Ortega Manuel A, Cogan Dillon P, Mukherjee Subha, Garg Neha, Li Bo, Thibodeaux Gabrielle N, Maffioli Sonia I, Donadio Stefano, Sosio Margherita, Escano Jerome, Smith Leif, Nair Satish K, van der Donk Wilfred A
Department of Biochemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory , 600 S. Mathews Ave., Urbana, Illinois 61801, United States.
Department of Chemistry, University of Illinois at Urbana-Champaign, Roger Adams Laboratory , 600 S. Mathews Ave., Urbana, Illinois 61801, United States.
ACS Chem Biol. 2017 Feb 17;12(2):548-557. doi: 10.1021/acschembio.6b01031. Epub 2017 Jan 13.
Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these cross-links, the clinical candidate lantibiotic NAI-107 also possesses a C-terminal S-[(Z)-2-aminovinyl]-d-cysteine (AviCys) and a unique 5-chloro-l-tryptophan (ClTrp) moiety linked to its potent bioactivity. Bioinformatic and genetic analyses on the NAI-107 biosynthetic gene cluster identified mibH and mibD as genes encoding flavoenzymes responsible for the formation of ClTrp and AviCys, respectively. The biochemical basis for the installation of these modifications on NAI-107 and the substrate specificity of either enzyme is currently unknown. Using a combination of mass spectrometry, liquid chromatography, and bioinformatic analyses, we demonstrate that MibD is an FAD-dependent Cys decarboxylase and that MibH is an FADH-dependent Trp halogenase. Most FADH-dependent Trp halogenases halogenate free Trp, but MibH was only active when Trp was embedded within its cognate peptide substrate deschloro NAI-107. Structural comparison of the 1.88-Å resolution crystal structure of MibH with other flavin-dependent Trp halogenases revealed that subtle amino acid differences within the MibH substrate binding site generates a solvent exposed crevice presumably involved in determining the substrate specificity of this unusual peptide halogenase.
羊毛硫抗生素是核糖体合成并经翻译后修饰的含有硫醚环的抗菌肽。除了这些交联结构外，临床候选羊毛硫抗生素NAI-107还具有一个C端S-[(Z)-2-氨基乙烯基]-D-半胱氨酸（AviCys）以及一个与其强大生物活性相关的独特的5-氯-L-色氨酸（ClTrp）部分。对NAI-107生物合成基因簇的生物信息学和遗传学分析确定mibH和mibD分别为编码负责ClTrp和AviCys形成的黄素酶的基因。目前尚不清楚在NAI-107上进行这些修饰的生化基础以及这两种酶的底物特异性。通过结合质谱、液相色谱和生物信息学分析，我们证明MibD是一种依赖FAD的半胱氨酸脱羧酶，而MibH是一种依赖FADH的色氨酸卤化酶。大多数依赖FADH的色氨酸卤化酶使游离色氨酸卤化，但MibH仅在色氨酸嵌入其同源肽底物去氯NAI-107时才有活性。将MibH分辨率为1.88 Å的晶体结构与其他黄素依赖性色氨酸卤化酶进行结构比较，结果显示MibH底物结合位点内细微的氨基酸差异产生了一个可能与确定这种不同寻常的肽卤化酶的底物特异性有关的溶剂暴露裂隙。
