The Resource Structural and enzymatic characterization of bacterial cell wall enzymes : focus on penicillin-binding proteins, Sudheer Bobba

Structural and enzymatic characterization of bacterial cell wall enzymes : focus on penicillin-binding proteins, Sudheer Bobba

Label
Structural and enzymatic characterization of bacterial cell wall enzymes : focus on penicillin-binding proteins
Title
Structural and enzymatic characterization of bacterial cell wall enzymes
Title remainder
focus on penicillin-binding proteins
Statement of responsibility
Sudheer Bobba
Creator
Contributor
Author
Thesis advisor
Subject
Genre
Language
eng
Summary
The research presented in this dissertation was aimed at understanding several structural and biochemical features underlying the bacterial cell wall biosynthetic process using statistical, biochemical and analytical tools. Penicillin-binding proteins (PBPs) are ubiquitous and essential enzymes necessary for cell wall biosynthesis. PBPs are broadly classified as low molecular mass (LMM) and high molecular mass (HMM) based on their molecular weight. Although all PBPs share a set of highly conserved active site residues, different PBPs have different propensities for catalyzing reactions. To understand the structural differences among PBPs, a global catalytic residue comparison was performed for all the PBPs available in the protein data bank. This dataset was analyzed using univariate and multivariate statistical methods, revealing several interesting relationships such as: (1) Distribution of the dihedral angle for the SXXK-motif Lys side chain was bimodal, and strongly correlated with HMM/transpeptidase vs LMM/hydrolase activity; (2) Distance between the SXXK-motif Lys-NZ atom and the Lys/His-nitrogen atom of the (K/H)T(S)G-motif was highly conserved. Unlike LMM PBPs, HMM PBPs when purified, give undetectable or weak enzyme activity that has impeded efforts to develop new inhibitors. In the next section of this dissertation, we described a fluorescently detected microtiter plate-based assay for HMM PBPs. Purified PBPs were immobilized onto microtiter plate wells and labeled with biotinylated-ampicillin (Bi--Amp). Treatment of Bi--Amp-labeled PBPs with a streptavidin--horseradish peroxidase conjugate followed by a fluorogenic HRP substrate allowed the detection of PBPs. The HMM PBP assay was extended to penicillin-binding protein 2a, the molecular determinant of high-level [beta]-lactam resistance in methicillin-resistant Staphylococcus aureus using Bio-Amp and biotinylated-cephalexin as tagging reagents. This assay was then demonstrated for use in competition assays for screening and characterizing both [beta]-lactam and non [beta]-lactam inhibitors. The cytoplasmic steps of cell wall biosynthesis involve a series of UDP-linked intermediates whose synthesis is catalyzed by Mur enzymes. The final section of the dissertation deals with developing an LC-MS/MS detection and quantification of UDP-peptidoglycan intermediates in the bacterial cell wall biosynthetic pathway. The assay was validated and then used to quantitate the in vivo levels of UDP-intermediates in S. aureus treated with different antibiotics acting at various stages of cell wall biosynthesis
Cataloging source
UMK
http://library.link/vocab/creatorDate
1986-
http://library.link/vocab/creatorName
Bobba, Sudheer
Degree
Ph. D. (School of Pharmacy and Dept. of Chemistry).
Dissertation year
2013.
Granting institution
University of Missouri-Kansas City,
Illustrations
illustrations
Index
no index present
Literary form
non fiction
Nature of contents
  • dictionaries
  • bibliography
  • theses
http://library.link/vocab/relatedWorkOrContributorName
Gutheil, William G.
http://library.link/vocab/subjectName
  • Penicillin
  • Bacterial cell walls
Label
Structural and enzymatic characterization of bacterial cell wall enzymes : focus on penicillin-binding proteins, Sudheer Bobba
Instantiates
Publication
Note
  • "A dissertation in Pharmaceutical Sciences and Chemistry."
  • Advisor: William G. Gutheil
  • Vita
Antecedent source
not applicable
Bibliography note
Includes bibliographical references (pages 213-244)
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier.
Color
black and white
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent.
Contents
Introduction and literature review -- Multivariate geometrical analysis of catalytic residues in the penicillin-binding proteins -- Substrate specificity studies of low molecular mass PBP actinomadura R39 -- A microtiter plate-based [beta]-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins -- A microtiter plate-based assay for inhibitors of penicillin-binding protein 2A from methicillin-resistant staphylococcus aureus -- Quantitative analysis of cytoplasmic UDP-peptidoglycan intermediates in s. aureus using LC-ESI-MS/MS -- Liquid chromatography-tandem mass spectrometry assay for detection and quantitation of endogenous bioactive peptides in rat brain -- Summary and conclusions -- Appendix
Control code
857794172
Dimensions
unknown
Extent
1 online resource (266 pages)
File format
one file format
Form of item
online
Level of compression
mixed
Media category
computer
Media MARC source
rdamedia.
Media type code
  • c
Other physical details
illustrations (some color)
Quality assurance targets
not applicable
Specific material designation
remote
System control number
(OCoLC)857794172
System details
  • The full text of the dissertation is available as an Adobe Acrobat .pdf file; Adobe Acrobat Reader required to view the file
  • Mode of access: World Wide Web
Label
Structural and enzymatic characterization of bacterial cell wall enzymes : focus on penicillin-binding proteins, Sudheer Bobba
Publication
Note
  • "A dissertation in Pharmaceutical Sciences and Chemistry."
  • Advisor: William G. Gutheil
  • Vita
Antecedent source
not applicable
Bibliography note
Includes bibliographical references (pages 213-244)
Carrier category
online resource
Carrier category code
  • cr
Carrier MARC source
rdacarrier.
Color
black and white
Content category
text
Content type code
  • txt
Content type MARC source
rdacontent.
Contents
Introduction and literature review -- Multivariate geometrical analysis of catalytic residues in the penicillin-binding proteins -- Substrate specificity studies of low molecular mass PBP actinomadura R39 -- A microtiter plate-based [beta]-lactam binding assay for inhibitors of high-molecular-mass penicillin-binding proteins -- A microtiter plate-based assay for inhibitors of penicillin-binding protein 2A from methicillin-resistant staphylococcus aureus -- Quantitative analysis of cytoplasmic UDP-peptidoglycan intermediates in s. aureus using LC-ESI-MS/MS -- Liquid chromatography-tandem mass spectrometry assay for detection and quantitation of endogenous bioactive peptides in rat brain -- Summary and conclusions -- Appendix
Control code
857794172
Dimensions
unknown
Extent
1 online resource (266 pages)
File format
one file format
Form of item
online
Level of compression
mixed
Media category
computer
Media MARC source
rdamedia.
Media type code
  • c
Other physical details
illustrations (some color)
Quality assurance targets
not applicable
Specific material designation
remote
System control number
(OCoLC)857794172
System details
  • The full text of the dissertation is available as an Adobe Acrobat .pdf file; Adobe Acrobat Reader required to view the file
  • Mode of access: World Wide Web

Library Locations

  • Curtis Laws Wilson LibraryBorrow it
    400 West 14th Street, Rolla, MO, 65409, US
    37.955220 -91.772210
  • Missouri University of Science & Technology Library DepositoryBorrow it
    2908 Lemone Blvd, Columbia, MO, 65201, US
    38.919360 -92.291620
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