Assay for peptidoglycan O-acetyltransferase: A potential new antibacterial target
Assay for peptidoglycan O-acetyltransferase: A potential new antibacterial target
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DOI：
10.1016/j.ab.2013.04.022
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发表时间：
2013-08-15
影响因子：
2.9
通讯作者：
Clarke, Anthony J.
中科院分区：
文献类型：
--
作者：
Moynihan, Patrick J.;Clarke, Anthony J.
在许多革兰氏阳性和革兰氏阴性的人类病原体中，肽聚糖的o -乙酰化发生在muramoyl残基的C-6羟基上，如金黄色葡萄球菌和各种弯曲杆菌、幽门螺杆菌、奈瑟菌和芽孢杆菌，包括炭疽芽孢杆菌。该过程是由革兰氏阳性细菌的整体膜o-乙酰肽聚糖转移酶（Oat）或革兰氏阴性细胞的双组分肽聚糖o-乙酰转移酶系统（PatA/PatB）催化的成熟事件。在这里，我们描述了使用淋病奈瑟菌pathb作为模型酶的任何肽聚糖o -乙酰转移酶的第一个体外测定的发展。该试验是基于使用显色对硝基苯醋酸酯作为供体底物和壳寡糖作为模型受体底物代替肽聚糖。采用基质辅助激光解吸/电离飞行时间质谱法对o -乙酰化壳寡糖进行鉴定。考虑到醋酸供体的自发和酶催化水解，通过监测对硝基苯酚的释放，分光光度法测定了转乙酰化的速率。建立了以微滴板形式使用该方法的条件，并通过测定pathb的第一Michaelis-Menten动力学参数证明了其适用性。该试验易于应用于高通量筛选潜在的肽聚糖o -乙酰转移酶抑制剂，这些抑制剂可能被证明是新型抗生素的先导。(C) 2013爱思唯尔公司版权所有。
The O-acetylation of peptidoglycan occurs at the C-6 hydroxyl group of muramoyl residues in many human pathogens, both gram positive and gram negative, such as Staphylococcus aureus and species of Campylobacter, Helicobacter, Neisseria, and Bacillus, including Bacillus anthracis. The process is a maturation event being catalyzed either by integral membrane O-acetylpeptidoglycan transferase (Oat) of gram-positive bacteria or by a two-component peptidoglycan O-acetyltransferase system (PatA/PatB) in gram-negative cells. Here, we describe the development of the first in vitro assay for any peptidoglycan O-acetyltransferase using PatB from Neisseria gonorrhoeae as the model enzyme. This assay is based on the use of chromogenic p-nitrophenyl acetate as the donor substrate and chitooligosaccharides as model acceptor substrates in place of peptidoglycan. The identity of the O-acetylated chitooligosaccharides was confirmed by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Rates of transacetylations were determined spectrophotometrically by monitoring p-nitrophenol release after accounting for both spontaneous and enzyme-catalyzed hydrolysis of the acetate donor. Conditions were established for use of the assay in microtiter plate format, and its applicability was demonstrated by determining the first Michaelis-Menten kinetic parameters for PatB. The assay is readily amenable for application in the high-throughput screening for potential inhibitors of peptidoglycan O-acetyltransferases that may prove to be leads for novel classes of antibiotics. (C) 2013 Elsevier Inc. All rights reserved.