Lantibiotic engineering: peptide engineering approach using the modification enzymes

努卡星ISK-1是由华纳葡萄球菌ISK-1产生的一种抗生素。核糖体合成的前体多肽Nuka通过一个单一的修饰酶NukM引入不寻常的氨基酸，并去除前导序列，由ABC转运蛋白NukT分泌成熟多肽。根据一个预测，NukM被分为两个结构域，NukMN和NukMC，每个结构域分别负责脱水(磷酸化和进一步的磷酸消除)和环化。NukM、NukMN和NukMC的体外重组表明，NukM可以完全修饰Nuka，NukMN可以部分磷酸化和脱水。构建了一系列NukT突变体，并对其体内转运活性和体外肽酶活性进行了研究。提示NukT的多肽酶活性依赖于ATP的水解，N端的多肽酶结构域可能与C端的三磷酸腺苷结合区协同作用。

Nukacin ISK-1 is a lantibiotic produced by Staphylococcus warneri ISK-1. Ribosomally synthesized precursor peptide, NukA undergoes introduction of unusual amino acids by a single modification enzyme, NukM, and the leader sequence is removed and the mature peptide is secreted by ABC transporter NukT. NukM was divided into two domains, NukMN and NukMC, based on a prediction that each domain is responsible for dehydration (phosphorylation and further phosphate elimination) and cyclization, respectively. The in vitro reconstitution of NukM, NukMN, and NukMC revealed that NukM can fully modify NukA, and NukMN could partially phosphorylate and dehydrate it. A series of NukT mutants were constructed and their transport activity in vivo and peptidase activity in vitro were investigated. It was suggested that peptidase activity of NukT depends on ATP hydrolysis, and the N-terminal peptidase domain of NukT may cooperatively function with the C-terminal ATP-binding domain.