Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-Aminovinyl-Cysteine during NAI-107 Biosynthesis.
Two Flavoenzymes Catalyze the Post-Translational Generation of 5-Chlorotryptophan and 2-Aminovinyl-Cysteine during NAI-107 Biosynthesis.
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DOI：
10.1021/acschembio.6b01031
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发表时间：
2017-02-17
影响因子：
4
通讯作者：
van der Donk WA
中科院分区：
文献类型：
--
作者：
Ortega MA;Cogan DP;Mukherjee S;Garg N;Li B;Thibodeaux GN;Maffioli SI;Donadio S;Sosio M;Escano J;Smith L;Nair SK;van der Donk WA
羊毛硫抗生素是由核糖体合成并经后修饰的含有硫醚环的抗菌肽。除了这些交联之外，临床候选lantibiotic NAI-107还具有C-末端S-[（Z）-2-氨基乙烯基]-D-半胱氨酸（AviCys）和与其有效生物活性相关的独特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分析鉴定了分别负责形成ClTrp和AviCys的编码黄素酶的mibH和mibD。NAI-107上这些修饰的生物化学基础和两种酶的底物特异性目前尚不清楚。使用质谱，液相色谱和生物信息学分析的组合，我们证明，MibD是一个FAD依赖的Cys脱羧酶和MibH是一个FADH 2依赖的色氨酸卤化酶。大多数FADH 2依赖性Trp卤化酶卤化游离Trp，但MibH仅在Trp嵌入其同源肽底物去氯NAI-107内时才有活性。MibH与其他黄素依赖性Trp卤化酶的1.85-kDa分辨率晶体结构的结构比较显示，MibH底物结合位点内的细微氨基酸差异产生了溶剂暴露的裂缝，推测参与确定其不寻常的底物特异性。
Lantibiotics are ribosomally synthesized and post-translationally modified antimicrobial peptides containing thioether rings. In addition to these crosslinks, 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 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 FADH2-dependent Trp halogenase. Most FADH2-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 1.85-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 its unusual substrate specificity.