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Title: Structural analysis of proteins from the radical SAM superfamily
Author: Dinis, Pedro Cleto Esteves Guerreiro
ISNI:       0000 0004 5917 0709
Awarding Body: University of Southampton
Current Institution: University of Southampton
Date of Award: 2016
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The Radical SAM superfamily is a large group of enzymes which use an iron‐sulfur cluster to catalyse the reductive cleavage of S‐adenosylmethionine (SAM), resulting in the formation of a highly reactive intermediate. This potent oxidant is used to functionalise relatively inert substrates to catalyse an extensive role of reactions: cofactor biosynthesis; anaerobic metabolism; methylation and post‐translational modifications. Most members of this family share some structural similarities, most notably a [4Fe‐4S] cluster, coordinated by a cysteine triad motif; some conserved motifs for the binding of SAM and an active site with a full or partial triose‐phosphate isomerase (TIM) barrel fold. However, despite a quarter of a million predicted protein sequences, the number of structures from which to draw conclusions is severely limited, not reaching 50 available X‐ray structures in the Protein Data Bank. This project aims to solve the structures of three proteins of this superfamily. HydG, an enzyme involved in the biosynthesis of the complex active‐site cofactor of the [Fe‐Fe] HydA hydrogenase, is proposed to use L‐tyrosine and an auxiliary cluster to form a distinctive Fe(CO)2CN synthon. The structure presented in this work allows a deeper understanding of the mechanism behind the complex product formation, by use of a never before reported [5Fe‐5S] cluster. LipA is an enzyme responsible for the last step on the production of the lipoyl cofactor, the insertion of two sulfur atoms, at C6 and C8 of an octanoyl subunit. The currently accepted mechanism of catalysis has LipA serving as both a catalyst and a substrate, by sacrificing its auxiliary [4Fe‐4S] cluster as the sulfur source. With a crystallographic structure recently published, it was possible to identify a residue (serine) coordinating the cluster. Previous mutagenesis studies showed the mutation of this serine has a strong impact on activity, and the crystallization of a serine to cysteine mutant in this project strengthens the mechanistic proposal being developed. The third enzyme, Cfr is responsible for the methylation of A2503 in the 23S ribosomal subunit, conferring resistance to several antibiotics, making it a major health concern. Co‐crystallization of the protein with an RNA fragment may help direct the efforts to design effective inhibitors, and the first steps in the expression and purification of a small rRNA fragment suitable for crystallization studies are expressed herein.
Supervisor: Roach, Peter Sponsor: Not available
Qualification Name: Thesis (Ph.D.) Qualification Level: Doctoral
EThOS ID:  DOI: Not available