Journal of Structural Biology, Vol.196, No.3, 479-486, 2016
Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by family 63 inverting alpha-glycosidase
Glycoside hydrolases are divided into two groups, known as inverting and retaining enzymes, based on their hydrolytic mechanisms. Glycoside hydrolase family 63 (GH63) is composed of inverting alpha-glycosidases, which act mainly on alpha-glucosides. We previously found that Escherichia coli GH63 enzyme, YgjK, can hydrolyze 2-O-alpha-D-glucosyl-D-galactose. Two constructed glycosynthase mutants, D324N and E727A, which catalyze the transfer of a p-glucosyl fluoride donor to galactose, lactose, and melibiose. Here, we determined the crystal structures of D324N and E727A soaked with a mixture of glucose and lactose at 1.8- and 2.1-angstrom resolutions, respectively. Because glucose and lactose molecules are found at the active sites in both structures, it is possible that these structures mimic the enzyme product complex of YgjK. A glucose molecule found at subsite -1 in both structures adopts an unusual S-1(3) skew-boat conformation. Comparison between these structures and the previously determined enzyme-substrate complex structure reveals that the glucose pyranose ring might be distorted immediately after nucleophilic attack by a water molecule. These structures represent the first enzyme-product complex for the GH63 family, as well as the structurally-related glycosidases, and it may provide insight into the catalytic mechanism of these enzymes. (C) 2016 Elsevier Inc. All rights reserved.