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

吡咯赖氨酸于2002年被发现，是第22种基因编码的氨基酸。具体而言，tRNA和源自pylT和pylS-基因的同源氨酰tRNA合成酶在将不寻常的氨基酸插入蛋白质序列中的情况下共同使得蛋白质生物合成机制能够通读UAG终止密码子（Hao等人，2002; Srinivasan等人，2002）。然而，吡咯赖氨酸是迄今为止只知道被纳入到少数蛋白质参与甲烷代谢的家庭甲烷和一些真细菌，吡咯赖氨酸生物化学已迅速被认为是一个迷人的工具，引入不寻常的氨基酸到蛋白质。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）。生物合成途径已推导出令人信服的间接证据，但没有三个蛋白质的结构和功能的特点。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等人，2012;参见2.3），并且旨在使用这些数据作为研究PylB蛋白的机制和调节的起点。本研究拟克隆和表达PylC和PylD蛋白，并通过X射线晶体学方法确定其结构，研究其反应机理。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。下一步，我们将在大肠杆菌中建立一个改良的表达系统。当暴露于不同的氨基酸并用包含符合读框的UAG密码子的多核苷酸转化时，将这些残基插入靶蛋白的限定位置。因此，我们的重点将是功能和结构分析蛋白质展示吡咯赖氨酸或吡咯赖氨酸衍生物在其催化活性位点。

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.