc. This compound was formed when the 9 hydroxy group at C6 of the glucose moiety of non-reducing end of cellobiose attacked the 10 anomeric carbon of the β-galactosyl formate intermediate. 11 The glycosidic linkages of the transfer products using PNP glucosides as acceptor 12 molecules were examined by methylation analysis.…”

“…Discussion 10 It is difficult to use an orthodox glycosynthase to synthesize an α-galactoside because of the 11 instability of β-galactosyl fluoride. In this study, we tested other known procedures for making α- 12 galactosides using a catalytic nucleophile mutant of GH97 α-galactosidase, BtGH97b-D415G.…”

Two Novel Glycoside Hydrolases Responsible for the Catabolism of Cyclobis-(1→6)-α-nigerosyl

“…The enzyme (1.8 units/ml) was incubated with 7.5 mM 4-nitrophenyl ␣-glucopyranoside in 5 mM sodium phosphate buffer (pH 8.0) at 30°C for 20 min. The anomeric form of glucose produced by the reaction was analyzed by HPLC using a previous report procedure (32).…”