Deciphering the Biosynthetic Origin of L-allo-Isoleucine
Deciphering the Biosynthetic Origin of L-allo-Isoleucine
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破译 L-别异亮氨酸的生物合成起源
DOI：
10.1021/jacs.5b11380
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发表时间：
2016-01-13
影响因子：
15
通讯作者：
Ju, Jianhua
中科院分区：
文献类型：
--
作者：
Li, Qinglian;Qin, Xiangjing;Ju, Jianhua
非蛋白质氨基酸L-别异亮氨酸（L-allo-Ile）在包括但不限于细菌、真菌、植物和哺乳动物系统（包括智人）的各种生命形式中具有特征。尽管它的普遍性和功能的重要性，这种独特的氨基酸的具体来源已经逃避表征。在这项研究中，我们描述了两个酶对组成的吡哆醛5 '-磷酸（PLP）-连接氨基转移酶和一个前所未有的异构酶协同负责的L-异亮氨酸（L-Ile）的生物合成L-异源-Ile在天然产物中的发现和表征。来自Streptomyces scopuliridis SCSIO ZJ 46中的去氧酰胺生物合成途径的DsaD/DsaE和来自Streptomyces drozdowiczii SCSIO 10141中的马弗霉素生物合成途径的MfnO/MfnH驱动每个相应系统中的L-异源-Ile生成。体内基因失活验证了DsaD/DsaE对和MfnO/MfnH对在L-allo-Ile单位生物合成中的重要性。PLP连接的氨基转移酶DsaD和MfnO的失活分别导致地索酰胺和马福霉素滴度显著降低。此外，异构酶基因dsaE和mfnH的失活完全消除了两种生物合成途径中所有含代谢物的产生。值得注意的是，体外生物化学测定显示DsaD/DsaE和MfnO/MfnH各自催化L-异源-Ile之间的双向反应，并且定点诱变实验显示酶促反应涉及PLP连接的酮亚胺中间体，并且使用来自每个异构酶的C-末端的精氨酸残基来差向异构化氨基酸β-位置。因此，这些数据为具有药用潜力的天然产物中Lallo-Ile的起源提供了重要的新见解，并为生物工具的开发提供了新的可能性。
The nonproteinogenic amino acid L-allo-isoleucine (L-allo-Ile) is featured in an assortment of life forms comprised of, but not limited to, bacteria, fungi, plants and mammalian systems including Homo sapiens. Despite its ubiquity and functional importance, the specific origins of this unique amino acid have eluded characterization. In this study, we describe the discovery and characterization of two enzyme pairs consisting of a pyridoxal 5'-phosphate (PLP)-linked aminotransferase and an unprecedented isomerase synergistically responsible for the biosynthesis of L-allo-Ile from L-isoleucine (L-Ile) in natural products. DsaD/DsaE from the desotamide biosynthetic pathway in Streptomyces scopuliridis SCSIO ZJ46, and MfnO/MfnH from the marformycin biosynthetic pathway in Streptomyces drozdowiczii SCSIO 10141 drive L-allo-Ile generation in each respective system. In vivo gene inactivations validated the importance of the DsaD/DsaE pair and MfnO/MfnH pair in L-allo-Ile unit biosynthesis. Inactivation of PLP-linked aminotransferases DsaD and MfnO led to significantly diminished desotamide and marformycin titers, respectively. Additionally, inactivation of the isomerase genes dsaE and mfnH completely abolished production of all containing metabolites in both biosynthetic pathways. Notably, in vitro biochemical assays revealed that DsaD/DsaE and MfnO/MfnH each catalyze a bidirectional reaction between L-allo-Ile and Site-directed mutagenesis experiments revealed that the enzymatic reaction involves a PLP-linked ketimine intermediate and uses an arginine residue from the C-terminus of each isomerase to epimerize the amino acid beta-position. Consequently, these data provide important new insight into the origins of Lallo-Ile in natural products with medicinal potential and illuminate new possibilities for biotool development.