Main content

Characterization of an aldolase found within the cholesterol degradation pathway of Mycobacterium tuberculosis.

Show full item record

Title: Characterization of an aldolase found within the cholesterol degradation pathway of Mycobacterium tuberculosis.
Author: Gilbert, Stephanie Elizabeth
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Seah, Stephen
Abstract: The heteromeric acyl-CoA dehydrogenase, FadE28-FadE29 and the enoyl-CoA hydratase ChsH1-ChsH2, respectively, catalyze the dehydrogenation and subsequent hydration of the 3-carbon side chain cholesterol metabolite to form 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA) in Mycobacterium tuberculosis. The gene downstream of chsH2, ltp2, was introduced into Rhodococcus jostii RHA1 and the recombinantly produced His-tagged Ltp2 co-purified with untagged ChsH1-ChsH2, ChsH2, or the C-terminal domain of ChsH2 that contains a domain of unknown function (DUF35). Ltp2-ChsH1-ChsH2 or Ltp2-DUF35 complexes catalyzed the retro-aldol cleavage of 17-HOPC-CoA to form androst-4-ene-3,17-dione and propionyl-CoA. Ltp2-DUF35 complex has optimal activity at pH 7.5 with Km of 6.54 ± 0.90 M and kcat of 159 ± 8.50 s-1. ChsH1-ChsH2 alone can only hydrate about 30% of its substrate, but its association with Ltp2 enables complete hydration. Conditions for crystallizing Ltp2-DUF35 were formulated which may facilitate future structure determination by X-ray crystallography.
URI: http://hdl.handle.net/10214/12109
Date: 2017-12
Terms of Use: All items in the Atrium are protected by copyright with all rights reserved unless otherwise indicated.


Files in this item

Files Size Format View Description
Gilbert_Stephanie_201712_Msc.pdf 3.292Mb PDF View/Open Thesis

This item appears in the following Collection(s)

Show full item record