Crystal Structures of Trehalose Synthase from Deinococcus Radiodurans Reveal a Closed Conformation for Intramolecular Isomerization Catalysis and Mutant Induction of an Active-Site Aperture

Trehalose has been used in food, cosmetic, and biotechnological industries due to its exceptional stability. Trehalose synthase (TS) catalyzes a simple conversion of inexpensive maltose into trehalose and hence has a great potential. TS consists of a catalytic (β/α)8 barrel, a subdomain B, a C-terminal β domain and two TS-unique subdomains (S7 and S8). The apo TS structures from Mycobacterium smegmatis and M. tuberculosis showed an unusual inactive conformation, in which the S7 loop blocks the substrate-binding pocket. Here we report structural and mutational studies of TS from Deinococcus radioduran (DrTS). The complex structures of DrTS with the inhibitor Tris share high homology with the substrate-bound sucrose hydrolase, amylosucrase, and sucrose isomerase, particularly virtually identical active-site architectures. A maltose was modelled into the active site and subsequent mutational analysis suggested that Tyr213, Glu320, and Glu324 are essential for the TS activity. In addition, the interaction networks between subdomains B and S7 seal the active-site entrance. Disruption of such networks through replacement of Arg148 and Asn253 with alanine resulted in a decreased isomerase activity but an increased hydrolase activity. The R148A and N253A structures showed a small pore created for water entry. Unexpectedly, the apo N253F mutant with ∼80% of hydrolase activity but no detectable isomerase activity showed a strikingly different conformation, in which the Bβ1-Bβ2 loop in subdomain B is disordered, and the subdomain B rotates away to create an open active site. Interestingly, this mutant displays a high structural similarity to the apo sucrose hydrolase. Therefore, our DrTS-N253F structure may represent an open conformation for the apo TS, while the DrTS-Tris may represent a substrate-induced closed conformation that will facilitate intramolecular isomerization and minimize disaccharide hydrolysis.