Spies M Ashley, Reese Joseph G, Dodd Dylan, Pankow Katherine L, Blanke Steven R, Baudry Jerome
Department of Biochemistry, Institute for Genomic Biology, University of Illinois, Urbana, Illinois 61801, USA.
J Am Chem Soc. 2009 Apr 15;131(14):5274-84. doi: 10.1021/ja809660g.
Glutamate racemases (EC 5.1.1.3) catalyze the cofactor-independent stereoinversion of D- and L-glutamate and are important for viability in several gram-negative and -positive bacteria. As the only enzyme involved in the stereoinversion of L- to D-glutamate for peptidoglycan biosynthesis, glutamate racemase is an attractive target for the design of antibacterial agents. However, the development of competitive tight-binding inhibitors has been problematic and highly species specific. Despite a number of recent crystal structures of cofactor-independent epimerases and racemases, cocrystallized with substrates or substrate analogues, the source of these enzymes' catalytic power and their ability to acidify the C alpha of amino acids remains unknown. The present integrated computational and experimental study focuses on the glutamate racemase from Bacillus subtilis (RacE). A particular focus is placed on the interaction of the glutamate carbanion intermediate with RacE. Results suggest that the reactive form of the RacE-glutamate carbanion complex, vis-à-vis proton abstraction from C alpha, is significantly different than the RacE-D-glutamate complex on the basis of the crystal structure and possesses dramatically stronger enzyme-ligand interaction energy. In silico and experimental site-directed mutagenesis indicates that the strength of the RacE-glutamate carbanion interaction energy is highly distributed among numerous electrostatic interactions in the active site, rather than being dominated by strong hydrogen bonds. Results from this study are important for laying the groundwork for discovery and design of high-affinity ligands to this class of cofactor-independent racemases.
谷氨酸消旋酶（EC 5.1.1.3）催化D-和L-谷氨酸的无辅因子立体异构化，对多种革兰氏阴性菌和革兰氏阳性菌的生存能力至关重要。作为肽聚糖生物合成中唯一参与L-谷氨酸向D-谷氨酸立体异构化的酶，谷氨酸消旋酶是抗菌剂设计的一个有吸引力的靶点。然而，竞争性紧密结合抑制剂的开发一直存在问题，且具有高度的物种特异性。尽管最近有许多无辅因子差向异构酶和消旋酶与底物或底物类似物共结晶的晶体结构，但这些酶的催化能力来源及其酸化氨基酸α-碳的能力仍然未知。目前的综合计算和实验研究聚焦于枯草芽孢杆菌的谷氨酸消旋酶（RacE）。特别关注谷氨酸碳负离子中间体与RacE的相互作用。结果表明，基于晶体结构，RacE-谷氨酸碳负离子复合物相对于从α-碳夺取质子的反应形式与RacE-D-谷氨酸复合物有显著不同，并且具有明显更强的酶-配体相互作用能。计算机模拟和实验定点诱变表明，RacE-谷氨酸碳负离子相互作用能的强度高度分布在活性位点的众多静电相互作用中，而非由强氢键主导。这项研究的结果对于为发现和设计针对这类无辅因子消旋酶高亲和力配体奠定基础很重要。
