Flügel Veronika, Vrabel Milan, Schneider Sabine
Department of Chemistry, TU Munich, Garching, Germany.
Department of Chemistry, Ludwig-Maximilians University, Munich, Germany.
PLoS One. 2014 Apr 23;9(4):e96198. doi: 10.1371/journal.pone.0096198. eCollection 2014.
Posttranslational modifications (PTMs) of proteins determine their structure-function relationships, interaction partners, as well as their fate in the cell and are crucial for many cellular key processes. For instance chromatin structure and hence gene expression is epigenetically regulated by acetylation or methylation of lysine residues in histones, a phenomenon known as the 'histone code'. Recently it was shown that these lysine residues can furthermore be malonylated, succinylated, butyrylated, propionylated and crotonylated, resulting in significant alteration of gene expression patterns. However the functional implications of these PTMs, which only differ marginally in their chemical structure, is not yet understood. Therefore generation of proteins containing these modified amino acids site specifically is an important tool. In the last decade methods for the translational incorporation of non-natural amino acids using orthogonal aminoacyl-tRNA synthetase (aaRS):tRNAaaCUA pairs were developed. A number of studies show that aaRS can be evolved to use non-natural amino acids and expand the genetic code. Nevertheless the wild type pyrrolysyl-tRNA synthetase (PylRS) from Methanosarcina mazei readily accepts a number of lysine derivatives as substrates. This enzyme can further be engineered by mutagenesis to utilize a range of non-natural amino acids. Here we present structural data on the wild type enzyme in complex with adenylated ε-N-alkynyl-, ε-N-butyryl-, ε-N-crotonyl- and ε-N-propionyl-lysine providing insights into the plasticity of the PylRS active site. This shows that given certain key features in the non-natural amino acid to be incorporated, directed evolution of this enzyme is not necessary for substrate tolerance.
蛋白质的翻译后修饰（PTM）决定了它们的结构-功能关系、相互作用伙伴以及它们在细胞中的命运，并且对于许多细胞关键过程至关重要。例如，染色质结构以及基因表达通过组蛋白中赖氨酸残基的乙酰化或甲基化进行表观遗传调控，这一现象被称为“组蛋白密码”。最近研究表明，这些赖氨酸残基还可以被丙二酰化、琥珀酰化、丁酰化、丙酰化和巴豆酰化，从而导致基因表达模式的显著改变。然而，这些在化学结构上仅有微小差异的PTM的功能意义尚未被理解。因此，位点特异性地生成包含这些修饰氨基酸的蛋白质是一种重要工具。在过去十年中，利用正交氨酰-tRNA合成酶（aaRS）：tRNAaaCUA对翻译掺入非天然氨基酸的方法得到了发展。许多研究表明，aaRS可以经过进化以使用非天然氨基酸并扩展遗传密码。尽管如此，来自马氏甲烷八叠球菌的野生型吡咯赖氨酸-tRNA合成酶（PylRS）很容易接受多种赖氨酸衍生物作为底物。这种酶可以通过诱变进一步改造以利用一系列非天然氨基酸。在此，我们展示了野生型酶与腺苷化的ε-N-炔基-、ε-N-丁酰-、ε-N-巴豆酰-和ε-N-丙酰-赖氨酸复合物的结构数据，为PylRS活性位点的可塑性提供了见解。这表明，鉴于要掺入的非天然氨基酸具有某些关键特征，对于底物耐受性而言，该酶的定向进化并非必要。
