Selection of peptide inhibitors against the Pseudomonas aeruginosa MurD cell wall enzyme

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• 作者：Catherine Paradis-Bleau ; Mé ; lanie Beaumont ; Lydia Boudreault ; Adrian Lloyd ; Franç ; ois Sanschagrin ; Timothy D.H. Bugg and Roger C. Levesque
• 关键词：Pseudomonas aeruginosa ; MurD ; UDP-N-acetylmuramyl-class=""smCaps"">l-alanine ; class=""smCaps"">d-glutamate ligase ; Phage display ; Inhibitory peptides
• 刊名：Peptides
• 出版年：2006
• 出版时间：July 2006
• 年：2006
• 卷：27
• 期：7
• 页码：1693-1700
• 全文大小：453 K

文摘

The purified Pseudomonas aeruginosa cell wall biosynthesis MurD amide ligase enzyme was used to screen C-7-C and 12 mers peptides from phage display libraries using competitive biopanning approaches with the specific substrates d-glutamate and ATP. From the 60 phage-encoded peptides identified, DNA was sequenced, deduced amino acid sequences aligned and two peptides were synthesized from consensus sequences identified. The UDP-N-acetylmuramyl-l-alanine MurD substrate was synthesized, purified and used to develop a spectrophotometric assay. One peptide synthesized was found to specifically inhibit ATPase activity of MurD. The IC₅₀ value was estimated at 4 ;c;M for the C-7-C MurDp1 peptide. The loop conformation of MurDp1 was shown to be important for the inhibition of the UDP-N-acetylmuramyl-l-alanine:d-glutamate MurD ligase. The linear 12 mers MurD2 peptide has an IC₅₀ value of 15 mM. A conserved amino acid motif was found between MurDp2 and the bacterial glyceraldehyde 3-phosphate dehydrogenase indicating that MurDp2 binds at a protein–protein interacting site. The approach proposed and results obtained suggest that efficient peptide inhibitors as well as protein–protein interaction domains can be identified by phage display.