Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by family 63 inverting α-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 α-glycosidases, which act mainly on α-glucosides. We previously found that Escherichia coli GH63 enzyme, YgjK, can hydrolyze 2-O-α-d-glucosyl-d-galactose. Two constructed glycosynthase mutants, D324N and E727A, which catalyze the transfer of a β-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-Å 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 unusual1S3skew-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.
Research Institute of Green Science and Technology, Shizuoka University, 836 Ohya, Suruga-ku, Shizuoka 422-8529 Japan; Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Takatsugu
T
Miyazaki
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan.
Atsushi
A
Nishikawa
Department of Applied Biological Science, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509, Japan. Electronic address: tonozuka@cc.tuat.ac.jp.
Takashi
T
Tonozuka
D000595:Amino Acid Sequence; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000737:chemistry; D029968:Escherichia coli Proteins / Q000737:chemistry* / Q000235:genetics; D005690:Galactose / Q000737:chemistry; D005947:Glucose / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000235:genetics; D007785:Lactose / Q000737:chemistry; D008958:Models, Molecular; D050505:Mutant Proteins / Q000737:chemistry*; D009699:N-Glycosyl Hydrolases / Q000737:chemistry* / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity; D000073893:Sugars / Q000737:chemistry
D029968:Escherichia coli Proteins; D050505:Mutant Proteins; D000073893:Sugars; D006026:Glycoside Hydrolases; D009699:N-Glycosyl Hydrolases; C497011:YgjK protein, E coli; D005947:Glucose; D007785:Lactose; D005690:Galactose
Conformational itinerary; Cremer-Pople; Glucosylgalactose; Glycoside hydrolase family 63; Inverting mechanism; Skew-boat conformation