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 ðβ∕αÞ 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.crystal structure complexes | exopolysaccharide | Lactobacillus reuteri | dental caries

Residue Leu940 Has a Crucial Role in the Linkage and Reaction Specificity of the Glucansucrase GTF180 of the Probiotic Bacterium Lactobacillus reuteri 180

“…This result may be explained by the fact that maltose (as an acceptor) binds "away" from the position of Leu 940 (19). In other words, the mutations at position Leu 940 are not close enough to the maltose binding site to affect the linkage specificity of oligosaccharides produced.…”

“…Docking Studies-Docking studies of wild-type GTF180-⌬N and GTF180-⌬N L940W were performed with isomaltotriose, essentially as described previously (19). Briefly, all the dockings were carried out using an energy-minimized model of the glucosyl-enzyme covalent intermediate of GTF180-⌬N.…”

“…In our present study, the linkage distribution of the ␣-glucans produced by Leu 940 mutants clearly suggests that Leu 940 of domain B plays an important role in linkage specificity of GTF180-⌬N. Although residue Leu 940 in GTF180-⌬N is not close to the catalytic residues (the distance to Asp 1025 /Glu 1063 is ϳ10 -11 Å), its side chain points toward the substrate/acceptor binding groove and is located at 6 -8 Å distance from the ϩ1 glucosyl moiety of the bound maltose (Protein Data Bank code 3KLL (19)). The clear influence of the size of the (mutant) side chain at position 940 on linkage specificity suggests that steric effects may affect acceptor binding and thus determine which linkage type is favored.…”

“…The crystal structures of N-terminally truncated GTF180 (GTF180-⌬N) in complex with the donor substrate sucrose and the acceptor substrate maltose show that the active site is delineated by residues from domain A and B (19). Residue Leu 940 is located in a loop of domain B, which shapes the binding groove near the acceptor binding site (19); it is close to the ϩ1 glucosyl unit of maltose (19). The crystal structures of GTFA-⌬N (28) and DSRE⌬N 123 -GBD-CD2 (12) showed that the corresponding phenylalanine residues (Phe 939 and Phe 2210 , respectively) are in the same position.…”

“…Three catalytically crucial residues of glucansucrases have been identified in previous studies (6,17,18). In glycoside hydrolase family 70 enzymes, Asp 1025 (catalytic nucleophile, GTF180 numbering) is involved in the formation of a covalent glucosyl-enzyme intermediate, Glu 1063 is the acid/base catalyst, and Asp 1136 is the transition state stabilizer (19). Glucansucrases produce ␣-glucans with different structures especially regarding the glycosidic linkages.…”