Gatlawi, Hana Bashir, authorCrick, Dean C., advisorQuackenbush, Sandra L., committee memberStargel, Laurie A., committee memberPerera, Rushika, committee member2021-06-072022-06-022021https://hdl.handle.net/10217/232633Characterization of GrcC1 and GrcC2 prenyl diphosphate synthases potentially involved in menaquinone synthesis in Mycobacterium tuberculosis, and a homologous enzyme (MS1133) in Mycobacterium smegmatis Biosynthesis pathways provide attractive drug targets in Mycobacterium tuberculosis. Understanding the biochemistry of the enzymes that are involved in those pathways is very important to make an achievement in this field. Menaquinones are the major lipoquinones found in M. tuberculosis and their synthesis has been suggested to be a valid drug target in this bacterium. Menaquinone is a key component of the M. tuberculosis respiratory chain and a major electron carrier during aerobic growth with many electron acceptors. It has also been reported that the isoprenoid side chain is an important antioxidant. The genes grcC1 and grcC2 in M. tuberculosis encode proteins, probably polyprenyl diphosphate synthases, but little information is known about these enzymes although previous studies suggested that polyprenyl diphosphate synthases could be involved in the menaquinone biosynthesis. In this study, we cloned, expressed, purified and biochemically characterized the enzymatic activity of enzymes encoded by grcC1, grcC2 in M. tuberculosis strain H37Rv, and the homologue polyprenyl diphosphate synthase ms1133 in Mycobacterium smegmatis. The enzymes were active and catalyzed the condensation reaction that added [14C]IPP to allylic substrates of varying chain-lengths, including dimethylallyl diphosphate (DMAPP), geranyl diphosphate (GPP), farnesyl diphosphate (FPP), and geranylgeranyl diphosphate (GGPP). The purified expressed proteins generally preferred the longer chains allylic substrates of 10 or more carbon atoms. GPP is likely the preferred substrate of GrcC1, whereas FPP is likely the preferred substrate of GrcC2 and MS1133. The values of Vmax, Km and Kcat have been calculated and the final products of these enzymes were determined. The final products of all of these enzymes are long chain polyprenyl diphosphates. GrcC1 catalyzed the formation of solanesyl diphosphate with nine isoprene units (C45) which is needed for the menaquinone biosynthesis in M. tuberculosis. Since the role of the enzyme in the menaquinone biosynthesis pathway has been suggested elucidation of the properties of this enzyme and obtaining more information on the biological role helps to demonstrate the involvement of the enzyme in menaquinone biosynthesis in M. tuberculosis. Our results support the hypothesis that the enzyme is integral part of menaquinone synthesis in mycobacteria. This dissertation also illustrates the essentiality of the studied genes. Our constructed knock-out mutations tested the essentiality of the genes grcC1, grcC2 and ms1133 for the survival and growth of the pathogen M. tuberculosis and its lab surrogate M. smegmatis. The results demonstrated that grcC1 is an essential gene for M. tuberculosis survival. grcC2 and ms1133 both are not essential for survival, but the absence of these genes delays bacterial growth in M. tuberculosis and M. smegmatis.born digitaldoctoral dissertationsengCopyright and other restrictions may apply. User is responsible for compliance with all applicable laws. For information about copyright law, please see https://libguides.colostate.edu/copyright.Text