Molina-López José, Sanschagrin François, Levesque Roger C
Departamento de Salud Pública, Facultad de Medicina, Universidad Nacional Autónoma de México, Mexico.
Peptides. 2006 Dec;27(12):3115-21. doi: 10.1016/j.peptides.2006.08.023. Epub 2006 Oct 9.
The MurA enzyme from Pseudomonas aeruginosa was purified to homogeneity and found to be biologically active as a UDP-N-acetylglucosamine (UNAG) enolpyruvyl transferase in a coupled enzyme assay where ATPase activity was measured by the release of inorganic phosphate. A microtiter plate assay coupled to competitive biopanning using the UDP-N-acetylglucosamine was used to screen 10(9) C-7-C and 12-mers peptides from phage display libraries. From 60 phage-encoded peptides identified after the fourth round of biopanning, deduced amino acid sequences were aligned and two peptides were synthesized and tested for inhibition of the MurA-catalyzed reaction. The PEP 1354 peptide inhibited the ATPase activity of MurA with an IC(50) value of 200muM and was found to be a competitive inhibitor of UNAG. The pre-incubation of MurA with inhibitor indicated a time-independent inhibition. This time-dependent inhibition is the first report of peptide inhibitors of MurA, which represent the scaffold for the synthesis of inhibitory peptidomimetic molecules.
铜绿假单胞菌的MurA酶被纯化至同质，并在一种偶联酶测定中被发现作为UDP-N-乙酰葡糖胺（UNAG）烯醇丙酮酸转移酶具有生物活性，在该测定中通过无机磷酸盐的释放来测量ATP酶活性。使用与UDP-N-乙酰葡糖胺偶联的竞争性生物淘选的微量滴定板测定法，从噬菌体展示文库中筛选了10⁹个C-7-C和12聚体肽。在第四轮生物淘选后鉴定出的60个噬菌体编码肽中，对推导的氨基酸序列进行比对，并合成了两个肽并测试其对MurA催化反应的抑制作用。PEP 1354肽以200μM的IC₅₀值抑制MurA的ATP酶活性，并被发现是UNAG的竞争性抑制剂。MurA与抑制剂的预孵育表明存在非时间依赖性抑制。这种时间依赖性抑制是MurA肽抑制剂的首次报道，这些抑制剂代表了合成抑制性拟肽分子的支架。
