Show simple item record

dc.contributor.advisorWalker, Suzanneen_US
dc.contributor.advisorKahne, Danielen_US
dc.contributor.authorQiao, Yuanen_US
dc.date.accessioned2017-07-25T14:40:07Z
dash.embargo.terms2017-05-01en_US
dc.date.created2016-05en_US
dc.date.issued2016-05-10en_US
dc.date.submitted2016en_US
dc.identifier.citationQiao, Yuan. 2016. Reconstitution of the Final Step of Peptidoglycan Assembly in Staphylococcus aureus. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.en_US
dc.identifier.urihttp://nrs.harvard.edu/urn-3:HUL.InstRepos:33493392
dc.description.abstractBacterial peptidoglycan (PG) is an exoskeleton structure that maintains cell shape and protects cells from lysis. Peptidoglycan is essential in bacteria but is not found in mammalian cells. Therefore, it is the target for several classes of antibiotics, including the beta-lactam family. Beta-lactams target the transpeptidase (TP) domain of penicillin-binding proteins (PBPs), the enzymes responsible for the final step of peptidoglycan biosynthesis. Mutations of transpeptidases in Staphylococcus aureus (S. aureus) have been implicated in beta-lactam resistance in the clinic (commonly known as MRSA infections). Despite the fact that PBPs are important antibiotic targets, there have been no direct assays to monitor their enzymatic activity, primarily due to inaccessibility to appropriate substrates. The PG precursor, Lipid II, is required to study transpeptidase activity. Lipid II contains a glycopeptide attached to a pyrophosphate lipid containing 55 carbons. It has poor physical properties and is present in low abundance in bacteria. This thesis describes the reconstitution of peptidoglycan assembly in Staphylococcus aureus by the essential Class A PBP, PBP2. This was enabled by several key advances, which are also described. The first advance is the discovery that PBP4, a low-molecular weight PBP in S. aureus, can use a Lipid II analogue as a transpeptidation substrate. It can incorporate biotin-D-Lys (BDL) and other non-canonical D-amino acids into the terminal position of the stem peptide in Lipid II. BDL labeling of Lipid II with S. aureus PBP4 has enabled the second advance: the direct detection of native Lipid II extracted from bacteria. This has facilitated elucidation of cellular mechanisms of antibiotics that target cell wall biosynthesis. The third advance is a general strategy to accumulate and isolate native Lipid II in bacteria in useful quantities. Access to substantial quantities of native S. aureus Lipid II has enabled reconstitution of PBP2 transpeptidase activity, as well as characterization of several beta-lactam antibiotics by monitoring enzymatic inhibition. In sum, this work establishes important tools for studying enzymatic mechanisms of bacterial transpeptidases and for characterizing inhibitors that target bacterial peptidoglycan biosynthesis.en_US
dc.description.sponsorshipChemical Biologyen_US
dc.format.mimetypeapplication/pdfen_US
dc.language.isoenen_US
dash.licenseLAAen_US
dc.subjectChemistry, Biochemistryen_US
dc.titleReconstitution of the Final Step of Peptidoglycan Assembly in Staphylococcus aureusen_US
dc.typeThesis or Dissertationen_US
dash.depositing.authorQiao, Yuanen_US
dc.date.available2017-07-26T07:31:04Z
thesis.degree.date2016en_US
thesis.degree.grantorGraduate School of Arts & Sciencesen_US
thesis.degree.levelDoctoralen_US
thesis.degree.nameDoctor of Philosophyen_US
dc.contributor.committeeMemberBalskus, Emily P.en_US
dc.contributor.committeeMemberStubbe, JoAnneen_US
dc.type.materialtexten_US
thesis.degree.departmentChemical Biologyen_US
dash.identifier.vireohttp://etds.lib.harvard.edu/gsas/admin/view/704en_US
dc.description.keywordspeptidoglycan; beta-lactams; transpeptidase; penicillin-binding proteins; Staphylococcus aureusen_US
dash.author.emailyuanqiao16@gmail.comen_US
dash.contributor.affiliatedQiao, Yuan


Files in this item

Thumbnail

This item appears in the following Collection(s)

Show simple item record