Advisory Committee Chair
William B Parker
Advisory Committee Members
Jennifer R King
Mahmoud H El Kouni
Michael E Niederweis
Andries Jc Steyn
Document Type
Dissertation
Date of Award
2011
Degree Name by School
Doctor of Philosophy (PhD) Heersink School of Medicine
Abstract
Tuberculosis (TB) is one of the leading infectious diseases in the world. An estimated one third of the world's population is infected with Mycobacterium tuberculosis, the causative pathogen of TB. With the emergence of drug resistant strains of the mycobacterium, and the HIV-TB coinfection epidemic, TB remains a global health emergency. Purine metabolism is an essential cellular component to all living cells. Previous studies have shown that differences exist between mycobacterial and human purine metabolism. One of the differences was in the metabolism of adenosine (Ado), whose cleavage was observed in mycobacterial cells whereas Ado cleavage is inefficient in human cells. While Ado cleavage activity had been observed in mycobacteria, the enzyme(s) responsible for this activity was unknown. The identification and understanding of the biochemical properties of Ado cleavage enzyme(s) could aid in the development of Ado analogs for the treatment of TB. There are several candidate enzymes in nature that can cleave Ado, and therefore the identification of endogenous Ado cleavage activity from M. smegmatis was preferred over a genetic approach. This strategy enabled the discovery of two Ado cleavage activities. The first enzyme was named adenosine-purine nucleoside phosphorylase (Ado-PNP), and could cleave Ado, inosine, and guanosine. Because of its ability to cleave Ado, Ado-PNP is different from the trimeric mycobacterial PNP that accepts 6-oxopurines but not 6-aminopurines as substrates. The second enzyme identified could cleave Ado, but 5'-methylthioadenosine (MTA) was the preferred substrate. MTA cleavage was phosphate dependent, and therefore the second Ado-cleaving enzyme identified in M. smegmatis was an MTA phosphorylase. This marked the identification of the first bacterial MTAP. Based on sequence homology, Rv0535 had been annotated as a probable MTAP in M. tuberculosis. Recombinant Rv0535 was expressed, purified, and characterized. While MTA was the preferred substrate, Rv0535 could also cleave Ado and S-adenosyl-L-homocysteine at 2% and 0.8% of the MTA cleavage activity, respectively. To identify other substrates of Rv0535, a structure-activity relationship study was conducted and the initial results are presented. This work has identified two mycobacterial Ado cleavage enzymes that could be exploited for the development of new anti-TB drugs.
Recommended Citation
Buckoreelall, Kajal, "Identification and characterization of novel adenosine cleavage enzymes in mycobacteria" (2011). All ETDs from UAB. 1282.
https://digitalcommons.library.uab.edu/etd-collection/1282