Lee M Violet, Ihnken Leigh Anne Furgerson, You Young Ok, McClerren Amanda L, van der Donk Wilfred A, Kelleher Neil L
Department of Chemistry, University of Illinois at Urbana-Champaign, 600 South Mathews Avenue, Urbana, Illinois 61801, USA.
J Am Chem Soc. 2009 Sep 2;131(34):12258-64. doi: 10.1021/ja9033507.
The lantibiotic synthetases LctM and HalM2 are bifunctional enzymes that catalyze both the dehydration of serine and threonine residues and the Michael-type additions of cysteine residues to the resulting dehydroamino acids in their substrate peptides. Using Fourier transform mass spectrometry to analyze these activities in vitro, the dehydration is shown to take place by a distributive mechanism, with build-up of intermediates observed in electrospray mass spectra. The cyclization activity of HalM2 was monitored through alkylation of free cysteines in intermediates, providing access to the regioselectivity of lanthionine ring formation using high-resolution tandem mass spectrometry. HalM2 is shown to catalyze the cyclization process in a largely N- to C-terminal directional fashion, forming a total of four lanthionine rings in its HalA2 substrate. These studies advance a model for lantibiotic production where substrate binding via an N-terminal leader results in dehydration and cyclization on similar time scales and with a high, though not strict, propensity for N-to-C directionality.
羊毛硫抗生素合成酶LctM和HalM2是双功能酶，可催化丝氨酸和苏氨酸残基的脱水反应，以及半胱氨酸残基对其底物肽中生成的脱氢氨基酸的迈克尔型加成反应。利用傅里叶变换质谱分析这些体外活性，结果表明脱水反应通过分布机制进行，在电喷雾质谱中观察到中间体的积累。通过中间体中游离半胱氨酸的烷基化来监测HalM2的环化活性，利用高分辨率串联质谱可了解羊毛硫氨酸环形成的区域选择性。结果表明，HalM2在很大程度上以从N端到C端的方向催化环化过程，在其HalA2底物中总共形成四个羊毛硫氨酸环。这些研究提出了一种羊毛硫抗生素产生的模型，即通过N端前导肽与底物结合，在相似的时间尺度上导致脱水和环化，并且具有较高（但不严格）的从N端到C端的方向性倾向。
Zotero 插件
