Department of Chemistry, Washington State University, Pullman, WA 99164, USA.
Paul G Allen School for Global Health, Washington State University, Pullman, WA 99164, USA.
Int J Mol Sci. 2022 May 7;23(9):5229. doi: 10.3390/ijms23095229.
Cefotaximase-Munich (CTX-M) extended-spectrum beta-lactamases (ESBLs) are commonly associated with Gram-negative, hospital-acquired infections worldwide. Several beta-lactamase inhibitors, such as clavulanate, are used to inhibit the activity of these enzymes. To understand the mechanism of CTX-M-15 activity, we have determined the crystal structures of CTX-M-15 in complex with two specific classes of beta-lactam compounds, desfuroylceftiofur (DFC) and ampicillin, and an inhibitor, clavulanic acid. The crystal structures revealed that Ser70 and five other residues (Lys73, Tyr105, Glu166, Ser130, and Ser237) participate in catalysis and binding of those compounds. Based on analysis of steady-state kinetics, thermodynamic data, and molecular docking to both wild-type and S70A mutant structures, we determined that CTX-M-15 has a similar affinity for all beta-lactam compounds (ceftiofur, nitrocefin, DFC, and ampicillin), but with lower affinity for clavulanic acid. A catalytic mechanism for tested β-lactams and two-step inhibition mechanism of clavulanic acid were proposed. CTX-M-15 showed a higher activity toward DFC and nitrocefin, but significantly lower activity toward ampicillin and ceftiofur. The interaction between CTX-M-15 and both ampicillin and ceftiofur displayed a higher entropic but lower enthalpic effect, compared with DFC and nitrocefin. DFC, a metabolite of ceftiofur, displayed lower entropy and higher enthalpy than ceftiofur. This finding suggests that compounds containing amine moiety (e.g., ampicillin) and the furfural moiety (e.g., ceftiofur) could hinder the hydrolytic activity of CTX-M-15.
头孢噻肟酶-慕尼黑（CTX-M）扩展谱β-内酰胺酶（ESBLs）通常与全球范围内的革兰氏阴性、医院获得性感染相关。几种β-内酰胺酶抑制剂，如克拉维酸，被用于抑制这些酶的活性。为了了解 CTX-M-15 的活性机制，我们已经确定了 CTX-M-15 与两种特定类别的β-内酰胺化合物（去呋喃基头孢噻肟（DFC）和氨苄西林）以及抑制剂克拉维酸复合物的晶体结构。晶体结构表明，Ser70 和其他五个残基（Lys73、Tyr105、Glu166、Ser130 和 Ser237）参与了这些化合物的催化和结合。基于对稳态动力学、热力学数据和对野生型和 S70A 突变体结构的分子对接的分析，我们确定 CTX-M-15 对所有β-内酰胺化合物（头孢噻肟、硝基头孢菌素、DFC 和氨苄西林）具有相似的亲和力，但对克拉维酸的亲和力较低。提出了测试的β-内酰胺的催化机制和克拉维酸的两步抑制机制。CTX-M-15 对 DFC 和硝基头孢菌素显示出更高的活性，但对氨苄西林和头孢噻肟的活性明显较低。与 DFC 和硝基头孢菌素相比，CTX-M-15 与氨苄西林和头孢噻肟的相互作用显示出更高的熵但更低的焓效应。DFC 是头孢噻肟的代谢物，其熵比头孢噻肟低，但焓比头孢噻肟高。这一发现表明，含有胺基部分（如氨苄西林）和呋喃基部分（如头孢噻肟）的化合物可能会阻碍 CTX-M-15 的水解活性。
