title: Involvement of Val 315 located in the C-terminal region of thermolysin in its expression in Escherichia coli and its thermal stability.
abstract: Thermolysin is a thermophilic and halophilic zinc metalloproteinase that consists of β-rich N-terminal (residues 1-157) and α-rich C-terminal (residues 158-316) domains. Expression of thermolysin variants truncated from the C-terminus was examined in E. coli culture. The C-terminal Lys316 residue was not significant in the expression, but Val315 was critical. Variants in which Val315 was substituted with fourteen amino acids were prepared. The variants substituted with hydrophobic amino acids such as Leu and Ile were almost the same as wild-type thermolysin (WT) in the expression amount, α-helix content, and stability. Variants with charged (Asp, Glu, Lys, and Arg), bulky (Trp), or small (Gly) amino acids were lower in these characteristics than WT. All variants exhibited considerably high activities (50-100% of WT) in hydrolyzing protein and peptide substrates. The expression amount, helix content, and stability of variants showed good correlation with hydropathy indexes of the amino acids substituted for Val315. Crystallographic study of thermolysin has indicated that V315 is a member of the C-terminal hydrophobic cluster. The results obtained in the present study indicate that stabilization of the cluster increases thermolysin stability and that the variants with higher stability are expressed more in the culture. Although thermolysin activity was not severely affected by the variation at position 315, the stability and specificity were modified significantly, suggesting the long-range interaction between the C-terminal region and active site.
authors.affiliation: Laboratory of Enzyme Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Kenji
authors.initials: K
authors.lastname: Kojima
authors.affiliation: Laboratory of Enzyme Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan.
authors.forename: Hiroki
authors.initials: H
authors.lastname: Nakata
authors.affiliation: Laboratory of Enzyme Chemistry, Division of Food Science and Biotechnology, Graduate School of Agriculture, Kyoto University, Sakyo-ku, Kyoto 606-8502, Japan. Electronic address: inouye@kais.kyoto-u.ac.jp.
authors.forename: Kuniyo
authors.initials: K
authors.lastname: Inouye
mesh_terms: D000179:Acrylates / Q000378:metabolism; D001407:Bacillus / Q000201:enzymology; D002364:Caseins / Q000378:metabolism; D004151:Dipeptides / Q000378:metabolism; D004795:Enzyme Stability; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D015971:Gene Expression Regulation, Enzymologic; D006868:Hydrolysis; D008958:Models, Molecular; D016297:Mutagenesis, Site-Directed; D017510:Protein Folding; D017434:Protein Structure, Tertiary; D013820:Thermolysin / Q000737:chemistry* / Q000235:genetics* / Q000378:metabolism; D014633:Valine / Q000502:physiology*
chemical_list: D000179:Acrylates; D002364:Caseins; D004151:Dipeptides; C108649:N-(3-(2-furyl)acryloyl)glycyl-leucinamide; D013820:Thermolysin; D014633:Valine
keywords: C-terminal region; CBB; CC; CD; Circular dichroism; Coomassie Brilliant Blue; Correlation coefficient; DA; Degree of activation; EA; Enzyme expression; Enzyme stability; Expression amount; FAGLA; HC; Hydropathy; N-[3-(2-Fury)acryloyl]-glycyl-l-leucine amide; PU; Proteolytic unit; REA; RHC; Relative expression amount; Relative α-helix content; Site-directed mutagenesis; Thermolysin; WT; Wild-type thermolysin; α-helix content