You Young Ok, Levengood Matthew R, Ihnken L A Furgerson, Knowlton Aaron K, van der Donk Wilfred A
Department of Biochemistry and Howard Hughes Medical Institute, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, IL 61801, USA.
ACS Chem Biol. 2009 May 15;4(5):379-85. doi: 10.1021/cb800309v.
Methods that introduce posttranslational modifications in a general, mild, and non-sequence-specific manner using biologically produced peptides have great utility for investigation of the functions of these modifications. In this study, the substrate promiscuity of a lantibiotic synthetase was exploited for the preparation of phosphopeptides, glycopeptides, and peptides containing analogs of methylated or acetylated lysine residues. Peptides attached to the C-terminus of the leader peptide of the lacticin 481 precursor peptide were phosphorylated on serine residues in a wide variety of sequence contexts by the R399M and T405A mutants of lacticin 481 synthetase (LctM). Serine residues located as many as 30 amino acids C-terminal to the leader peptide were phosphorylated. Wild-type LctM was shown to dehydrate these peptides to generate dehydroalanine-containing products that can be conveniently modified with external nucleophiles including thiosaccharides, 2-(dimethylamino)ethanethiol, and N-acetyl cysteamine, resulting in mimics of O-linked glycopeptides and acetylated and methylated lysines.
使用生物合成肽以通用、温和且非序列特异性的方式引入翻译后修饰的方法，对于研究这些修饰的功能具有很大的实用价值。在本研究中，利用羊毛硫抗生素合成酶的底物混杂性来制备磷酸肽、糖肽以及含有甲基化或乙酰化赖氨酸残基类似物的肽。乳酸乳球菌素481前体肽前导肽C末端连接的肽，在多种序列背景下，被乳酸乳球菌素481合成酶（LctM）的R399M和T405A突变体在丝氨酸残基上磷酸化。位于前导肽C末端多达30个氨基酸处的丝氨酸残基被磷酸化。野生型LctM被证明可使这些肽脱水生成含脱氢丙氨酸的产物，这些产物可方便地用包括硫代糖、2-（二甲氨基）乙硫醇和N-乙酰半胱胺在内的外部亲核试剂进行修饰，从而生成O-连接糖肽以及乙酰化和甲基化赖氨酸的模拟物。
