Department of Biochemical Sciences "A. Rossi Fanelli", Istituto Pasteur-Fondazione Cenci Bolognetti, Sapienza University of Rome, Italy.
Institute of Molecular Biology and Pathology, CNR - National Research Council of Italy, Rome, Italy.
FEBS J. 2017 Sep;284(18):2981-2999. doi: 10.1111/febs.14163. Epub 2017 Aug 7.
We determined the crystal structure of Thr1, the self-standing adenylation domain involved in the nonribosomal-like biosynthesis of free 4-chlorothreonine in Streptomyces sp. OH-5093. Thr1 shows two monomers in the crystallographic asymmetric unit with different relative orientations of the C- and N-terminal subdomains both in the presence of substrates and in the unliganded form. Cocrystallization with substrates, adenosine 5'-triphosphate and l-threonine, yielded one monomer containing the two substrates and the other in complex with l-threonine adenylate, locked in a postadenylation state. Steady-state kinetics showed that Thr1 activates l-Thr and its stereoisomers, as well as d-Ala, l- and d-Ser, albeit with lower efficiency. Modeling of these substrates in the active site highlighted the molecular bases of substrate discrimination. This work provides the first crystal structure of a threonine-activating adenylation enzyme, a contribution to the studies on conformational rearrangement in adenylation domains and on substrate recognition in nonribosomal biosynthesis.
Structural data are available in the Protein Data Bank under the accession number 5N9W and 5N9X.
我们确定了 Thr1 的晶体结构，Thr1 是一种独立的腺苷酸化结构域，参与链霉菌 OH-5093 中非核糖体样的游离 4-氯苏氨酸的生物合成。Thr1 在晶体学的不对称单位中显示出两个单体，它们的 C 端和 N 端亚结构的相对取向在有底物和无配体的形式下都不同。与底物、腺苷 5'-三磷酸和 l-苏氨酸共结晶，得到一个单体，其中包含两个底物，另一个单体与 l-苏氨酸腺苷酸复合物结合，处于后腺苷酸化状态。稳态动力学研究表明，Thr1 可激活 l-Thr 及其立体异构体，以及 d-Ala、l-和 d-Ser，但效率较低。在活性位点对这些底物进行建模，突出了底物识别的分子基础。这项工作提供了第一个苏氨酸激活的腺苷酸化酶的晶体结构，为研究腺苷酸化结构域中的构象重排和非核糖体生物合成中的底物识别做出了贡献。
结构数据可在蛋白数据库（PDB）中以 5N9W 和 5N9X 的编号获取。
