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.
Booth
Michael P S
MP
School of Cellular and Molecular Medicine, University of Bristol, University Walk, Bristol, UK.
Challand
Martin R
MR
Emery
David C
DC
Roach
Peter L
PL
Spencer
James
J
D016948:Azotobacter vinelandii / Q000235:genetics; D024881:Drug Resistance, Bacterial; D004926:Escherichia coli / Q000235:genetics; D008780:Methyltransferases / Q000235:genetics* / Q000378:metabolism*; D009876:Operon; D012335:RNA, Ribosomal / Q000378:metabolism*; D012436:S-Adenosylmethionine / Q000378:metabolism*
D012335:RNA, Ribosomal; D012436:S-Adenosylmethionine; D008780:Methyltransferases