Département de biochimie, Université de Montréal, Montréal, Québec, H3C 3J7, Canada.
Appl Microbiol Biotechnol. 2014 Jan;98(1):219-30. doi: 10.1007/s00253-013-4886-x. Epub 2013 Apr 25.
The great importance of amide bonds in industrial synthesis has encouraged the search for efficient catalysts of amide bond formation. Microbial transglutaminase (MTG) is heavily utilized in crosslinking proteins in the food and textile industries, where the side chain of a glutamine reacts with the side chain of a lysine, forming a secondary amide bond. Long alkylamines carrying diverse chemical entities can substitute for lysine as acyl-acceptor substrates, to link molecules of interest onto peptides or proteins. Here, we explore short and chemically varied acyl-acceptor substrates, to better understand the nature of nonnatural substrates that are tolerated by MTG, with the aim of diversifying biocatalytic applications of MTG. We show, for the first time, that very short-chain alkyl-based amino acids such as glycine can serve as acceptor substrates. The esterified α-amino acids Thr, Ser, Cys, and Trp--but not Ile--also showed reactivity. Extending the search to nonnatural compounds, a ring near the amine group--particularly if aromatic--was beneficial for reactivity, although ring substituents reduced reactivity. Overall, amines attached to a less hindered carbon increased reactivity. Importantly, very small amines carrying either the electron-rich azide or the alkyne groups required for click chemistry were highly reactive as acyl-acceptor substrates, providing a robust route to minimally modified, "clickable" peptides. These results demonstrate that MTG is tolerant to a variety of chemically varied natural and nonnatural acyl-acceptor substrates, which broadens the scope for modification of Gln-containing peptides and proteins.
酰胺键在工业合成中的重要性促使人们寻找高效的酰胺键形成催化剂。微生物谷氨酰胺转胺酶（MTG）在食品和纺织工业中大量用于交联蛋白质，其中谷氨酰胺的侧链与赖氨酸的侧链反应，形成次级酰胺键。带有各种化学实体的长链烷基胺可以替代赖氨酸作为酰基受体底物，将感兴趣的分子连接到肽或蛋白质上。在这里，我们探索了短而化学变化多样的酰基受体底物，以更好地了解 MTG 所容忍的非天然底物的性质，旨在使 MTG 的生物催化应用多样化。我们首次表明，像甘氨酸这样的非常短链烷基氨基酸可以作为受体底物。酯化的α-氨基酸 Thr、Ser、Cys 和 Trp——但不是 Ile——也表现出反应性。将搜索范围扩大到非天然化合物，靠近胺基的环——特别是如果是芳香族的——有利于反应性，尽管环取代基会降低反应性。总的来说，连接在较少阻碍的碳原子上的胺增加了反应性。重要的是，带有点击化学所需的富电子叠氮基或炔基的非常小的胺作为酰基受体底物具有很高的反应性，为最小修饰的“点击”肽提供了一种稳健的途径。这些结果表明 MTG 能够容忍各种化学变化多样的天然和非天然酰基受体底物，从而扩大了修饰含谷氨酰胺肽和蛋白质的范围。
