Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China; Biology Institute, Qilu University of Technology (Shandong Academy of Sciences), Key Laboratory for Drug Screening Technology of Shandong Academy of Sciences, Jinan, Shandong province, China.
Key Laboratory of Experimental Marine Biology, Institute of Oceanology, Chinese Academy of Sciences, Qingdao, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao, China.
Biochimie. 2020 Apr-May;171-172:213-222. doi: 10.1016/j.biochi.2020.03.011. Epub 2020 Mar 13.
β-Lactams are the most widely used antibiotics in treating bacterial infections. However, they are rarely applied in infections caused by Vibrio parahaemolyticus, as the bacterium is intrinsically resistant to penicillins by expressing β-lactamase. Here we report structural characterization of the CARB β-lactamase from V. parahaemolyticus (CARB-20). CARB-20 is a class A β-lactamase, belonging to subclass A1 (containing 70STFKAL75, 130SDNTAANL137, 164RXEXXLN170, 231VGDKTG236, etc.), group LSBL2 (with the disulfide bridge C77-C123, motif 231IADRSGAG238 and R244). CARB-20 adopts a typical subclass A1 β-lactamase fold consisting of two domains. Its active site is constituted by four conserved motifs, similar to that of known subclass A1 β-lactamases. Analysis of the active site structure reveals its substrate preference for penicillin, ampicillin and carbenicillin but not for latterly developed cephalosporins. Meanwhile, β-lactamase inhibitors such as clavulanate and sulbactam can well fit into the active site, supporting β-lactams combined with β-lactamase inhibitors as a potential approach for treating infection of V. parahaemolyticus. The residues around the active site show certain variations, which can be useful for specific inhibitor design. In the directed evolution experiment, CARB-20 exhibited plasticity in developing significant resistance to inhibitors by accumulated residue substitutions. Therefore, careful monitoring of enzyme mutations is necessary for successfully applying β-lactam/β-lactamase inhibitor combination therapy. Taken together, our results open up an avenue of inhibitor design targeting vibrio β-lactamases, facilitating the application of β-lactams in treating vibrio infections.
β-内酰胺类抗生素是治疗细菌感染最广泛使用的抗生素。然而，由于副溶血性弧菌本质上对青霉素具有抗性，通过表达β-内酰胺酶，它们很少用于由副溶血性弧菌引起的感染。在这里，我们报道了来自副溶血性弧菌（CARB-20）的 CARB β-内酰胺酶的结构特征。CARB-20 是一种 A 类β-内酰胺酶，属于 A1 亚类（包含 70STFKAL75、130SDNTAANL137、164RXEXXLN170、231VGDKTG236 等），LSBL2 组（具有 C77-C123 二硫键、 motif 231IADRSGAG238 和 R244）。CARB-20 采用典型的 A1 亚类β-内酰胺酶折叠结构，由两个结构域组成。其活性位点由四个保守基序组成，与已知的 A1 亚类β-内酰胺酶相似。活性位点结构分析表明，其底物对青霉素、氨苄西林和卡巴西林具有偏好性，但对后来开发的头孢菌素没有偏好性。同时，β-内酰胺酶抑制剂如克拉维酸和舒巴坦可以很好地适应活性位点，支持将β-内酰胺与β-内酰胺酶抑制剂联合使用作为治疗副溶血性弧菌感染的一种潜在方法。活性位点周围的残基显示出一定的变化，这对于特定抑制剂的设计可能有用。在定向进化实验中，CARB-20 通过积累的残基取代表现出对抑制剂产生显著抗性的可塑性。因此，成功应用β-内酰胺/β-内酰胺酶抑制剂联合治疗需要仔细监测酶突变。总的来说，我们的研究结果为设计针对弧菌β-内酰胺酶的抑制剂开辟了一条新途径，有助于β-内酰胺类抗生素在治疗弧菌感染中的应用。
