Functional Analysis of Butyrolactone Autoregulator Receptor from Streptomyces virginiae

1996 Fiscal Year Final Research Report Summary

• Project/Area Number: 07680684
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Functional biochemistry
• Research Institution: Osaka University
• Principal Investigator: NIHIRA Takuya Osaka University, Dept.Biotechnology Associate Professor, 工学部, 助教授 (70144441)
• Co-Investigator(Kenkyū-buntansha): IHARA Fumio Osaka University, Dept.Biotechnology Assistant Professor, 工学部, 助手 (70252583)
• Project Period (FY): 1995 – 1996
• Keywords: butyrolactone autoregulator / receptor / Streptomyces

Research Abstract

To roughly determine the essential regions on the butyrolactone autoregulator receptor, we at first prepared several deletion mutants by means of PCR.However, the one lacking N-terminal 47 amino acid residues formed high-molecular weight aggregates, while the ones lacking either C-terminal 49 or 96 amino acid resildues formed inclusion bodies when expressed in E.coli. Fusion proteins with thioredoxin also showed the same phenomena, indicating that both the N-terminal and C-terminal 50 amino acid residues are important in maintaining the higher structure of the receptor protein.
Next, to identify the essential amino acid residues, we modified the receptor by chemical reagents. DTNB which can specifically react with Cys residues could modify two Cys residues of the receptor. However, the resulting receptor retained the full ligand binding activity and also showed the intact dimeric structure as revealed by gel-filtration experiments, indicating that Cys residues did not play any important roles in the receptor. Dye-sensitized photooxidation resulted in the loss of ligand binding activity, but the modified receptor was present as high-molecular weight aggregates, indicating that the modified residues participate in structure stability.