Use this URL to cite or link to this record in EThOS: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.651258
Title: Structural and functional studies into the sugar fermentation stimulation protein SfsA
Author: Bliss, Sophie J.
ISNI:       0000 0004 5357 848X
Awarding Body: University of Sheffield
Current Institution: University of Sheffield
Date of Award: 2015
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Abstract:
The sugar fermentation stimulation protein A (SfsA) is ubiquitous among all kingdoms of life. The structures of E. coli and P. furiosus SfsAs show the protein is unique among DNA-binding proteins, containing both an oligonucleotide-binding (OB) fold and a restriction endonuclease-like domain. This domain contains the semi-conserved PD-(D/E)xK motif that is the catalytic center of type II restriction endonucleases, and which binds metal ion cofactors to coordinate a cleavage reaction at the substrate. Accordingly, activity assays have indicated that the enzyme acts as a non-specific nuclease. The structure of a P. furiosus SfsA-DNA complex has shown that a conserved OB-fold arginine and phenylalanine interdigitate the DNA duplex at a gap position and so may be essential for function. This thesis presents the results of a structure-function investigation into the roles of highly conserved residues from both SfsA domains. Site-directed mutants of the key OB-fold residues and the PD-(D/E)xK active site have been produced from E. coli and P. furiosus SfsAs and their structures determined by X-ray crystallography. This has resulted in the highest resolution structures of SfsA for both species. Together with these structures, substrate precipitation and DNA degradation activity assays have shown that the active site PD-(D/E)xK residues D117, E130 and K132 are essential for function in P. furiosus SfsA, with the acidic residues coordinating the active site metals and the lysine polarizing the nucleophilic water molecule. In E. coli SfsA the equivalent E135 residue is also essential, but the D121A mutant retains some residual activity. The OB-fold R18A, F19A P. furiosus SfsA double mutant, and their E. coli counterparts show that these two residues are essential for binding covalently closed DNA, and for the initial endonuclease event, but not for subsequent exonuclease activity. Additionally, the first structure of an E. coli SfsA-DNA complex has been determined to 2.0 Å resolution. This shows a small conformational change between the two domains and a significant bend in the substrate, which is stablilised through binding of the RF motif. The transcription factors CRP and TBP bend DNA by a comparable amount, perhaps alluding to the cellular function of SfsA.
Supervisor: Baker, Patrick Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID: uk.bl.ethos.651258  DOI: Not available
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