Crystal structure of inactivated Thermotoga maritima invertase in complex with the trisaccharide substrate raffinose.
Thermotoga maritima invertase (beta-fructosidase), a member of the glycoside hydrolase family GH-32, readily releases beta-D-fructose from sucrose, raffinose and fructan polymers such as inulin. These carbohydrates represent major carbon and energy sources for prokaryotes and eukaryotes. The invertase cleaves beta-fructopyranosidic linkages by a double-displacement mechanism, which involves a nucleophilic aspartate and a catalytic glutamic acid acting as a general acid/base. The three-dimensional structure of invertase shows a bimodular enzyme with a five bladed beta-propeller catalytic domain linked to a beta-sandwich of unknown function. In the present study we report the crystal structure of the inactivated invertase in interaction with the natural substrate molecule alpha-D-galactopyranosyl-(1,6)-alpha-D-glucopyranosyl-beta-D-fructofuranoside (raffinose) at 1.87 A (1 A=0.1 nm) resolution. The structural analysis of the complex reveals the presence of three binding-subsites, which explains why T. maritima invertase exhibits a higher affinity for raffinose than sucrose, but a lower catalytic efficiency with raffinose as substrate than with sucrose.
Alberto
François
F
Architecture et Fonction des Macromolécules Biologiques, UMR6098, CNRS, Universités Aix-Marseille I & II, Case 932, 163 Avenue de Luminy, 13288 Marseille Cedex 9, France.
Jordi
Emmanuelle
E
Henrissat
Bernard
B
Czjzek
Mirjam
M
D001230:Aspergillus / Q000201:enzymology; D001665:Binding Sites; D002236:Carbohydrate Conformation; D020134:Catalytic Domain; D018651:Cichorium intybus / Q000201:enzymology; D018360:Crystallography, X-Ray; D004789:Enzyme Activation; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism; D006860:Hydrogen Bonding; D008958:Models, Molecular; D011485:Protein Binding; D020836:Protein Structure, Quaternary; D011887:Raffinose / Q000737:chemistry* / Q000378:metabolism*; D040681:Structural Homology, Protein; D013379:Substrate Specificity; D020124:Thermotoga maritima / Q000201:enzymology* / Q000235:genetics; D043324:beta-Fructofuranosidase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*
D006026:Glycoside Hydrolases; D043324:beta-Fructofuranosidase; C023086:inulinase; D011887:Raffinose