Department of Biophysics, Johns Hopkins University, 3400 North Charles Street, Baltimore, MD 21218, USA.
Proc Natl Acad Sci U S A. 2012 Feb 14;109(7):2308-13. doi: 10.1073/pnas.1113633109. Epub 2012 Jan 30.
ThnT is a pantetheine hydrolase from the DmpA/OAT superfamily involved in the biosynthesis of the β-lactam antibiotic thienamycin. We performed a structural and mechanistic investigation into the cis-autoproteolytic activation of ThnT, a process that has not previously been subject to analysis within this superfamily of enzymes. Removal of the γ-methyl of the threonine nucleophile resulted in a rate deceleration that we attribute to a reduction in the population of the reactive rotamer. This phenomenon is broadly applicable and constitutes a rationale for the evolutionary selection of threonine nucleophiles in autoproteolytic systems. Conservative substitution of the nucleophile (T282C) allowed determination of a 1.6-Å proenzyme ThnT crystal structure, which revealed a level of structural flexibility not previously observed within an autoprocessing active site. We assigned the major conformer as a nonreactive state that is unable to populate a reactive rotamer. Our analysis shows the system is activated by a structural rearrangement that places the scissile amide into an oxyanion hole and forces the nucleophilic residue into a forbidden region of Ramachandran space. We propose that conformational strain may drive autoprocessing through the destabilization of nonproductive states. Comparison of our data with previous reports uncovered evidence that many inactivated structures display nonreactive conformations. For penicillin and cephalosporin acylases, this discrepancy between structure and function may be resolved by invoking the presence of a hidden conformational state, similar to that reported here for ThnT.
ThnT 是 DmpA/OAT 超家族中的一种泛酰巯基乙胺水解酶，参与 β-内酰胺抗生素噻霉素的生物合成。我们对 ThnT 的顺式-自动蛋白水解激活进行了结构和机制研究，这一过程在该酶超家族中以前尚未进行分析。去除苏氨酸亲核基团的 γ-甲基导致反应速率降低，我们认为这是由于活性旋转体的比例减少。这种现象具有广泛的适用性，构成了在自动蛋白水解系统中选择苏氨酸亲核基团的进化选择的基础。保守取代亲核基团（T282C）允许确定一个 1.6 Å 的前酶 ThnT 晶体结构，该结构显示出在自动加工活性位点中以前未观察到的结构灵活性水平。我们将主要构象指定为非反应状态，无法进入反应旋转体。我们的分析表明，该系统通过结构重排激活，将裂解酰胺置于氧阴离子穴中，并迫使亲核残基进入 Ramachandran 空间的禁止区域。我们提出构象应变可能通过破坏非生产性状态来驱动自动加工。将我们的数据与以前的报告进行比较，发现有证据表明许多失活结构显示非反应构象。对于青霉素和头孢菌素酰基酶，结构和功能之间的这种差异可以通过引入隐藏的构象状态来解决，类似于这里报道的 ThnT。
