The Norwegian Structural Biology Centre (NorStruct), Department of Chemistry, Faculty of Science and Technology, UiT the Arctic University of Norway, Tromsø, Norway.
FEBS J. 2020 Nov;287(21):4641-4658. doi: 10.1111/febs.15265. Epub 2020 Mar 18.
l-2,4-diaminobutyric acid (DABA) aminotransferases can catalyze the formation of amines at the distal ω-position of substrates, and is the intial and rate-limiting enzyme in the biosynthesis pathway of the cytoprotecting molecule (S)-2-methyl-1,4,5,6-tetrahydro-4-pyrimidine carboxylic acid (ectoine). Although there is an industrial interest in the biosynthesis of ectoine, the DABA aminotransferases remain poorly characterized. Herein, we present the crystal structure of EctB (2.45 Å), a DABA aminotransferase from Chromohalobacter salexigens DSM 3043, a well-studied organism with respect to osmoadaptation by ectoine biosynthesis. We investigate the enzyme's oligomeric state to show that EctB from C. salexigens is a tetramer of two functional dimers, and suggest conserved recognition sites for dimerization that also includes the characteristic gating loop that helps shape the active site of the neighboring monomer. Although ω-transaminases are known to have two binding pockets to accommodate for their dual substrate specificity, we herein provide the first description of two binding pockets in the active site that may account for the catalytic character of DABA aminotransferases. Furthermore, our biochemical data reveal that the EctB enzyme from C. salexigens is a thermostable, halotolerant enzyme with a broad pH tolerance which may be linked to its tetrameric state. Put together, this study creates a solid foundation for a deeper structural understanding of DABA aminotransferases and opening up for future downstream studies of EctB's catalytic character and its redesign as a better catalyst for ectoine biosynthesis. In summary, we believe that the EctB enzyme from C. salexigens can serve as a benchmark enzyme for characterization of DABA aminotransferases. DATABASE: Structural data are available in PDB database under the accession number 6RL5.
l-2,4-二氨基丁酸（DABA）氨基转移酶可以催化底物远端ω位的胺形成，是细胞保护分子（S）-2-甲基-1,4,5,6-四氢-4-嘧啶羧酸（章鱼胺）生物合成途径中的初始限速酶。尽管人们对章鱼胺的生物合成具有工业兴趣，但 DABA 氨基转移酶的特性仍知之甚少。在此，我们展示了 Chromohalobacter salexigens DSM 3043 的 DABA 氨基转移酶 EctB（2.45Å）的晶体结构，该菌是通过章鱼胺生物合成进行渗透适应的研究得很好的生物体。我们研究了酶的寡聚状态，表明来自 C.salexigens 的 EctB 是两个功能二聚体的四聚体，并提出了保守的二聚化识别位点，其中还包括有助于形成相邻单体活性位点的特征门控环。尽管 ω-转氨酶已知具有两个结合口袋以适应其双重底物特异性，但我们在此首次描述了活性位点中的两个结合口袋，这些口袋可能解释了 DABA 氨基转移酶的催化特性。此外，我们的生化数据表明，来自 C.salexigens 的 EctB 酶是一种耐热、耐盐的酶，具有广泛的 pH 耐受性，这可能与其四聚体状态有关。综上所述，这项研究为深入了解 DABA 氨基转移酶的结构提供了坚实的基础，并为未来研究 EctB 的催化特性及其作为更好的章鱼胺生物合成催化剂的重新设计奠定了基础。总之，我们认为 C.salexigens 的 EctB 酶可以作为 DABA 氨基转移酶特征描述的基准酶。数据库：结构数据可在 PDB 数据库中以 6RL5 的登录号获得。
