The University of Southampton
University of Southampton Institutional Repository

Structural analysis of proteins from the radical SAM superfamily

Structural analysis of proteins from the radical SAM superfamily
Structural analysis of proteins from the radical SAM superfamily
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.
Dinis, Pedro Cleto Esteves Guerreiro
40a6c6d3-c9f3-44fc-809b-c865d203ce74
Dinis, Pedro Cleto Esteves Guerreiro
40a6c6d3-c9f3-44fc-809b-c865d203ce74
Roach, Peter
ca94060c-4443-482b-af3e-979243488ba9

(2016) Structural analysis of proteins from the radical SAM superfamily. University of Southampton, Department of Chemistry, Doctoral Thesis, 478pp.

Record type: Thesis (Doctoral)

Abstract

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.

PDF
PDinis_25878034_PhD_Dissertation.pdf - Other
Download (40MB)

More information

Published date: 25 January 2016
Organisations: University of Southampton, Chemistry

Identifiers

Local EPrints ID: 387225
URI: http://eprints.soton.ac.uk/id/eprint/387225
PURE UUID: 11790fa0-1d75-4951-a7c8-ebb5ee509b35
ORCID for Peter Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 18 Feb 2016 11:49
Last modified: 25 Jan 2019 05:01

Export record

Contributors

Author: Pedro Cleto Esteves Guerreiro Dinis
Thesis advisor: Peter Roach ORCID iD

University divisions

Download statistics

Downloads from ePrints over the past year. Other digital versions may also be available to download e.g. from the publisher's website.

View more statistics

Atom RSS 1.0 RSS 2.0

Contact ePrints Soton: eprints@soton.ac.uk

ePrints Soton supports OAI 2.0 with a base URL of http://eprints.soton.ac.uk/cgi/oai2

This repository has been built using EPrints software, developed at the University of Southampton, but available to everyone to use.

We use cookies to ensure that we give you the best experience on our website. If you continue without changing your settings, we will assume that you are happy to receive cookies on the University of Southampton website.

×