Yamanaka H, Nomura T, Okamoto K
Department of Biochemistry, Faculty of Pharmaceutical Sciences, Tokushima Bunri University, Yamashiro, Tokushima, 770-8514, Japan.
Microb Pathog. 1998 Mar;24(3):145-54. doi: 10.1006/mpat.1997.0182.
Escherichia coli heat-stable enterotoxin Ip (STIp) is a small peptide toxin composed of 18 amino acid residues containing three intramolecular disulfide bonds. We found previously that the bonds are formed by the catalysis of DsbA (a oxidoreductase) in periplasm [1]. To interact with DsbA, the STIp in periplasm must have a structure suitable as substrate. However, the amino acid residues contributing to the construction of this structure have not been elucidated. We mutated the codon for the glutamic acid at position 7 of STIp by oligonucleotide site-specific mutagenesis in vivo and analysed the STIp produced from the mutant gene. The intramolecular disulfide bonds were not formed in mutant STIp (Glu-7-->Ala), but were formed in mutant STIp (Glu-7-->Asp). Furthermore, we found that replacing the asparagine residue at position 11 and the proline residue at position 12 did not affect the disulfide bond formation of STIp. The results indicate that a negative charge at position 7 in the sequence of STIp is necessary for STIp to interact with DsbA in periplasm.
大肠杆菌热稳定肠毒素Ip（STIp）是一种由18个氨基酸残基组成的小肽毒素，含有三个分子内二硫键。我们之前发现这些二硫键是由周质中的DsbA（一种氧化还原酶）催化形成的[1]。为了与DsbA相互作用，周质中的STIp必须具有适合作为底物的结构。然而，构成该结构的氨基酸残基尚未阐明。我们通过体内寡核苷酸位点特异性诱变对STIp第7位的谷氨酸密码子进行突变，并分析了突变基因产生的STIp。突变型STIp（Glu-7→Ala）中未形成分子内二硫键，但突变型STIp（Glu-7→Asp）中形成了二硫键。此外，我们发现替换第11位的天冬酰胺残基和第12位的脯氨酸残基并不影响STIp二硫键的形成。结果表明，STIp序列中第7位的负电荷对于STIp在周质中与DsbA相互作用是必需的。
