Neckles Carla, Pschibul Annica, Lai Cheng-Tsung, Hirschbeck Maria, Kuper Jochen, Davoodi Shabnam, Zou Junjie, Liu Nina, Pan Pan, Shah Sonam, Daryaee Fereidoon, Bommineni Gopal R, Lai Cristina, Simmerling Carlos, Kisker Caroline, Tonge Peter J
Rudolf Virchow Center for Experimental Biomedicine, Institute for Structural Biology, University of Würzburg , D-97080 Würzburg, Germany.
William A. Shine Great Neck South High School , Great Neck, New York 11020, United States.
Biochemistry. 2016 May 31;55(21):2992-3006. doi: 10.1021/acs.biochem.5b01301. Epub 2016 May 17.
The enoyl-ACP reductase (ENR) catalyzes the last reaction in the elongation cycle of the bacterial type II fatty acid biosynthesis (FAS-II) pathway. While the FabI ENR is a well-validated drug target in organisms such as Mycobacterium tuberculosis and Staphylococcus aureus, alternate ENR isoforms have been discovered in other pathogens, including the FabV enzyme that is the sole ENR in Yersinia pestis (ypFabV). Previously, we showed that the prototypical ENR inhibitor triclosan was a poor inhibitor of ypFabV and that inhibitors based on the 2-pyridone scaffold were more potent [Hirschbeck, M. (2012) Structure 20 (1), 89-100]. These studies were performed with the T276S FabV variant. In the work presented here, we describe a detailed examination of the mechanism and inhibition of wild-type ypFabV and the T276S variant. The T276S mutation significantly reduces the affinity of diphenyl ether inhibitors for ypFabV (20-fold → 100-fold). In addition, while T276S ypFabV generally displays an affinity for 2-pyridone inhibitors higher than that of the wild-type enzyme, the 4-pyridone scaffold yields compounds with similar affinity for both wild-type and T276S ypFabV. T276 is located at the N-terminus of the helical substrate-binding loop, and structural studies coupled with site-directed mutagenesis reveal that alterations in this residue modulate the size of the active site portal. Subsequently, we were able to probe the mechanism of time-dependent inhibition in this enzyme family by extending the inhibition studies to include P142W ypFabV, a mutation that results in a gain of slow-onset inhibition for the 4-pyridone PT156.
烯脂酰-ACP还原酶（ENR）催化细菌II型脂肪酸生物合成（FAS-II）途径延长循环中的最后一步反应。虽然FabI ENR是结核分枝杆菌和金黄色葡萄球菌等生物体中经过充分验证的药物靶点，但在其他病原体中也发现了替代的ENR同工型，包括鼠疫耶尔森菌（ypFabV）中唯一的ENR——FabV酶。此前，我们发现典型的ENR抑制剂三氯生对ypFabV的抑制作用较差，而基于2-吡啶酮支架的抑制剂更有效[赫施贝克，M.（2012年）《结构》20（1），89 - 100]。这些研究是针对T276S FabV变体进行的。在本文介绍的工作中，我们详细研究了野生型ypFabV和T276S变体的作用机制及抑制情况。T276S突变显著降低了二苯醚抑制剂对ypFabV的亲和力（从20倍降至100倍）。此外，虽然T276S ypFabV对2-吡啶酮抑制剂的亲和力通常高于野生型酶，但4-吡啶酮支架产生的化合物对野生型和T276S ypFabV的亲和力相似。T276位于螺旋底物结合环的N端，结构研究结合定点诱变表明，该残基的改变会调节活性位点入口的大小。随后，我们通过将抑制研究扩展到包括P142W ypFabV，探究了该酶家族中时间依赖性抑制的机制，P142W ypFabV突变导致对4-吡啶酮PT156产生缓慢起效的抑制作用。
