Nagao Jun-Ichi, Morinaga Yoshiko, Islam Mohammad R, Asaduzzaman Sikder M, Aso Yuji, Nakayama Jiro, Sonomoto Kenji
Laboratory of Microbial Technology, Division of Microbial Science and Technology, Department of Bioscience and Biotechnology, Faculty of Agriculture, Graduate School, Kyushu University, 6-10-1 Hakozaki, Higashi-ku, Fukuoka 812-8581, Japan.
Peptides. 2009 Aug;30(8):1412-20. doi: 10.1016/j.peptides.2009.05.021. Epub 2009 May 27.
The prepeptide NukA of the lantibiotic nukacin ISK-1 consists of an N-terminal leader peptide followed by a propeptide moiety that undergoes post-translational modifications, that is, formation of unusual amino acids by NukM, cleavage of the leader peptide and transport by NukT to yield a mature peptide. To identify the region and conformation required for the maturation of prepeptide, we expressed a series of NukA mutants, mutants with the N-terminus-truncated leader peptide and site-directed mutants with conserved residues in the leader peptide of type A(II) lantibiotics, which were evaluated on the basis of the production of nukacin ISK-1. In addition, the secondary structure data of NukA mutants or fragments were obtained by circular dichroism spectra. The results indicated the importance of the alpha-helical leader peptide with hydrophobic and hydrophilic orientation consisting of the conserved residues in type A(II) lantibiotics. The expression data from various combinations of the chimeric prepeptides consisting of NukA and LctA (the prepeptide of lacticin 481, which shows high identity with NukA) further revealed that the amino acid difference at the C-terminus of the propeptide moiety between NukA and LctA, especially His at position 15 and Phe at position 19, was important for the maturation processes by the nukacin ISK-1 biosynthetic enzymes. Our findings suggest that the determinants in NukA were critically involved in the biosynthesis of nukacin ISK-1 and would thus be important for recognition by the nukacin ISK-1 biosynthetic enzymes.
羊毛硫抗生素 nukacin ISK - 1 的前肽 NukA 由 N 端前导肽和一个前肽部分组成，该前肽部分会经历翻译后修饰，即由 NukM 形成不寻常氨基酸、前导肽的切割以及由 NukT 转运，从而产生成熟肽。为了确定前肽成熟所需的区域和构象，我们表达了一系列 NukA 突变体、N 端截短前导肽的突变体以及 A(II)型羊毛硫抗生素前导肽中保守残基的定点突变体，并根据 nukacin ISK - 1 的产生情况对其进行评估。此外，通过圆二色光谱获得了 NukA 突变体或片段的二级结构数据。结果表明，具有 A(II)型羊毛硫抗生素中保守残基组成的具有疏水和亲水取向的α - 螺旋前导肽很重要。由 NukA 和 LctA（乳酸乳球菌素 481 的前肽，与 NukA 具有高度同源性）组成的嵌合前肽的各种组合的表达数据进一步表明，NukA 和 LctA 前肽部分 C 端的氨基酸差异，尤其是第 15 位的 His 和第 19 位的 Phe，对于 nukacin ISK - 1 生物合成酶的成熟过程很重要。我们的研究结果表明，NukA 中的决定因素在 nukacin ISK - 1 的生物合成中起关键作用，因此对于 nukacin ISK - 1 生物合成酶的识别很重要。
