Crystal structure of a 117 kDa glucansucrase fragment provides insight into evolution and product specificity of GH70 enzymes.
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.
Laboratory of Biophysical Chemistry, University of Groningen, Nijenborgh 4, 9747 AG Groningen, The Netherlands.
Andreja
A
Vujicic-Zagar
Tjaard
T
Pijning
Slavko
S
Kralj
Cesar A
CA
López
Wieger
W
Eeuwema
Lubbert
L
Dijkhuizen
Bauke W
BW
Dijkstra
D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D016695:Glycosyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D052200:Limosilactobacillus reuteri / Q000201:enzymology; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010446:Peptide Fragments / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017434:Protein Structure, Tertiary; D013379:Substrate Specificity
D001426:Bacterial Proteins; D010446:Peptide Fragments; D016695:Glycosyltransferases; C033837:alternansucrase