Browsing by Subject "Isoprenoid biosynthesis"
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Item Mechanistic studies of two enzymes that employ common coenzymes in uncommon ways(2010-08) Thibodeaux, Christopher James; Liu, Hung-wen, 1952-Enzymes are biological catalysts which greatly accelerate the rates of chemical reactions, oftentimes by many orders of magnitude over the uncatalyzed reaction. The remarkable catalytic rate enhancement afforded by enzymes derives ultimately from the structure and chemical properties of the enzyme active sites, which allow enzymes to selectively bind to their substrates and to stabilize high energy chemical species and unstable intermediates along the reaction coordinate. To enhance their catalytic ability, many enzymes have also evolved to require coenzymes for optimal activity. These coenzymes often provide chemical functionality and reactivity that are not accessible by the twenty canonical amino acids and, hence, coenzymes serve to greatly enhance the diversity of chemical reactions that can be mediated by enzymes. The work described in this dissertation focuses on mechanistic studies of two enzymes that use common coenzymes in unusual ways. In the first section of this work, studies will focus on the type II isopentenyl diphosphate:dimethylallyl diphosphate isomerase (IDI-2), an essential enzyme in isoprenoid biosynthesis that employs a flavin mononucleotide (FMN) coenzyme for catalysis. In most biological systems, flavin coenzymes mediate electron transfer reactions. However, the IDI-2 catalyzed reaction involves no net redox change, raising questions as to the role of the flavin in the chemical mechanism. The chemical mechanism of IDI-2 will be interrogated with a combination of spectroscopic studies and biochemical techniques. Our studies suggest that the flavin coenzyme of IDI-2 may employ a novel mode of flavin-dependent catalysis involving acid/base chemistry. In the second section of this dissertation, attention will be focused on elucidating the chemical mechanism of 1-aminocyclopropane-1-carboxylate deaminase (ACCD), an enzyme that plays a role in regulating the production of the potent plant hormone, ethylene. ACCD is a pyridoxal-5ʹ-phosphate (PLP)-dependent enzyme that catalyzes a C-C bond cleavage event that is unique among the catalytic cycles of PLP-dependent enzymes. Altogether, our mechanistic studies of IDI-2 and ACCD help to illustrate the catalytic diversity of common coenzymes, and demonstrate that some enzymes have evolved to exploit readily available coenzymes for atypical reactions.Item The Biochemical Investigation and Isolation of Small Molecule Inhibitors for Two Essential Proteins of Mycobacterium tuberculosis H37Rv: IspD and Wag31(2014-06-12) Joseph, SoniaTuberculosis is one of the leading causes of death due to infectious disease. The causative agent, Mycobacterium tuberculosis, is a facultative intracellular parasite with a slow regeneration rate. Though there is a decline in the overall TB incidence since 2005, the emergence of resistant strains that are impervious to existing treatment regimens make the discovery and development of new drug leads crucial. To this end, exploiting key differences between the biology of the host and the pathogen can generate novel lead molecules with minimal side effects. This thesis details the study of two proteins that are essential for the survival of M. tuberculosis (M.tb) but are not present in the host, making them potential drug targets. The first protein, IspD (2-C-methyl-D-erythritol-4-phosphate (MEP) cytidyl transferase), is a part of the non-mevalonate pathway for the synthesis of isoprenoids and catalyzes the condensation of MEP and cytidine triphosphate (CTP) to form 4-diphosphocytidyl-2-C-methylerythritol (CDP-ME) and pyrophosphate (PPi). A medium-throughput enzyme assay was developed to identify inhibitors for this protein. It was screened against 3550 compounds drawn from five different M. tb whole-cell active small molecule libraries generating a total of five hits. These molecules were then assessed for their potency against IspD as measured by their IC50, their activity against M. tb whole cells and their cytotoxicity. Of the five hits, two compounds inhibited M. tb whole cell growth at a concentration below 50 ?M while exhibiting no general cytotoxicity to human dermal fibroblasts (HDF). They each had an IC50 of 26.1 ?M and 37.8?M and preliminary SAR studies were performed on the latter. These molecules could prove to be a viable starting point for the rational design of IspD inhibitors. The second protein, Wag31 is a cell division associated protein that regulates mycobacterial cell size and septum formation. Wag31 exhibited a propensity for gel formation both alone and in association with other cellular proteins. A purification strategy was developed to circumvent this tendency and generate soluble protein. It was found that mutations within the wag31 protein coding sequence conferred resistance to a whole-cell active small molecule (MIC99=6.25 ?g/ml) in both M. tuberculosis and M. smegmatis. Moreover, all of the discovered mutations were clustered within the C-terminal coiled-coil domain of the protein. It was established that this compound binds to Wag31 and seems to shift the equilibrium of the protein solution towards gel formation. The mutated protein does not form gel and seems to bind to the compound at a significantly reduced rate. To further confirm that Wag31 was indeed the target of this small molecule, whole cell viability assays were performed to establish whether the over-expression of Wag31 in M. smegmatis would shift the EC50 and MIC99 values. Wag31 over-expression reduced both the EC50 and MIC99 values providing further proof that Wag31 is the target for this compound. The compound appears to act by shifting the equilibrium of the protein towards a gelatinous state which proves inhibitory for cell growth.