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

被引:23
作者
Paradis-Bleau, Catherine
Beaumont, Melanie
Boudreault, Lydia
Lloyd, Adrian
Sanschagrin, Francois
Bugg, Timothy D. H.
Leuesque, Roger C. [1 ]
机构
[1] Univ Laval, Fac Med, Dept Med Biol, CREFSIP, Ste Foy, PQ G1K 7P4, Canada
[2] Univ Warwick, Dept Chem, Coventry CV4 7AL, W Midlands, England
关键词
Pseudomonas aeruginosa; MurD; UDP-N-acetylmuramyl-L-alanine; D-glutamate ligase; phage display; inhibitory peptides;
D O I
10.1016/j.peptides.2006.01.017
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 UDPN-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 mu 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. (c) 2006 Elsevier Inc. All rights reserved.
引用
收藏
页码:1693 / 1700
页数:8
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