casseliflavus肠球菌SSK中vanC2 d-Ala-d-Ser连接酶的过表达、纯化、生化特性及恶二唑衍生物对其的抑制作用

IF 4.3 3区 化学 Q2 CHEMISTRY, MULTIDISCIPLINARY
Sneha B. Paymal, Sagar S. Barale, Shirishkumar V. Supanekar*, Kailas D. Sonawane* and Kiran D. Pawar*, 
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引用次数: 0

摘要

细菌细胞壁和参与肽聚糖生物合成的酶是发现新型抗菌剂的主要目标。在这些酶中,d-丙氨酸-d-丙氨酸连接酶(Ddl)尤其重要,因为它们利用特定底物(d-氨基酸)对细菌的存活至关重要。在耐万古霉素的革兰氏阳性细菌中发现了利用替代底物(如 d-乳酸或 d-丝氨酸)的 Ddl 等位酶,最初是在肠球菌中发现的,现在已成为临床环境中日益严重的问题。在本研究中,我们分离并鉴定了耐万古霉素的卡氏肠球菌(E. casseliflavus)菌株 SSK,并利用它扩增、克隆和纯化了 vanC2 型 d-丙氨酸-d-丝氨酸连接酶(EcfDdls)。对底物特异性和酶动力学的研究有助于深入了解该酶的作用机理。使用无机磷酸盐检测法评估了之前几乎已筛选过的噁二唑衍生物 1-[(5-甲基-1,2-恶唑-3-基)甲基]-4-{[3-(丙-2-基)-1,2,4-恶二唑-5-基]甲基}哌嗪(CID 45805715)的抑制潜力,结果表明该衍生物对纯化的 EcfDdls 具有完全的酶抑制作用。在测试中,CID 45805715 显著抑制了 Ddl 的活性,其 IC50 值为 76.7 μM,而参考化合物 DCS 的 IC50 值为 313 μM。此外,该化合物还对耐万古霉素的卡氏伊红菌株 SSK 具有抗菌活性。因此,这些研究结果为了解 vanC2 EcfDdls 的活性和抑制作用提供了有价值的见解,为解决肠球菌对万古霉素的耐药性问题,尤其是影响免疫力低下患者的院内感染问题,提供了一种前景广阔的途径。
本文章由计算机程序翻译,如有差异,请以英文原文为准。
Overexpression, Purification, and Biochemical Characterization of the vanC2 d-Ala-d-Ser Ligase from Enterococcus casseliflavus SSK and Its Inhibition by an Oxadiazole Derivative

The bacterial cell wall and enzymes involved in peptidoglycan biosynthesis are prime targets for the discovery of novel antibacterial agents. Among these enzymes, d-alanine-d-alanine ligases (Ddl) are particularly significant due to their utilization of specific substrates (d-amino acids) essential for bacterial viability. Isozymes of Ddl that utilize alternative substrates such as d-lactate or d-serine are found in vancomycin-resistant Gram-positive bacteria, initially identified in Enterococcus species, and now represent a growing concern in clinical settings. In this study, we isolated and identified vancomycin-resistant Enterococcus casseliflavus (E. casseliflavus) strain SSK and used it for amplification, cloning, and purification of the vanC2 type of d-alanine-d-serine ligase (EcfDdls). Investigations of substrate specificity and enzyme kinetics provided insights into the enzyme’s mechanistic action. Evaluation of the inhibitory potential of the previously virtually screened oxadiazole derivative 1-[(5-methyl-1,2-oxazol-3-yl)methyl]-4-{[3-(propan-2-yl)-1,2,4-oxadiazol-5-yl]methyl}piperazine (CID 45805715) was carried out using an inorganic phosphate detection assay, which demonstrated complete enzymatic inhibition of purified EcfDdls. When tested, CID 45805715 significantly inhibited activity of Ddl, with an IC50 of 76.7 μM, compared to 313 μM for the reference compound DCS. Moreover, this compound also exhibited antimicrobial activity against vancomycin-resistant E. casseliflavus strain SSK. Thus, these findings provide valuable insights into the activity and inhibition of vanC2 EcfDdls, offering a promising avenue for addressing vancomycin resistance in enterococci, particularly in nosocomial infections affecting immunocompromised patients.

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来源期刊
ACS Omega
ACS Omega Chemical Engineering-General Chemical Engineering
CiteScore
6.60
自引率
4.90%
发文量
3945
审稿时长
2.4 months
期刊介绍: ACS Omega is an open-access global publication for scientific articles that describe new findings in chemistry and interfacing areas of science, without any perceived evaluation of immediate impact.
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