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Pseudomonas aeruginosa MurE amide ligase : enzyme kinetics and peptide inhibitor
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Paradis-Bleau, Catherine, Lloyd, Adrian, Sanschagrin, Francois, Maaroufi, Halim, Clarke, Thomas B., Blewett, Ann, Dowson, Christopher G., Roper, David I., Bugg, Tim and Levesque, Roger C. (2009) Pseudomonas aeruginosa MurE amide ligase : enzyme kinetics and peptide inhibitor. Biochemical Journal, Vol.421 (No. 2). pp. 263-272. doi:10.1042/BJ20081395 ISSN 0264-6021.
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Official URL: http://dx.doi.org/10.1042/BJ20081395
Abstract
The enzyme kinetics of the amide ligase MurE, a cell wall biosynthesis enzyme, from Pseudomonas aeruginosa were determined using the synthesized nucleotide substrate UDP-MurNAc-Ala-Glu (uridine 5'-diphosphoryl N-acetylmuramoyl-L- alanyl-D-glutamate). When Coupled to a competitive bio-panning technique using a M 13 phage display library encoding similar to 2.7 x 10(9) random peptide permutations and the specific Substrates meso-A2pm (meso-diaminopimelic acid) and ATP, a peptide inhibitor of MurE was identified. The MurEp1 dodecamer selected and synthesized inhibited MurE ATPase activity with all IC50 value of 500 mu M. The inhibition was shown to be time-dependent and was reversed by the addition of meso-A2pm or UDP-MurNAc-Ala-Glu during the pre-incubation step. Kinetic analysis defined MurEp 1 as a mixed inhibitor against both substrates with K-i values of 160 and 80 mu M respectively. MurEp 1 was found to interfere in meso-A2pm and UDP-MurNAc-Ala-Glu binding necessary for amide bond formation. Modelling of Ps. aeruginosa MurE and docking of MurEp 1 oil the Ps. aeruginosa MurE surface indicated that MurEp I binds at the juxtaposition of both meso-A2pm- and UDF-MurNAc-Ala-Glu-binding sites ill the closed conformational state of the enzyme. Identification of the MurEp 1 residues involved ill MurE binding and inhibition will allow the development of a novel class of: inhibitors having a novel mode of action against MurE.
| Item Type: | Journal Article | ||||
|---|---|---|---|---|---|
| Subjects: | Q Science > QD Chemistry | ||||
| Divisions: | Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- ) > Biological Sciences ( -2010) Faculty of Science, Engineering and Medicine > Science > Chemistry |
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| Journal or Publication Title: | Biochemical Journal | ||||
| Publisher: | Portland Press | ||||
| ISSN: | 0264-6021 | ||||
| Official Date: | 15 July 2009 | ||||
| Dates: |
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| Volume: | Vol.421 | ||||
| Number: | No. 2 | ||||
| Number of Pages: | 10 | ||||
| Page Range: | pp. 263-272 | ||||
| DOI: | 10.1042/BJ20081395 | ||||
| Status: | Peer Reviewed | ||||
| Publication Status: | Published | ||||
| Access rights to Published version: | Restricted or Subscription Access | ||||
| Funder: | Quebecois de la Recherche sur la Nature et les Technologies, Natural Sciences and Engineering Research Council of Canada | ||||
| Grant number: | FQRNT:FT085722, NSERC:341729 |
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