title: Two Fe-S clusters catalyze sulfur insertion by radical-SAM methylthiotransferases.
abstract: How living organisms create carbon-sulfur bonds during the biosynthesis of critical sulfur-containing compounds is still poorly understood. The methylthiotransferases MiaB and RimO catalyze sulfur insertion into tRNAs and ribosomal protein S12, respectively. Both belong to a subgroup of radical-S-adenosylmethionine (radical-SAM) enzymes that bear two [4Fe-4S] clusters. One cluster binds S-adenosylmethionine and generates an Ado• radical via a well-established mechanism. However, the precise role of the second cluster is unclear. For some sulfur-inserting radical-SAM enzymes, this cluster has been proposed to act as a sacrificial source of sulfur for the reaction. In this paper, we report parallel enzymological, spectroscopic and crystallographic investigations of RimO and MiaB, which provide what is to our knowledge the first evidence that these enzymes are true catalysts and support a new sulfation mechanism involving activation of an exogenous sulfur cosubstrate at an exchangeable coordination site on the second cluster, which remains intact during the reaction.
authors.affiliation: Northeast Structural Genomics Consortium, New York, New York, USA.
authors.forename: Farhad
authors.initials: F
authors.lastname: Forouhar
authors.forename: Simon
authors.initials: S
authors.lastname: Arragain
authors.forename: Mohamed
authors.initials: M
authors.lastname: Atta
authors.forename: Serge
authors.initials: S
authors.lastname: Gambarelli
authors.forename: Jean-Marie
authors.initials: JM
authors.lastname: Mouesca
authors.forename: Munif
authors.initials: M
authors.lastname: Hussain
authors.forename: Rong
authors.initials: R
authors.lastname: Xiao
authors.forename: Sylvie
authors.initials: S
authors.lastname: Kieffer-Jaquinod
authors.forename: Jayaraman
authors.initials: J
authors.lastname: Seetharaman
authors.forename: Thomas B
authors.initials: TB
authors.lastname: Acton
authors.forename: Gaetano T
authors.initials: GT
authors.lastname: Montelione
authors.forename: Etienne
authors.initials: E
authors.lastname: Mulliez
authors.forename: John F
authors.initials: JF
authors.lastname: Hunt
authors.forename: Marc
authors.initials: M
authors.lastname: Fontecave
mesh_terms: D055162:Biocatalysis; D018360:Crystallography, X-Ray; D005609:Free Radicals / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry* / Q000378:metabolism*; D008958:Models, Molecular; D015394:Molecular Structure; D012436:S-Adenosylmethionine / Q000378:metabolism*; D013455:Sulfur / Q000737:chemistry / Q000378:metabolism*; D013466:Sulfurtransferases / Q000737:chemistry / Q000378:metabolism*; D020124:Thermotoga maritima / Q000201:enzymology / Q000378:metabolism*
chemical_list: D005609:Free Radicals; D007506:Iron-Sulfur Proteins; D013455:Sulfur; D012436:S-Adenosylmethionine; D013466:Sulfurtransferases
references.citation: Crit Rev Biochem Mol Biol. 2008 Jan-Feb;43(1):63-88
references.pmid: 18307109
references.citation: Nucleic Acids Res. 2001 Mar 1;29(5):1097-106
references.pmid: 11222759
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Curr Opin Chem Biol. 2009 Feb;13(1):74-83
references.pmid: 19269883
references.citation: Nat Rev Microbiol. 2011 Mar;9(3):153-65
references.pmid: 21283116
references.citation: Biochemistry. 2006 Mar 21;45(11):3824-34
references.pmid: 16533066
