Bacillus licheniformistrehalose-6-phosphate hydrolase structures suggest keys to substrate specificity

Trehalose-6-phosphate hydrolase (TreA) belongs to glycoside hydrolase family 13 (GH13) and catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to yield glucose and glucose 6-phosphate. The products of this reaction can be further metabolized by the energy-generating glycolytic pathway. Here, crystal structures of Bacillus licheniformis TreA ( Bl TreA) and its R201Q mutant complexed with p -nitrophenyl-α-D-glucopyranoside (R201Q– p PNG) are presented at 2.0 and 2.05 Å resolution, respectively. The overall structure of Bl TreA is similar to those of other GH13 family enzymes. However, detailed structural comparisons revealed that the catalytic site of Bl TreA contains a long loop that adopts a different conformation from those of other GH13 family members. Unlike the homologous regions of Bacillus cereus oligo-1,6-glucosidase ( Bc Ogl) and Erwinia rhapontici isomaltulose synthase (NX-5), the surface potential of the Bl TreA active site exhibits a largely positive charge contributed by the four basic residues His281, His282, Lys284 and Lys292. Mutation of these residues resulted in significant decreases in the enzymatic activity of Bl TreA. Strikingly, the 281 HHLK 284 motif and Lys292 play critical roles in substrate discrimination by Bl TreA.