TY - JOUR
T1 - Role of N- and C-terminal domains and non-homologous region in co-refolding of Thermotoga maritima β-glucosidase
AU - Kim, Bong Jo
AU - Mangala, Selanere L.
AU - Muralidhara, B. K.
AU - Hayashi, Kiyoshi
N1 - Funding Information:
B.J. Kim and S.L. Mangala thank the Japan Science and Technology Corporation (JST) for the award of an STA fellowship. This work was supported in part by a grant from the Program for Promotion of Basic Research Activities for Innovative Biosciences in Japan.
PY - 2005/12/1
Y1 - 2005/12/1
N2 - Thermotoga maritima β-glucosidase consists of three structural regions with 721 amino acids: the N-terminal domain, middle non-homologous region and a C-terminal domain. To investigate the role of these domains in the co-refolding of two fragments into catalytically active form, five sites coding the amino acid residue at 244, 331 in the N-terminal domain, 403 in the non-homologous region, 476 and 521 in the C-terminal domain were selected to split the gene. All the 10 resultant individual fragments were obtained as insoluble inclusion bodies and found to be catalytically inactive. However, the catalytic activity was recovered when the two fragments derived from N-terminal and C-terminal peptides were co-refolded together. It is quite interesting to find that not only the complement polypeptides such as N476/477C but also the truncated combination (N476/522C, amino acid residues from 477 to 521 is truncated) and overlapped combination (N476/245C and N476/404C, amino acid residues from 245 to 476 and from 404 to 476 are overlapped) also gave catalytically active enzymes. Our results showed that folding motifs consisted of the complete N-terminal domain play an important role in the co-refolding of the polypeptides into the catalytically active form.
AB - Thermotoga maritima β-glucosidase consists of three structural regions with 721 amino acids: the N-terminal domain, middle non-homologous region and a C-terminal domain. To investigate the role of these domains in the co-refolding of two fragments into catalytically active form, five sites coding the amino acid residue at 244, 331 in the N-terminal domain, 403 in the non-homologous region, 476 and 521 in the C-terminal domain were selected to split the gene. All the 10 resultant individual fragments were obtained as insoluble inclusion bodies and found to be catalytically inactive. However, the catalytic activity was recovered when the two fragments derived from N-terminal and C-terminal peptides were co-refolded together. It is quite interesting to find that not only the complement polypeptides such as N476/477C but also the truncated combination (N476/522C, amino acid residues from 477 to 521 is truncated) and overlapped combination (N476/245C and N476/404C, amino acid residues from 245 to 476 and from 404 to 476 are overlapped) also gave catalytically active enzymes. Our results showed that folding motifs consisted of the complete N-terminal domain play an important role in the co-refolding of the polypeptides into the catalytically active form.
KW - Co-refolding
KW - Gene splitting
KW - N- and C-terminal domains
KW - Non-homologous region
KW - Thermotoga maritima
KW - β-Glucosidase
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U2 - 10.1016/j.molcatb.2005.10.002
DO - 10.1016/j.molcatb.2005.10.002
M3 - Article
AN - SCOPUS:27944474302
SN - 1381-1177
VL - 37
SP - 101
EP - 108
JO - Journal of Molecular Catalysis B: Enzymatic
JF - Journal of Molecular Catalysis B: Enzymatic
IS - 1-6
ER -