references.citation: Curr Opin Struct Biol. 2010 Dec;20(6):684-92
references.pmid: 20951571
references.citation: J Biol Chem. 2011 Sep 2;286(35):30245-30252
references.pmid: 21771780
references.citation: J Am Chem Soc. 2009 Jul 8;131(26):9184-5
references.pmid: 19566093
references.citation: J Biol Chem. 2010 Feb 19;285(8):5792-801
references.pmid: 20007320
references.citation: Curr Opin Chem Biol. 2007 Oct;11(5):543-52
references.pmid: 17936058
references.citation: Nucleic Acids Res. 2010 Jul;38(Web Server issue):W545-9
references.pmid: 20457744
references.citation: Anal Biochem. 1983 Jun;131(2):373-8
references.pmid: 6614472
references.citation: J Struct Biol. 1999 Apr-May;125(2-3):156-65
references.pmid: 10222271
references.citation: J Biol Chem. 2004 Nov 12;279(46):47555-63
references.pmid: 15339930
references.citation: Methods Enzymol. 2003;374:492-509
references.pmid: 14696386
references.citation: Biochemistry. 2009 Oct 27;48(42):10162-74
references.pmid: 19736993
references.citation: FEBS Lett. 1996 Apr 22;384(3):219-21
references.pmid: 8617357
references.citation: Chem Rev. 2003 Jun;103(6):2149-66
references.pmid: 12797827
references.citation: Chem Rev. 2011 Apr 13;111(4):2487-506
references.pmid: 21370834
references.citation: J Biomol NMR. 1996 Mar;7(2):131-41
references.pmid: 8616269
references.citation: Methods Enzymol. 2005;394:210-43
references.pmid: 15808222
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: J Chromatogr. 1989 Jun 2;471:3-36
references.pmid: 2670985
references.citation: Protein Sci. 1996 Aug;5(8):1625-32
references.pmid: 8844851
references.citation: Phys Rev B Condens Matter. 1986 Jun 15;33(12):8822-8824
references.pmid: 9938299
references.citation: J Am Chem Soc. 2012 Apr 11;134(14):6479-90
references.pmid: 22424175
references.citation: Science. 2004 Jan 2;303(5654):76-9
references.pmid: 14704425
references.citation: J Comput Chem. 2006 Sep;27(12):1385-97
references.pmid: 16788911
references.citation: Proc Natl Acad Sci U S A. 2004 Aug 31;101(35):12870-5
references.pmid: 15317939
references.citation: FEMS Microbiol Lett. 2001 Apr 13;197(2):215-21
references.pmid: 11313137
references.citation: J Clin Invest. 2011 Sep;121(9):3598-608
references.pmid: 21841312
references.citation: Biochemistry. 2001 Jul 27;40(28):8352-8
references.pmid: 11444982
references.citation: Biochemistry. 1996 Aug 27;35(34):11053-61
references.pmid: 8780507
references.citation: J Biol Chem. 2003 Aug 8;278(32):29515-24
references.pmid: 12766153
references.citation: Biochemistry. 2007 May 1;46(17):5140-7
references.pmid: 17407324
references.citation: Nat Struct Mol Biol. 2010 Sep;17(9):1072-8
references.pmid: 20694005
references.citation: Phys Rev A Gen Phys. 1988 Sep 15;38(6):3098-3100
references.pmid: 9900728
references.citation: Biochemistry. 2004 Oct 26;43(42):13496-509
references.pmid: 15491157
references.citation: Nucleic Acids Res. 2008 Jan;36(Database issue):D419-25
references.pmid: 18000004
references.citation: Inorg Chem. 1999 Jul 26;38(15):3569-3574
references.pmid: 11671106
references.citation: Acta Crystallogr Sect F Struct Biol Cryst Commun. 2008 Dec 1;64(Pt 12):1096-100
references.pmid: 19052358
references.citation: Nat Struct Biol. 2002 Oct;9(10):771-8
references.pmid: 12352954
references.citation: J Biol Chem. 2010 Sep 10;285(37):28425-33
references.pmid: 20584901
references.citation: Methods Enzymol. 1988;158:357-64
references.pmid: 3374387