Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes

Abstract

Glucansucrases are large enzymes belonging to glycoside hydrolase family 70, which catalyze the cleavage of sucrose into fructose and glucose, with the concomitant transfer of the glucose residue to a growing α-glucan polymer. Among others, plaque-forming oral bacteria secrete these enzymes to produce α-glucans, which facilitate the adhesion of the bacteria to the tooth enamel. We determined the crystal structure of a fully active, 1,031-residue fragment encompassing the catalytic and C-terminal domains of GTF180 from Lactobacillus reuteri 180, both in the native state, and in complexes with sucrose and maltose. These structures show that the enzyme has an α-amylase-like (β/α)(8)-barrel catalytic domain that is circularly permuted compared to the catalytic domains of members of glycoside hydrolase families 13 and 77, which belong to the same GH-H superfamily. In contrast to previous suggestions, the enzyme has only one active site and one nucleophilic residue. Surprisingly, in GTF180 the peptide chain follows a "U"-path, such that four of the five domains are made up from discontiguous N- and C-terminal stretches of the peptide chain. Finally, the structures give insight into the factors that determine the different linkage types in the polymeric product.

Fig. 1.

Overall structure of L. reuteri 180 GTF180-ΔN. (A) Crystal structure of GTF180-ΔN, the N- and the C-terminal ends of the polypeptide chain are indicated, the Ca²⁺ ion is shown as a magenta sphere; (B) Schematic presentation of the “U-shaped” course of the polypeptide chain. Domains A, B, C, IV, and V are coloured in blue, green, magenta, yellow, and red, respectively, with dark and light colors for the N- and C-terminal stretches of the peptide chain.

Fig. 2.

Stereo views of sucrose and maltose binding in the active site of GTF180(D1025N)-ΔN. (A) Sucrose (white stick representation) bound at subsites -1 and +1 in the D1025N mutant. Residues of the 980–984 loop are depicted as a transparent cartoon for clarity. Strictly conserved residues in the α-amylase superfamily are labeled in bold, except for D1504 which is not shown; (B) Maltose M1 (yellow stick presentation) bound in subsites +1 and +2. Maltose M2 bound at subsites +2 and +3 is also shown. The glucosyl moiety of the sucrose from the GTF180-ΔN-sucrose complex (white stick representation) is superimposed. Leu938 in front of the +1 glucosyl residue was omitted for clarity. Residues from domains A and B are colored blue and green, respectively. The Ca²⁺ ion is shown as a magenta sphere, water molecules are shown as smaller red spheres. Hydrogen bonding interactions are shown as black dashed lines.

Fig. 3.

Docking of isomaltose (A), isomaltotriose (B), and nigerose (C) in the active site of a modeled glucosyl-GTF180 intermediate. For modelling and docking procedure see SI text. The covalently bound glucosyl moiety is shown in white stick representation, isomaltose, nigerose, and isomaltotriose are shown in yellow stick representation. Dashed lines show hydrogen bonding distances of less than 3.5 Å, solid yellow lines show where the chain may extend; subsites are numbered with Arabic and Roman white numerals. Domains A and B of GTF180 are shown in surface presentation and are coloured blue and green, respectively.

Fig. 4.

Hypothetical evolutionary pathway based on the “permutation per duplication model” (28) leading to the unusual domain organization of GH70 family GTF180 starting from a putative GH13 α-amylase precursor. Sequence segments forming domains A, B, C, IV, and V are colored as before in blue, green, magenta, yellow, and red, respectively.