Iino T, Sako T
Yakult Central Institute for Microbiological Research, Tokyo, Japan.
J Biol Chem. 1988 Dec 15;263(35):19077-82.
Escherichia coli strains carrying certain prlA mutations (prlA4 and prlA401) could not support the processing and export of staphylokinase, resulting in the accumulation of the precursor form under high-level synthesis conditions. In order to clarify the cause of the defect in the structure of staphylokinase, we constructed signal peptide mutations of sak which suppressed the processing defect in the prlA4 cells by site-directed mutagenesis. The processing defect was suppressed when glycine or asparagine was introduced in place of the serine residue at position 17 from the amino terminus of the signal peptide. Substitutions of glycine for the leucine residue at position 15 and for the serine residue at position 19 were also effective. Other mutations we constructed had no suppression activity. Taking account of the correlation between the suppression activity and the parameter value of each substituted amino acid for the beta-turn probability, we predict that the staphylokinase signal peptide requires a more bending structure at the end of the hydrophobic core to act efficiently in the prlA4 cells than in the prl+ cells and that a function of the PrlA protein necessary to recognize the staphylokinase signal peptide has become deficient through the prlA4 mutation.
携带某些prlA突变（prlA4和prlA401）的大肠杆菌菌株无法支持葡萄球菌激酶的加工和输出，导致在高水平合成条件下前体形式的积累。为了阐明葡萄球菌激酶结构缺陷的原因，我们通过定点诱变构建了sak的信号肽突变体，该突变体抑制了prlA4细胞中的加工缺陷。当在信号肽氨基末端第17位的丝氨酸残基被甘氨酸或天冬酰胺取代时，加工缺陷被抑制。将第15位的亮氨酸残基和第19位的丝氨酸残基替换为甘氨酸也有效。我们构建的其他突变没有抑制活性。考虑到抑制活性与每个取代氨基酸的β-转角概率参数值之间的相关性，我们预测，与prl+细胞相比，葡萄球菌激酶信号肽在prlA4细胞中高效发挥作用需要在疏水核心末端有更弯曲的结构，并且通过prlA4突变，识别葡萄球菌激酶信号肽所需的PrlA蛋白功能已缺失。
