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The Investigation of Novel Acyl-CoA Dehydrogenases Involved in Bacterial Steroid Degradation

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Title: The Investigation of Novel Acyl-CoA Dehydrogenases Involved in Bacterial Steroid Degradation
Author: Ruprecht, Amanda
Department: Department of Molecular and Cellular Biology
Program: Molecular and Cellular Biology
Advisor: Seah, Stephen
Abstract: FadE28-FadE29 is a heterotetrameric acyl-CoA dehydrogenase (ACAD) that catalyzes the dehydrogenation of the 3-carbon side chained 3-oxo-23,24-bisnorchol-4-en-22-oyl-CoA. FadE34 and CasC are also steroid degrading ACADs, however, they are a fusion of two ACAD domains encoded within one gene. FadE34 and CasC form homodimers and contain only two FAD molecules. Steady-state kinetic assays show that both CasC and FadE34 can utilize the 5- carbon side chained cholyl-CoA, deoxycholyl-CoA, and 3β-hydroxy-5-cholen-24-oyl-CoA as substrates, with varying catalytic efficiencies. This suggests these ACADs recognize both the side chain length and steroid ring structure. Replacement of a catalytic glutamic acid residue to a glutamine (FadE28-FadE29E241Q, FadE34E581Q, CasCE598Q) resulted in inactive variants, and replacement of an arginine residue to an alanine (FadE28R227A, FadE34R236A, CasCR239A) resulted in the decreased ability to bind FAD. These novel ACADs that utilize steroid substrates have half of the number of active sites compared to the previously characterized ACADs that utilize aliphatic substrates.
URI: http://hdl.handle.net/10214/8204
Date: 2014-06
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