DOI
https://doi.org/10.25772/AJ60-2936
Defense Date
2013
Document Type
Thesis
Degree Name
Master of Science
Department
Biochemistry
First Advisor
William Barton
Abstract
Sortases have been known to be essential in Gram-positive bacteria for attaching proteins onto the peptidoglycan layer of the bacterium. Sortase A has been found to be useful as a “molecular stapler”, although; in vivo, the enzyme is responsible for attaching proteins to the peptidoglycan layer of Gram-positive bacteria. It accomplishes both of these tasks by joining two proteins together via an LPXTG sorting sequence. The enzyme has been proven to be very useful in attaching any two proteins together without worrying about recombinant techniques to generate the fusion protein. The problem with this enzyme is that the catalytic diad, which is composed of Cys-184 and His-120, has to be in a certain form that exists .2% of the time at pH 7.0. There is also a hydrolytic shunt that the enzyme can undergo instead of the productive transpeptidase reaction. These issues lead to groups attempting to place S.aureus SrtA through directed evolution in order to increase the catalytic efficiency of the enzyme. Although mutants have been generated that increase the catalytic efficiency 13-fold and 130-fold, the structural basis behind this increase is poorly understood. Using crystallography, we will attempt to discover the structural basis behind the rate enhancement as well as understand more about different species of SrtA. We also will attempt to kinetically characterize the S.aureus SrtA enzyme, its mutants, and different strains of SrtA. Thus far G.moribillorum SrtA has been crystallized and its structure shows that there is a distinction in the β6/β7 loop which has been implied to be important to catalysis. Furthermore, the pentaglycine kinetics shone some light on how the different mutants interact with the pentaglycine substrate of S.aureus SrtA.
Rights
© The Author
Is Part Of
VCU University Archives
Is Part Of
VCU Theses and Dissertations
Date of Submission
May 2013