Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.
Species differences in alternative substrate utilization by the antibacterial target undecaprenyl pyrophosphate synthase.
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DOI：
10.1021/bi500545g
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发表时间：
2014-08-05
期刊：
影响因子：
2.9
通讯作者：
Troutman, Jerry M.
中科院分区：
文献类型：
--
作者：
Dodbele, Samantha;Martinez, Christina D.;Troutman, Jerry M.
十一碳烯基焦磷酸合成酶(UPPS)是细菌生存所必需的多糖生物合成所必需的关键酶。在这份报告中，我们测试了来自哺乳动物共生菌脆弱类杆菌、人类病原体创伤弧菌和典型的良性但有机会的病原体大肠杆菌的UPPS的底物选择性。含有邻氨基苯胺二磷酸的底物2-氨基苯丙二磷酸(2AA-GPP)只是脆弱芽孢杆菌UPPS蛋白的有效底物，但用腈[2-氰基苯丙二磷酸(2CNA-GPP)]取代酰胺得到了一种完全具有UPPS功能的化合物，来自所有三种目标生物。这些荧光底物类似物还被发现在类异戊二烯链延长时经历荧光增加，这种荧光增加可用于在96孔板分析中监测UPPS的活性和抑制。随着链的延长，2CNA-GPP的荧光强度增加了2.5倍，而2AA-GPP的荧光强度仅增加了1.2倍。因此，2CNA-GPP化合物在筛选多个细菌物种的UPPS活性方面更加通用，并且经历了更大的荧光增加，从而提高了其检测链长增加的能力。总之，这项工作描述了UPPS新的检测方法的发展，并展示了不同物种UPPS之间底物利用的差异，这对UPPS抑制剂的开发、检测方法的构建和多糖生物合成探针的开发具有重要意义。
Undecaprenyl pyrophosphate synthase (UPPS) is a critical enzyme required for the biosynthesis of polysaccharides essential for bacterial survival. In this report, we have tested the substrate selectivity of UPPS derived from the mammalian symbiont Bacteroides fragilis, the human pathogen Vibrio vulnificus, and the typically benign but opportunistic pathogen Escherichia coli. An anthranilamide-containing substrate, 2-amideanilinogeranyl diphosphate (2AA-GPP), was an effective substrate for only the B. fragilis UPPS protein, yet replacing the amide with a nitrile [2-nitrileanilinogeranyl diphosphate (2CNA-GPP)] led to a compound that was fully functional for UPPS from all three target organisms. These fluorescent substrate analogues were also found to undergo increases in fluorescence upon isoprenoid chain elongation, and this increase in fluorescence can be utilized to monitor the activity and inhibition of UPPS in 96-well plate assays. The fluorescence of 2CNA-GPP increased by a factor of 2.5-fold upon chain elongation, while that of 2AA-GPP increased only 1.2-fold. The 2CNA-GPP compound was therefore more versatile for screening the activity of UPPS from multiple species of bacteria and underwent a larger increase in fluorescence that improved its ability to detect increases in chain length. Overall, this work describes the development of new assay methods for UPPS and demonstrates the difference in substrate utilization between forms of UPPS from different species, which has major implications for UPPS inhibitor development, assay construction, and the development of polysaccharide biosynthesis probes.
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