Structural and biochemical characterization of novel bacterial α-galactosidases belonging to glycoside hydrolase family 31

Abstract

Glycoside hydrolase family 31 (GH31) proteins have been reportedly identified as exo-α-glycosidases with activity for α-glucosides and α-xylosides. We focused on a GH31 subfamily, which contains proteins with low sequence identity (<24%) to the previously reported GH31 glycosidases and characterized two enzymes from Pedobacter heparinus and Pedobacter saltans. The enzymes unexpectedly exhibited α-galactosidase activity, but were not active on α-glucosides and α-xylosides. The crystal structures of one of the enzymes, PsGal31A, in unliganded form and in complexes with D-galactose or L-fucose and the catalytic nucleophile mutant in unliganded form and in complex with p-nitrophenyl-α-D-galactopyranoside, were determined at 1.85-2.30 Å (1 Å=0.1 nm) resolution. The overall structure of PsGal31A contains four domains and the catalytic domain adopts a (β/α)8-barrel fold that resembles the structures of other GH31 enzymes. Two catalytic aspartic acid residues are structurally conserved in the enzymes, whereas most residues forming the active site differ from those of GH31 α-glucosidases and α-xylosidases. PsGal31A forms a dimer via a unique loop that is not conserved in other reported GH31 enzymes; this loop is involved in its aglycone specificity and in binding L-fucose. Considering potential genes for α-L-fucosidases and carbohydrate-related proteins within the vicinity of Pedobacter Gal31, the identified Gal31 enzymes are likely to function in a novel sugar degradation system. This is the first report of α-galactosidases which belong to GH31 family.

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

“…8A). The structure was almost identical with the free wild-type enzyme with root 17 mean square deviations (r.m.s.d.) of 0.20 Å for 641 Cα atoms, indicating that the mutation had no 18 noticeable effect on the three-dimensional structure of the protein.…”

“…4D). This result implied that the trisaccharide has three 17 anomeric protons with α, α, and β anomeric configurations, respectively, and is the non-reducing 18 sugar, β-maltosyl α-D-galactopyranoside [α-D-Galp-(1↔1)-β-D-Glcp-(4←1)α-D-Glcp]. 19 The formation of two saccharides from cellobiose was confirmed by HPLC.…”

“…Kinetic parameters were 14 determined with varying concentrations of lactose under fixed concentrations of α-GalF (10 mM) 15 and an external nucleophile (100 mM sodium formate or 300 mM sodium azide). The velocity of 16 fluoride ion release at various lactose concentrations in the presence of formate displayed a 17 saturation curve, which did not pass through the origin (Fig. 6C).…”

“…13 15 The transfer products were identical among all reactions with homologous substrates and 16 acceptors. The products from reactions using α-GalF in the presence of formate ion were isolated 17 by HPLC and the main products were subjected to structural analyses. Using glucose as the 18 acceptor generated one major and three minor products.…”

“…Kinetic analysis of the chemical-rescued transglycosylation 16 Kinetic parameters of the reaction by BtGH97b-D415G in the presence of 100 mM sodium 17 formate or 300 mM sodium azide were determined with or without lactose ( Table 2). The initial 18 velocities of fluoride ion release from various concentrations of α-GalF as the sole substrate were 19 measured.…”