Crystal structure and identification of a key amino acid for glucose tolerance, substrate specificity, and transglycosylation activity of metagenomic β-glucosidase Td2F2.
β-Glucosidase Td2F2 isolated from a compost metagenome has high glucose tolerance and transglycosylation activity. In this study, we determined the high-resolution crystal structure of Td2F2. It has a unique structure at the -1 subsite that is important for substrate specificity but not for glucose tolerance. To elucidate the mechanism(s) of glucose tolerance, we isolated a glucose-sensitive Td2F2 mutant using random mutagenesis. In this mutant, Asn223 residue located between subsites +1 and +2 was mutated. The Asn223 mutation resulted in reduced glucose tolerance and transglycosylation activity, and drastically changed substrate specificity. These results indicate that the structure between subsites +1 and +2 is critical for the glucose tolerance and substrate specificity of Td2F2. Our findings shed light on the glucose tolerance and transglycosylation activity mechanisms of glycoside hydrolase family 1 β-glucosidases.



The atomic coordinates and structure factors (codes 3WH5, 3WH6, 3WH8, 3WH7, 5AYB, and 5AYI) have been deposited in the Protein Data Bank (http://wwpdb.org/).
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Tomohiko
T
Matsuzawa
Department of Biotechnology, The University of Tokyo, Japan.
Toshinori
T
Jo
Department of Biomaterial Sciences, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Japan.
Taku
T
Uchiyama
Department of Biotechnology, The University of Tokyo, Japan.
Jenny A
JA
Manninen
Department of Biotechnology, The University of Tokyo, Japan.
Takatoshi
T
Arakawa
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.|Department of Computational Biology and Medical Sciences, Graduate School of Frontier Sciences, The University of Tokyo, Chiba, Japan.
Kentaro
K
Miyazaki
Department of Biotechnology, The University of Tokyo, Japan.
Shinya
0000-0003-1346-6435
S
Fushinobu
Bioproduction Research Institute, National Institute of Advanced Industrial Science and Technology (AIST), Tsukuba, Ibaraki, Japan.
Katsuro
K
Yaoi
D000595:Amino Acid Sequence / Q000235:genetics; D000596:Amino Acids / Q000737:chemistry* / Q000235:genetics; D018360:Crystallography, X-Ray; D005947:Glucose / Q000737:chemistry / Q000378:metabolism; D006031:Glycosylation; D007700:Kinetics; D054892:Metagenome; D008958:Models, Molecular; D011487:Protein Conformation; D013379:Substrate Specificity; D001617:beta-Glucosidase / Q000737:chemistry* / Q000235:genetics
D000596:Amino Acids; D001617:beta-Glucosidase; D005947:Glucose
X-ray crystallography; glycoside hydrolase; metagenomics; substrate specificity; β-glucosidase