title: A substrate radical intermediate in catalysis by the antibiotic resistance protein Cfr.
abstract: Cfr-dependent methylation of C8 of A2503 in 23S ribosomal RNA confers bacterial resistance to an array of clinically important antibiotics that target the large subunit of the ribosome, including the synthetic oxazolidinone antibiotic linezolid. The key element of the proposed mechanism for Cfr, a radical S-adenosylmethionine enzyme, is the addition of a methylene radical, generated by hydrogen-atom abstraction from the methyl group of an S-methylated cysteine, onto C8 of A2503 to form a protein-nucleic acid crosslinked species containing an unpaired electron. Herein we use continuous-wave and pulsed EPR techniques to provide direct spectroscopic evidence for this intermediate, showing a spin-delocalized radical with maximum spin density at N7 of the adenine ring. In addition, we use rapid freeze-quench EPR to show that the radical forms and decays with rate constants that are consistent with the rate of formation of the methylated product.
authors: Department of Chemistry, Pennsylvania State University, University Park, Pennsylvania, USA.
authors: Tyler L
authors: TL
authors: Grove
authors: Jovan
authors: J
authors: Livada
authors: Erica L
authors: EL
authors: Schwalm
authors: Michael T
authors: MT
authors: Green
authors: Squire J
authors: SJ
authors: Booker
authors: Alexey
authors: A
authors: Silakov
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry*; D002384:Catalysis; D004352:Drug Resistance, Microbial; D004574:Electromagnetic Fields; D004578:Electron Spin Resonance Spectroscopy; D004926:Escherichia coli / Q000378:metabolism; D005609:Free Radicals; D006859:Hydrogen / Q000737:chemistry; D008956:Models, Chemical; D009696:Nucleic Acids / Q000737:chemistry; D012338:RNA, Ribosomal, 23S / Q000737:chemistry*; D013053:Spectrophotometry; D013211:Staphylococcus aureus / Q000737:chemistry*
chemical_list: D001426:Bacterial Proteins; C539943:CFR protein, Staphylococcus aureus; D005609:Free Radicals; D009696:Nucleic Acids; D012338:RNA, Ribosomal, 23S; D006859:Hydrogen
references: Crit Rev Biochem Mol Biol. 2008 Jan-Feb;43(1):63-88
references: 18307109
references: RNA. 2008 Jan;14(1):98-106
references: 18025251
references: EMBO J. 2010 Sep 15;29(18):3108-17
references: 20676057
references: Anal Biochem. 1976 May 7;72:248-54
references: 942051
references: Methods Enzymol. 1995;258:278-303
references: 8524156
references: Antimicrob Agents Chemother. 2008 Jun;52(6):2244-6
references: 18391032
references: Mol Microbiol. 2005 Aug;57(4):1064-73
references: 16091044
references: Curr Opin Chem Biol. 2009 Feb;13(1):58-73
references: 19297239
references: J Clin Microbiol. 2010 Aug;48(8):3041-3
references: 20519471
references: JAMA. 2010 Jun 9;303(22):2260-4
references: 20530779
references: Lancet. 2001 Dec 8;358(9297):1975-82
references: 11747939
references: RNA. 2009 Feb;15(2):327-36
references: 19144912
references: Arch Biochem Biophys. 1977 Jan 30;178(2):662-70
references: 13738
references: Antimicrob Agents Chemother. 2000 Sep;44(9):2530-3
references: 10952608
references: Science. 2006 Sep 29;313(5795):1935-42
references: 16959973
references: J Am Chem Soc. 2012 Oct 31;134(43):18074-81
references: 23088750
references: J Am Chem Soc. 2011 Dec 14;133(49):19586-9
references: 21916495
references: Mol Microbiol. 2007 Jun;64(6):1506-14
references: 17555436
references: Phys Rev B Condens Matter. 1988 Jan 15;37(2):785-789
references: 9944570
references: Science. 2011 May 27;332(6033):1089-92
references: 21527678
references: Science. 2005 Nov 4;310(5749):827-34
references: 16272117
references: J Antimicrob Chemother. 2010 Nov;65(11):2329-35
references: 20841419
references: Cell. 2001 Nov 30;107(5):679-88
references: 11733066
references: Antimicrob Agents Chemother. 2012 Jul;56(7):3917-22
references: 22491691
references: Nat Rev Microbiol. 2005 Nov;3(11):870-81
references: 16261170
references: Radiat Res. 1989 Mar;117(3):367-78
references: 2538857
references: J Biol Chem. 1995 Jul 28;270(30):17758-64
references: 7629075
references: Proc Natl Acad Sci U S A. 2011 Mar 8;108(10):3930-4
references: 21368151
references: Clin Infect Dis. 2010 Oct 1;51(7):796-800
references: 20726771
references: J Am Chem Soc. 2010 Mar 24;132(11):3953-64
references: 20184321
references: Science. 2011 Apr 29;332(6029):604-7
references: 21415317
references: J Phys Chem A. 2007 Jul 26;111(29):6806-12
references: 17388361
references: Antimicrob Agents Chemother. 2006 Jul;50(7):2500-5
references: 16801432
references: Phys Rev A Gen Phys. 1988 Sep 15;38(6):3098-3100
references: 9900728
references: Biochemistry. 2004 Oct 26;43(42):13496-509
references: 15491157
references: Clin Infect Dis. 2010 Mar 15;50(6):821-5
references: 20144045
references: Antimicrob Agents Chemother. 2008 May;52(5):1703-12
references: 18299405
references: Science. 2009 Aug 28;325(5944):1089-93
references: 19713519
references: Cell. 1998 Jun 26;93(7):1105-15
references: 9657144
references: J Magn Reson. 2006 Jan;178(1):42-55
references: 16188474
references: Nat Prod Rep. 2011 Oct;28(10):1696-721
references: 21779595
references: Antimicrob Agents Chemother. 1995 Mar;39(3):577-85
references: 7793855
references: Angew Chem Int Ed Engl. 2010 Jun 14;49(26):4381-98
references: 20509130
references: Antimicrob Agents Chemother. 2010 Dec;54(12):4978-84
references: 20921317
references: Antimicrob Agents Chemother. 2011 Aug;55(8):3684-90
references: 21670176
references: Phys Rev B Condens Matter. 1988 Dec 15;38(17):12228-12232
references: 9946160
references: Diagn Microbiol Infect Dis. 2009 Dec;65(4):392-403
references: 19913682
references: Nucleic Acids Res. 2010 Mar;38(5):1652-63
references: 20007606
references: Antimicrob Agents Chemother. 2012 Jul;56(7):3563-7
references: 22547628