Chlorogenic acid (CGA) is a phenolic compound with well-known antibacterial properties against pathogens. In this study, structural and biochemical characterization was used to show the inhibitory role of CGA against the enzyme of the shikimate pathway, a well-characterized drug target in several pathogens. Here, we report the crystal structures of dehydroquinate synthase (DHQS), the second enzyme of the shikimate pathway, from Providencia alcalifaciens (PaDHQS), in binary complex with NAD and ternary complex with NAD and CGA. Structural analyses reveal that CGA occupies the substrate position in the active site of PaDHQS, which disables domain movements, leaving the enzyme in an open and catalysis-incompetent state. The binding analyses by isothermal titration calorimetry (ITC) and surface plasmon resonance (SPR) show that CGA binds to PaDHQS with K_D (equilibrium dissociation constant) values of 6.3 μM and 0.5 μM, respectively. In vitro enzyme inhibition studies show that CGA inhibits PaDHQS with a K_i of 235 ± 21 μM, while it inhibits the growth of Providencia alcalifaciens, Moraxella catarrhalis, Staphylococcus aureus, and Escherichia coli with MIC values of 60 to 100 μM. In the presence of aromatic amino acids supplied externally, CGA does not show the toxic effect. These results, along with the observations of the inhibition of the 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) regulatory domain by CGA in our previous study, suggest that CGA binds to shikimate pathway enzymes with high affinity and inhibits their catalysis and can be further exploited for designing novel drug-like molecules.

中文翻译：

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结构和生化分析表明绿原酸抑制莽草酸途径。


绿原酸 (CGA) 是一种酚类化合物，具有众所周知的针对病原体的抗菌特性。在本研究中，结构和生化表征用于显示 CGA 对莽草酸途径酶的抑制作用，莽草酸途径是多种病原体中已充分表征的药物靶点。在这里，我们报道了来自普罗威登斯菌( Pa DHQS) 的脱氢奎酸合酶 (DHQS) 的晶体结构，DHQS 是莽草酸途径的第二种酶，与 NAD 形成二元复合物，以及与 NAD 和 CGA 形成三元复合物。结构分析表明，CGA 占据了Pa DHQS 活性位点的底物位置，这使得结构域运动失效，使酶处于开放且无催化能力的状态。通过等温滴定量热法 (ITC) 和表面等离振子共振 (SPR) 进行的结合分析表明，CGA 与Pa DHQS 结合， K _D （平衡解离常数）值分别为 6.3 μM 和 0.5 μM。体外酶抑制研究表明，CGA 抑制Pa DHQS， K _i为 235 ± 21 μM，同时抑制普罗维登斯菌、卡他莫拉菌、金黄色葡萄球菌和大肠杆菌的生长，MIC 值为 60 至 100 μM。在外部提供的芳香族氨基酸存在下，CGA不表现出毒性作用。 这些结果，以及我们之前研究中 CGA 对 3-脱氧-d-阿拉伯-庚酮酸-7-磷酸 (DAHP) 调节结构域的抑制作用的观察结果表明，CGA 以高亲和力与莽草酸途径酶结合，并抑制它们的催化作用可以进一步用于设计新型药物分子。