Characterization of an operon involved in mycobacterial cholesterol metabolism
Başlık çevirisi mevcut değil.
- Tez No: 400762
- Danışmanlar: PROF. EDITH SIM
- Tez Türü: Doktora
- Konular: Eczacılık ve Farmakoloji, Pharmacy and Pharmacology
- Anahtar Kelimeler: Belirtilmemiş.
- Yıl: 2009
- Dil: İngilizce
- Üniversite: University of Oxford
- Enstitü: Yurtdışı Enstitü
- Ana Bilim Dalı: Eczacılık Ana Bilim Dalı
- Bilim Dalı: Belirtilmemiş.
- Sayfa Sayısı: 215
Özet
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Özet (Çeviri)
In the recently identified cholesterol catabolic pathway of Mycobacterium tuberculosis, 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate hydrolase (HsaD) is proposed to catalyze the hydrolysis of a C-C bond in the cholesterol metabolite 4,9-DSHA (4,5- 9,10-diseco-3-hydroxy-5,9,17-trioxoandrosta-1(10),2-diene-4-oic acid). The work in this thesis describes the cloning and expression of the hsaD gene from M. tuberculosis. Using a heterologous expression system in Psuedomonas putida and affinity chromatography, HsaD was purified to apparent homogeneity. To test the hydrolytic activity of the enzyme, the coloured substrate 2-hydroxy-6-oxo-6-phenylhexa- 2,4-dienoic acid was synthesized and a 96-well assay was developed for HsaD. With the purified enzyme and hydrolytic assay, the enzymatic and physical properties of the protein were characterized. Using the purified protein and an enzymatically inactive form of HsaD, the structure of this enzyme either alone or in complex with several different substrates, including 4,9-DSHA, was determined to 1.8-2.35Å. In addition to this work, a series of genetic knockouts of the nat/hsa operon were created in Mycobacterium smegmatis and Mycobacterium bovis BCG. Deletion of hsaD in M. smegmatis had no effect on the growth rate of the bacteria with cholesterol, while the deletion of the gene hsaC was found to cause the bacterial supernatant to turn pink when grown with cholesterol. With ?hsaD M. bovis BCG the mutant strain was found to grow much slower on minimal medium supplemented with cholesterol. Deletion of hsaD in M. bovis BCG was also found to inhibit intracellular growth in macrophages. Further to this work, using antibodies raised against proteins encoded by this operon and genetic knockouts, the nat/hsa operon was found to contain an additional promoter. Furthermore, M. bovis BCG was found to significantly alter the level and distribution of several lipid species in macrophages, including cholesterol. These effects shed light on the mechanism of mycobacterial survival inside macrophages.
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