Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.492524
Title: Molecular Studies on tRNA-Dependent Ligase MurN from Penicillin-Resistant S. pneumoniae
Author: De Pascale, Gianfranco
ISNI:       0000 0001 3420 5686
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2007
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Abstract:
StreptocoCClIS pnell1110niae is a common cause of invasive disease and respiratory-tract infections. High-level penicillin resistant S. pnell1110niae were found to produce abnormal branched structured muropeptides carrying L-Ala-L-Ala or L-Ser-L-Ala cross-link. MurM and MurN are tRNA-dependent ligases that catalyse the addition of the first (L-AlaiL-Ser) and second (L-Ala) amino acid into the branched-structured cell wall. This work has developed a chemo-enzymatic route to synthesize MurM and MurN substrates. A single incubation containing MurA-F results in the synthesis of ~85 mg of UDP-MurNAc-pentapeptide. This was then converted to lipid II (MurM substrate) using MicrococclIS j!avlIs membranes, undecaprenyl phosphate and UDP-GlcNac. In order to synthesize the branched lipid II (MurN substrate), one alanine or serine was attached to the e-NH2 of the lysine in position 3 of the UDPMurNAc- pentapeptide via a carbodiimide coupling reaction in aqueous solution. Subsequently the UDP-MurNAc-hexapeptide was converted to the lipid-linked precursor. The llwrN genes from S. pnellll10niae penicillin-resistant (159) and sensitive (Pn16) were cloned and expressed as MBP fusion proteins. The activity of the purified MurN 159 and Pn16 was measured using a radiochemical assay. The specific activity of MurN 159 was 11.0 and 0.7 nmol min-I mg-I of MurN versus lipid II-L-Ala and lipid II-L-Ser respectively. Similar values were measured for MurN Pn16. The kinetic parameters were also measured for MurN 159 and Pn16 versus both lipid-linked substrates, showing a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-L-Ser. These results concur with the peptidoglycan analysis of S. pnelll1loniae, in which the major cross-linked species in the peptidoglycan is lipid II-L-Ala-L-Ala. The MurN data also agrees with the in vitro results obtained with MurM that has higher catalytic efficiency for alanine compared to serine.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.492524  DOI: Not available
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