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High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics

High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics
High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics
Cfr is a radical-SAM (S-adenosyl-l-methionine) enzyme that methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome. Radical-SAM enzymes use an Fe–S cluster to generate the 5?-deoxyadenosyl (DOA) radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8. However, despite its mechanistic interest and clinical relevance, until recently Cfr remained little characterised. Accordingly we have used co-expression with the Azotobacter vinelandii isc operon, encoding genes responsible for Fe–S cluster biosynthesis, to express hexahistidine-tagged Cfr in Escherichia coli BL21Star, and purified the recombinant protein in a yield more than 20 times greater than has been previously reported. As aerobically purified, Cfr contains secondary structure, is monomeric in solution and has an absorbance spectrum suggestive of a 2Fe–2S cluster. After anaerobic purification a 4Fe–4S cluster is indicated, while on reconstitution with excess iron and sulphide a further increase in metal content suggests that an additional, most likely 4Fe–4S, cluster is formed. Acquisition of additional secondary structure under these conditions indicates that Fe–S clusters are of structural, as well as functional, importance to Cfr. In the presence of sodium dithionite reconstituted Cfr is both reducible and able to cleave SAM to 5?-deoxyadeonsine (DOA), demonstrating that the purified reconstituted enzyme has radical-SAM activity. Co-expression with isc proteins thus enables recombinant active Cfr to be obtained in yields that facilitate its future spectroscopic and structural characterisation.

radical-sam, co-expression, reconstitution, fe–s cluster, oxazolidinone, antibiotic resistance
1046-5928
204-210
Booth, Michael P.S.
12f6e821-653c-4b61-af79-4fe5f4766fbb
Challand, Martin R.
03bb1ded-9274-4525-8377-8e5930f30d65
Emery, David C.
2ed22481-a75c-4aa4-bff3-835cdc594395
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Spencer, James
e1c8688f-4021-43ea-a92a-2ec703543fa0
Booth, Michael P.S.
12f6e821-653c-4b61-af79-4fe5f4766fbb
Challand, Martin R.
03bb1ded-9274-4525-8377-8e5930f30d65
Emery, David C.
2ed22481-a75c-4aa4-bff3-835cdc594395
Roach, Peter L.
ca94060c-4443-482b-af3e-979243488ba9
Spencer, James
e1c8688f-4021-43ea-a92a-2ec703543fa0

Booth, Michael P.S., Challand, Martin R., Emery, David C., Roach, Peter L. and Spencer, James (2010) High-level expression and reconstitution of active Cfr, a radical-SAM rRNA methyltransferase that confers resistance to ribosome-acting antibiotics. Protein Expression and Purification, 74 (2), 204-210. (doi:10.1016/j.pep.2010.07.010). (PMID:20678576)

Record type: Article

Abstract

Cfr is a radical-SAM (S-adenosyl-l-methionine) enzyme that methylates the 8 position of 23S rRNA residue A2503 to confer resistance to multiple antibiotic classes acting upon the large subunit of the bacterial ribosome. Radical-SAM enzymes use an Fe–S cluster to generate the 5?-deoxyadenosyl (DOA) radical from SAM, enabling them to modify intrinsically unreactive centres such as adenosine C8. However, despite its mechanistic interest and clinical relevance, until recently Cfr remained little characterised. Accordingly we have used co-expression with the Azotobacter vinelandii isc operon, encoding genes responsible for Fe–S cluster biosynthesis, to express hexahistidine-tagged Cfr in Escherichia coli BL21Star, and purified the recombinant protein in a yield more than 20 times greater than has been previously reported. As aerobically purified, Cfr contains secondary structure, is monomeric in solution and has an absorbance spectrum suggestive of a 2Fe–2S cluster. After anaerobic purification a 4Fe–4S cluster is indicated, while on reconstitution with excess iron and sulphide a further increase in metal content suggests that an additional, most likely 4Fe–4S, cluster is formed. Acquisition of additional secondary structure under these conditions indicates that Fe–S clusters are of structural, as well as functional, importance to Cfr. In the presence of sodium dithionite reconstituted Cfr is both reducible and able to cleave SAM to 5?-deoxyadeonsine (DOA), demonstrating that the purified reconstituted enzyme has radical-SAM activity. Co-expression with isc proteins thus enables recombinant active Cfr to be obtained in yields that facilitate its future spectroscopic and structural characterisation.

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More information

Published date: December 2010
Keywords: radical-sam, co-expression, reconstitution, fe–s cluster, oxazolidinone, antibiotic resistance

Identifiers

Local EPrints ID: 177021
URI: https://eprints.soton.ac.uk/id/eprint/177021
ISSN: 1046-5928
PURE UUID: af8b7d74-b7d8-4a00-b80a-2f05716a3259
ORCID for Peter L. Roach: ORCID iD orcid.org/0000-0001-9880-2877

Catalogue record

Date deposited: 28 Mar 2011 15:11
Last modified: 14 Aug 2018 00:34

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