Crystal Structures of 4-α-Glucanotransferase from Thermococcus litoralis and Its Complex with an Inhibitor

Abstract

Thermococcus litoralis 4-␣-glucanotransferase (TLGT) belongs to glucoside hydrolase family 57 and catalyzes the disproportionation of amylose and the formation of large cyclic ␣-1,4-glucan (cycloamylose) from linear amylose. We determined the crystal structure of TLGT with and without an inhibitor, acarbose. TLGT is composed of two domains: an N-terminal domain (domain I), which contains a (␤/␣) 7 barrel fold, and a C-terminal domain (domain II), which has a twisted ␤-sandwich fold. In the structure of TLGT complexed with acarbose, the inhibitor was bound at the cleft within domain I, indicating that domain I is a catalytic domain of TLGT. The acarbose-bound structure also clarified that Glu 123 and Asp 214 were the catalytic nucleophile and acid/base catalyst, respectively, and revealed the residues involved in substrate binding. It seemed that TLGT produces large cyclic glucans by preventing the production of small cyclic glucans by steric hindrance, which is achieved by three lids protruding into the active site cleft, as well as an extended active site cleft. Interestingly, domain I of TLGT shares some structural features with the catalytic domain of Golgi ␣-mannosidase from Drosophila melanogaster, which belongs to glucoside hydrolase family 38. Furthermore, the catalytic residue of the two enzymes is located in the same position. These observations suggest that families 57 and 38 evolved from a common ancestor.

New Structural Insights on Carbohydrate-active Enzymes

“…6) Our group has been trying to solve crystal structures of useful enzymes for use in bioindustry. Two of these were the first crystal structures within the GH42 and GH57 families, 7,8) and we then began to focus on CAZy enzymes belonging to structurally unknown families. We have since solved two more GH families, and provided the first crystal structures within GH54 and GH94.…”

A Novel Branching Enzyme of the GH-57 Family

“…This length is close to that (6.7 A ) of T. litoralis 4 α glucanotransferase, the 3D structure of which is the only reported in the GH 57 family. 14) We found five residues (His10, His145, Trp270, Trp407 and Trp416) near the catalytic residues. These residues are all conserved in the TK1436 orthologs along with two catalytic residues.…”

“…Regions A, B, C, E and F essentially correspond to the five conserved sequence motifs in the GH 57 family. 13) Two catalytic residues of the GH 57 family, initially identified in a series of biochemical and structural studies on Thermococcus litoralis 4 α glucanotransferase 14,15) and later shown to be conserved in other family members, 13,16) are located in Region C (Glu183, a nucleophile) and Region E (Asp354, an acid base catalyst). On the other hand, Region D is a region only conserved in the TK1436 subfamily.…”

Structural Similarity between a Starch-hydrolyzing Enzyme and an N-Glycan-Hydrolyzing Enzyme: Exohydrolases Cleaving α-1,X-Glucosidic Linkages to Produce β-Glucose

“…In the SCOP database (http://scop.mrclmb.cam.ac.uk/scop/) (21), the N-terminal domain has been classified into a galactose mutarotase-like superfamily with a super β-sandwich fold. Although the physiological function of the N-terminal domain is unclear, numerous glycosidases possessing the domain, such as GH57 4-α-glucanotransferase from Thermococcus litoralis (22), are classified into the same superfamily, suggesting that the super β-sandwich domain could bind to sugars.…”