Phospholipid biosynthesis begins with the acylation of glycerol 3-phosphate (G3P). In most Gram-positive bacteria including many pathogens, a membrane protein called PlsY is the only acyltransferase that catalyzes this essential step, making it a potential target for the development of antibiotics. A convenient enzymatic assay should facilitate such drug discovery activities. Previously, we developed a continuous assay by monitoring phosphate, one of the enzymatic product, using a fluorescently labeled phosphate binding protein in a bilayer environment called lipid cubic phase (LCP). However, some intrinsic characteristics of LCP, such as high viscosity, make the assay incompatible with common high-throughput liquid-handling platforms. Here, we adapted the assay by hosting PlsY in detergent micelles, enabling us to conduct the assay using standard multi-channel pipets in a high-throughput manner. With optimal enzyme loading, the reaction velocity was linear up to 30 min. PlsY showed Michaelis-Menten kinetics behavior in micelles with a Vmax of 57.5 μmol min-1 mg-1, and Km of 1.14 mM G3P and 6.2 μM acyl phosphate. The inhibitory product lysophosphatidic acid inhibited PlsY with the IC50 of 19 μM. The results principally demonstrated the feasibility of using the assay for high-throughput screening, and the protocol provided an encouraging starting point for further optimization and validation of the assay for automated platforms.

中文翻译：

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为整体膜甘油3-磷酸酰基转移酶开发高通量检测方法。

磷脂的生物合成从甘油3-磷酸（G3P）的酰化开始。在包括许多病原体在内的大多数革兰氏阳性细菌中，称为PlsY的膜蛋白是唯一催化这一重要步骤的酰基转移酶，使其成为开发抗生素的潜在目标。方便的酶法测定应促进此类药物发现活动。以前，我们通过在称为脂质立方相（LCP）的双层环境中使用荧光标记的磷酸盐结合蛋白来监测磷酸盐（一种酶促产物）来开发连续测定法。但是，LCP的某些固有特性（例如高粘度）使该测定与常见的高通量液体处理平台不兼容。在这里，我们通过将PlsY容纳在去污剂胶束中来适应该测定，使我们能够以高通量方式使用标准多通道移液器进行测定。在最佳酶负载下，反应速度在30分钟内呈线性。PlsY在Vmax为57.5μmolmin-1 mg-1，Km为1.14 mM G3P和6.2μM酰基磷酸酯的胶束中显示Michaelis-Menten动力学行为。抑制产物溶血磷脂酸抑制PlsY，IC50为19μM。结果主要证明了使用该测定进行高通量筛选的可行性，并且该方案为进一步优化和验证自动化平台的测定提供了令人鼓舞的起点。5μmolmin-1 mg-1，Km为1.14 mM G3P和6.2μM酰基磷酸酯。抑制产物溶血磷脂酸抑制PlsY，IC50为19μM。结果主要证明了使用该测定进行高通量筛选的可行性，并且该方案为进一步优化和验证自动化平台的测定提供了令人鼓舞的起点。5μmolmin-1 mg-1，Km为1.14 mM G3P和6.2μM酰基磷酸酯。抑制产物溶血磷脂酸抑制PlsY，IC50为19μM。结果主要证明了使用该测定进行高通量筛选的可行性，并且该方案为进一步优化和验证自动化平台的测定提供了令人鼓舞的起点。