Ectoine is a solute compatible with the physiologies of both prokaryotic and eukaryotic cells and is widely synthesized by bacteria as an osmotic stress protectant. Because it preserves functional attributes of proteins and macromolecular complexes, it is considered a chemical chaperone and has found numerous practical applications. However, the mechanism of its biosynthesis is incompletely understood. The second step in ectoine biosynthesis is catalyzed by l-2,4-diaminobutyrate acetyltransferase (EctA; EC 2.3.1.178), which transfers the acetyl group from acetyl-CoA to EctB-formed l-2,4-diaminobutyrate (DAB), yielding N-γ-acetyl-l-2,4-diaminobutyrate (N-γ-ADABA), the substrate of ectoine synthase (EctC). Here, we report the biochemical and structural characterization of the EctA enzyme from the thermotolerant bacterium Paenibacillus lautus (Pl). We found that (Pl)EctA forms a homodimer whose enzyme activity is highly regiospecific by producing N-γ-ADABA but not the ectoine catabolic intermediate N-α-acetyl-l-2,4-diaminobutyric acid. High-resolution crystal structures of (Pl)EctA (at 1.2-2.2 Å resolution) (i) for its apo-form, (ii) in complex with CoA, (iii) in complex with DAB, (iv) in complex with both CoA and DAB, and (v) in the presence of the product N-γ-ADABA were obtained. To pinpoint residues involved in DAB binding, we probed the structure-function relationship of (Pl)EctA by site-directed mutagenesis. Phylogenomics shows that EctA-type proteins from both Bacteria and Archaea are evolutionarily highly conserved, including catalytically important residues. Collectively, our biochemical and structural findings yielded detailed insights into the catalytic core of the EctA enzyme that laid the foundation for unraveling its reaction mechanism.

中文翻译：

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二氨基丁酸乙酰基转移酶活性位点的结构为化学分子伴侣外生素的生物合成提供了机械原理。

Ectoine是一种与原核和真核细胞生理相容的溶质，被细菌广泛合成为渗透压保护剂。由于它保留了蛋白质和大分子复合物的功能属性，因此被视为化学分子伴侣，并已发现了许多实际应用。但是，其生物合成的机制尚未完全了解。ectoine生物合成的第二步是由1-2-2,4-二氨基丁酸酯乙酰基转移酶（EctA; EC 2.3.1.178）催化，该酶将乙酰基从乙酰基CoA转移到EctB形成的1-2,4-二氨基丁酸酯（DAB）产生N-γ-乙酰基-1,2,4-二氨基丁酸酯（N-γ-ADABA），这是一种植物蛋白合酶（EctC）的底物。在这里，我们报告了来自耐热细菌月桂枯萎病菌（P1）的EctA酶的生化和结构表征。我们发现，（Pl）EctA通过产生N-γ-ADABA而不是胞外分解代谢的中间体N-α-乙酰基-1,2,4-二氨基丁酸形成一种酶活性具有高度区域特异性的同型二聚体。（Pl）EctA（1.2-2.2Å分辨率）的高分辨率晶体结构（i）其脱辅基形式；（ii）与CoA形成复合体；（iii）与DAB形成复合体；（iv）两者都复合得到CoA和DAB，以及（v）在产物N-γ-ADABA存在下。为了查明参与DAB结合的残基，我们通过定点诱变探讨了（Pl）EctA的结构-功能关系。系统生物学研究表明，细菌和古细菌中的EctA型蛋白质在进化上都是高度保守的，包括具有催化作用的重要残基。总的来说，