Treating Mycobacterium tuberculosis (M. tb) infections in the 21st century is an increasingly daunting task, as antibiotic resistance has become more prevalent and treatment accordingly more complex. One property that makes M. tb so difficult to treat is its extremely hydrophobic, highly impermeable cell wall, most notably characterized by the presence of long chain, &agr;-branched mycolic acids. The Antigen 85 (Ag85) family of enzymes catalyze the transfer of mycolic acids from a single donor, trehalose monomycolate (TMM), to different acceptors located within the cell wall. Although there are three different pathways in M. tb to produce trehalose, only one, the otsAB pathway, leads to the production of TMM. Thus, inhibiting the Ag85 enzymes or the otsAB pathway may seriously damage the ability of M. tb to build the cell wall, and ultimately could affect the viability of the organism itself.
The first part of this work describes the development of a real-time, high-throughput assay to assess enzyme activity and determine the steady-state parameters for one enzyme in the otsAB pathway, trehalose phosphate phosphatase (TPP), which catalyzes the hydrolysis of trehalose-6-phosphate (T6P) to produce trehalose. The assay was determined to be rapid, highly producible, and amenable for high-throughput screening.
The second part of this work describes work done to kinetically and structurally characterize the second step of the Ag85 ping-pong mechanism, since previous studies have not elucidated the processes that govern recognition and transfer of mycolic acids to carbohydrate acceptors. A single-enzyme mediated, high-throughput assay using esterified fluorophores as acyl donors was developed to evaluate acyl acceptor selectivity, as well as a means to screen potential inhibitors. Although the assay produced promising results for Ag85C, a combination of high background and low activity hampered the ability to assess the second half-reaction of the Ag85A and Ag85B enzymes. However, the assay was successful in identifying and assessing compounds that inhibit acyl transfer catalyzed by Ag85C. Additionally, a serine to alanine active site mutant of Ag85C (S124A) was structurally characterized with and without the presence of carbohydrate-based detergents, providing insight regarding the acyl-enzyme intermediate form of the enzyme and its interactions with substrate and product analogs.
|Advisor:||Ronning, Donald R.|
|Commitee:||Choong-Min, Kang, James, Slama, Steve, Sucheck|
|School:||The University of Toledo|
|School Location:||United States -- Ohio|
|Source:||DAI-B 74/06(E), Dissertation Abstracts International|
|Subjects:||Microbiology, Chemistry, Biochemistry, Pharmacy sciences|
|Keywords:||Antibiotic resistance, Mycobacterium tuberculosis, Mycolic acids, Trehalose monomycolate|
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