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Title: Structural enzymology of peptidoglycan biosynthetic D-amino acid dipeptide ligases
Author: Batson, Sarah
ISNI:       0000 0004 2690 2766
Awarding Body: University of Warwick
Current Institution: University of Warwick
Date of Award: 2010
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This thesis describes approaches to further understand the structure and enzymology of D-Ala-D-Ala ligases (DDL) and those ligases with altered second substrate specificity, which confer glycopeptide antibiotic resistance. DDL is an essential enzyme in the biosynthetic pathway of the bacterial cell wall peptidoglycan. The approaches described are based primarily on the previous transition state mimic; VanA and EcDdlB, co-crystal structures. Active site mapping of VanA by site directed mutagenesis yielded VanA mutants that were expressed and purified for kinetic studies. The active site mapping of VanA supports the predictions about catalysis from the X-ray structure of VanA, but failed to identify a catalytic base. Additionally, the first enzymatic characterisation of a VanD ligase was performed. This analysis revealed that VanD4 ligase is a less efficient D-Ala-D-Lac ligase, and selected for a wider variety of second substrates in comparison to VanA. EcDdlB was expressed in E.coli, purified and crystallised. Three structures of EcDdlB have been solved to 1.4-1.7Å resolutions, representing a product inhibition complex, a ternary complex, and a D-cycloserine inhibited complex. Based upon the latter EcDdlB structure, we propose a novel suicide-substrate mechanism for the Dcycloserine mediated inhibition of DDLs, involving phosphorylation of D-cycloserine. Future studies will validate this mechanism. Finally, contributions towards rational design of EcDdlB and VanA inhibitors have been made. This work has significantly increased knowledge in the field of DDL enzymes, progressing towards the exploitation of these targets for antibiotic development. The deposition of PDB files of three new EcDdlB structures will provide the tools for further rational based drug design campaigns against EcDdlB. The novel mechanism of Dcycloserine inhibition may also have implications for the further development of inhibitors of DDLs.
Supervisor: Not available Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available
Keywords: QP Physiology