Crystal structure of the enzyme-product complex reveals sugar ring distortion during catalysis by family 63 inverting α-glycosidase

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• 作者：Takatsugu Miyazaki^a ; ^b ; Atsushi Nishikawa^b ; Takashi Tonozuka^b ; ^{tonozuka@cc.tuat.ac.jp" class="auth_mail" title="E-mail the corresponding author}
• 关键词：EcYgjK ; Escherichia coli YgjK ; GH63 ; glycoside hydrolase family 63 ; Glc1 ; 2Gal ; 2-O-α-d-glucopyranosyl-d-galactose ; β-GlcF ; β-glucopyranosyl fluoride ; PDB ; Protein Data Bank ; RMSD ; root mean squared deviation
• 刊名：Journal of Structural Biology
• 出版年：2016
• 出版时间：December 2016
• 年：2016
• 卷：196
• 期：3
• 页码：479-486
• 全文大小：2312 K

文摘

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 unusual ¹S₃ 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.