A high-throughput solid-phase extraction assay capable of measuring diverse polyprenyl transferases as exemplified by the phosphate: sugar-1-phosphate WecA, MraY, and MurG proteins
A high-throughput solid-phase extraction assay capable of measuring diverse polyprenyl transferases as exemplified by the phosphate: sugar-1-phosphate WecA, MraY, and MurG proteins
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DOI：
10.1016/s0003-2697(03)00088-5
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发表时间：
2003-06-15
影响因子：
2.9
通讯作者：
Anderson, MS
中科院分区：
文献类型：
--
作者：
Hyland, SA;Anderson, MS
细菌蛋白 WecA 和 MraY 是聚异戊二烯磷酸酯：N-乙酰己糖胺-1-磷酸转移酶家族的成员，它们各自催化特定的己糖胺 1-P 从可溶性 UDP-己糖胺底物转移到膜表面的细菌异戊二烯磷酸酯载体。目前，旨在定量这些酶活性的测定依赖于纸色谱或液液萃取，或者专门针对该家族的少数成员。我们描述了一种针对这些活动的通用、高通量、一锅测定法，该测定法使用基于固液珠的分离系统来选择性地吸附高度疏水性的反应产物。通过明智地选择放射性标记的 UDP-己糖胺前体，相同的格式不仅可以用于定量该转移酶家族的不同成员，还可以用于定量催化这些转移酶产物进一步修饰的酶。这种可能性的例子是细菌细胞壁合成的 MurG 蛋白，它催化 N-乙酰氨基葡萄糖残基添加到 MraY 反应的产物中。因此，使用这种灵活的检测工具将允许在类似的环境中对许多此类膜结合转移酶进行关键的生化和酶学分析。 (C) 2003 年爱思唯尔科学（美国）。版权所有。
The bacterial proteins WecA and MraY are members of the polyprenyl phosphate:N-acetylhexosamine-1-phosphate transferase family, each of which catalyzes the transfer of a specific hexosamine 1-P from a soluble UDP-hexosamine substrate to a bactoprenyl phosphate carrier at the membrane surface. Currently, assays designed to quantitate the activity of these enzymes rely on paper chromatography or liquid-liquid extractions or are specialized to a few members of the family. We describe a generalizable, high-throughput, one-pot assay for these activities that uses a solid-liquid bead-based separation system to selectively adsorb the highly hydrophobic products of reaction. By judicious choice of radiolabeled UDP-hexosamine precursor, the same format can be used to quantitate not only diverse members of this transferase family, but also enzymes that catalyze the further modification of these transferase products. This possibility is exemplified by the MurG protein of bacterial cell wall synthesis, which catalyzes the addition of an N-acetylglucosamine residue to the product of the MraY reaction. Thus, the use of this flexible assay tool will allow a critical biochemical and enzymologic analysis of many such membrane-bound transferases in a similar setting. (C) 2003 Elsevier Science (USA). All rights reserved.