Enzymes for biosynthesis and utilization of the 22nd genetically encoded amino acid, pyrrolysine. Crystal structures, reaction mechanisms and applications in biotechnology

用于生物合成和利用第 22 种基因编码氨基酸吡咯赖氨酸的酶。

• 批准号: 226560260
• 负责人: Professor Dr. Michael Groll
• 金额: --
• 依托单位: Lehrstuhl für Biochemie
• 依托单位国家: 德国
• 项目类别: Research Grants
• 财政年份: 2012
• 资助国家: 德国
• 起止时间: 2011-12-31 至 2014-12-31
• 项目状态: 已结题
• 来源: https://gepris.dfg.de/gepris/projekt/226560260?language=en
• 关键词: Enzymes; biosynthesis; utilization; 22nd; genetically

Pyrrolysine was discovered in 2002 as the 22nd genetically encoded amino acid. Specifically, the tRNA and the cognate aminoacyl tRNA synthetase, derived from the pylT and pylS-genes, jointly enable the read-through of UAG stop codons by the protein biosynthetic machinery under insertion of the unusual amino acid into protein sequences (Hao et al. 2002; Srinivasan et al. 2002). Whereas pyrrolysine is so far only known to be incorporated into few proteins involved in the methane metabolism in the family Methanosarcinaceae and a few eubacteria, the pyrrolysine biochemistry has been rapidly recognized as a fascinating tool for introducing unusual amino acids into proteins. Pyrrolysine has been shown to be synthesized by the consecutive action of three proteins specified by the pylB, pylC and pylD genes (Gaston et al. 2011). The biosynthetic pathway has been deduced from compelling indirect evidence, but none of the three proteins had been characterized structurally and functionally. We have determined the X-ray structure of the S-adenoslymethionine iron sulfur protein PylB (Quitterer et al., 2012; see 2.3) and aim to use these data as a starting point for investigating the mechanism and the regulation of PylB protein. In this research project we would like to clone and express PylC and PylD protein as well, determine their structures by X-ray crystallography and investigate their reaction mechanisms. In addition, our goal is to prepare pyrrolysine and pyrrolysine derivatives in large quantities by using either PylBCD proteins or chemical synthesis. Next, we will establish a modified expression system in E. coli that, when exposed to the distinct amino acids and transformed with a polynucleotide comprising an in-frame UAG codon, inserts these residues into the target protein at defined positions. Hereby, our focus will be to functionally and structurally analyze proteins exhibiting pyrrolysine or pyrrolysine derivatives in their catalytic active sites.

吡咯赖氨酸于 2002 年被发现，是第 22 个基因编码氨基酸。具体来说，tRNA 和衍生自 pylT 和 pylS 基因的同源氨酰 tRNA 合成酶，在将不常见的氨基酸插入到蛋白质序列中时，共同实现了蛋白质生物合成机制对 UAG 终止密码子的通读（Hao 等人，2002 年；Srinivasan 等人，2002 年）。虽然迄今为止仅知道吡咯赖氨酸被纳入甲烷八叠球菌科和一些真细菌中涉及甲烷代谢的少数蛋白质中，但吡咯赖氨酸生物化学已迅速被认为是一种将不寻常氨基酸引入蛋白质中的迷人工具。吡咯赖氨酸已被证明是通过 pylB、pylC 和 pylD 基因指定的三种蛋白质的连续作用合成的（Gaston 等人，2011）。生物合成途径是从令人信服的间接证据中推断出来的，但这三种蛋白质都没有在结构和功能上得到表征。我们已经确定了 S-腺苷甲硫氨酸铁硫蛋白 PylB 的 X 射线结构（Quitterer 等，2012；参见 2.3），并旨在利用这些数据作为研究 PylB 蛋白机制和调节的起点。在这个研究项目中，我们还想克隆和表达PylC和PylD蛋白，通过X射线晶体学确定它们的结构并研究它们的反应机制。此外，我们的目标是通过使用PylBCD蛋白或化学合成大量制备吡咯赖氨酸和吡咯赖氨酸衍生物。接下来，我们将在大肠杆菌中建立一个修饰的表达系统，当暴露于不同的氨基酸并用包含框内 UAG 密码子的多核苷酸转化时，将这些残基插入到目标蛋白的指定位置。因此，我们的重点将是对在其催化活性位点中表现出吡咯赖氨酸或吡咯赖氨酸衍生物的蛋白质进行功能和结构分析。