Molecular Genetics Department, University of Groningen, Groningen, The Netherlands ; Synthetic Biology Centre, University of Groningen, Groningen, The Netherlands.
PLoS One. 2013 Sep 9;8(9):e74890. doi: 10.1371/journal.pone.0074890. eCollection 2013.
Precursor nisin is a model posttranslationally modified precursor lantibiotic that can be structurally divided into a leader peptide sequence and a modifiable core peptide part. The nisin core peptide clearly plays an important role in the precursor nisin-nisin modification enzymes interactions, since it has previously been shown that the construct containing only the nisin leader sequence is not sufficient to pull-down the nisin modification enzymes NisB and NisC. Serines and threonines in the core peptide part are the residues that NisB specifically dehydrates, and cysteines are the residues that NisC stereospecifically couples to the dehydrated amino acids. Here, we demonstrate that increasing the number of negatively charged residues in the core peptide part of precursor nisin, which are absent in wild-type nisin, does not abolish binding of precursor nisin to the modification enzymes NisB and NisC, but dramatically decreases the antimicrobial potency of these nisin mutants. An unnatural precursor nisin variant lacking all serines and threonines in the core peptide part and an unnatural precursor nisin variant lacking all cysteines in the core peptide part still bind the nisin modification enzymes NisB and NisC, suggesting that these residues are not essential for direct interactions with the nisin modification enzymes NisB and NisC. These results are important for lantibiotic engineering studies.
前体乳链菌肽是一种经过翻译后修饰的前体类菌素，可以结构上分为一个前导肽序列和一个可修饰的核心肽部分。乳链菌肽核心肽显然在前体乳链菌肽-乳链菌肽修饰酶相互作用中起着重要作用，因为以前已经表明，仅包含乳链菌肽前导序列的构建体不足以下拉乳链菌肽修饰酶 NisB 和 NisC。核心肽部分中的丝氨酸和苏氨酸是 NisB 特异性脱水的残基，而半胱氨酸是 NisC 立体特异性偶联到脱水氨基酸的残基。在这里，我们证明增加前体乳链菌肽核心肽部分中带负电荷的残基的数量（野生型乳链菌肽中不存在这些残基），不会破坏前体乳链菌肽与修饰酶 NisB 和 NisC 的结合，但会显著降低这些乳链菌肽突变体的抗菌效力。一种缺乏核心肽部分中所有丝氨酸和苏氨酸的非天然前体乳链菌肽变体和一种缺乏核心肽部分中所有半胱氨酸的非天然前体乳链菌肽变体仍然与乳链菌肽修饰酶 NisB 和 NisC 结合，这表明这些残基对于与乳链菌肽修饰酶 NisB 和 NisC 的直接相互作用不是必需的。这些结果对于类菌素工程研究很重要。
