title: Biochemical and structural studies of the Mycobacterium tuberculosis O6-methylguanine methyltransferase and mutated variants.
abstract: Mycobacterium tuberculosis displays remarkable genetic stability despite continuous exposure to the hostile environment represented by the host's infected macrophages. Similarly to other organisms, M. tuberculosis possesses multiple systems to counteract the harmful potential of DNA alkylation. In particular, the suicidal enzyme O(6)-methylguanine-DNA methyltransferase (OGT) is responsible for the direct repair of O(6)-alkylguanine in double-stranded DNA and is therefore supposed to play a central role in protecting the mycobacterial genome from the risk of G · C-to-A · T transition mutations. Notably, a number of geographically widely distributed M. tuberculosis strains shows nonsynonymous single-nucleotide polymorphisms in their OGT-encoding gene, leading to amino acid substitutions at position 15 (T15S) or position 37 (R37L) of the N-terminal domain of the corresponding protein. However, the role of these mutations in M. tuberculosis pathogenesis is unknown. We describe here the in vitro characterization of M. tuberculosis OGT (MtOGT) and of two point-mutated versions of the protein mimicking the naturally occurring ones, revealing that both mutated proteins are impaired in their activity as a consequence of their lower affinity for alkylated DNA than the wild-type protein. The analysis of the crystal structures of MtOGT and MtOGT-R37L confirms the high level of structural conservation of members of this protein family and provides clues to an understanding of the molecular bases for the reduced affinity for the natural substrate displayed by mutated MtOGT. Our in vitro results could contribute to validate the inferred participation of mutated OGTs in M. tuberculosis phylogeny and biology.
authors.affiliation: Dipartimento di Scienze del Farmaco, University of Piemonte Orientale A Avogadro, Novara, Italy.
authors.forename: Riccardo
authors.initials: R
authors.lastname: Miggiano
authors.forename: Valentina
authors.initials: V
authors.lastname: Casazza
authors.forename: Silvia
authors.initials: S
authors.lastname: Garavaglia
authors.forename: Maria
authors.initials: M
authors.lastname: Ciaramella
authors.forename: Giuseppe
authors.initials: G
authors.lastname: Perugino
authors.forename: Menico
authors.initials: M
authors.lastname: Rizzi
authors.forename: Franca
authors.initials: F
authors.lastname: Rossi
mesh_terms: D000595:Amino Acid Sequence; D018360:Crystallography, X-Ray; D004252:DNA Mutational Analysis; D004269:DNA, Bacterial / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D020125:Mutation, Missense; D009169:Mycobacterium tuberculosis / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D019853:O(6)-Methylguanine-DNA Methyltransferase / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D017354:Point Mutation; D011485:Protein Binding; D011487:Protein Conformation
chemical_list: D004269:DNA, Bacterial; D050505:Mutant Proteins; D019853:O(6)-Methylguanine-DNA Methyltransferase
references.citation: J Biol Chem. 2004 Sep 17;279(38):40174-84
references.pmid: 15247240
references.citation: Biochem J. 1998 Feb 1;329 ( Pt 3):545-50
references.pmid: 9445381
references.citation: DNA Repair (Amst). 2011 Jun 10;10(6):595-602
references.pmid: 21570366
references.citation: FEMS Microbiol Rev. 2009 May;33(3):471-87
references.pmid: 19385996
references.citation: Mutat Res. 1996 May 15;363(1):15-25
references.pmid: 8632775
references.citation: Magn Reson Chem. 2006 Jul;44 Spec No:S71-82
references.pmid: 16826543
references.citation: PLoS One. 2011 Jan 20;6(1):e16020
references.pmid: 21283803
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: FEMS Microbiol Rev. 2009 Nov;33(6):1044-78
references.pmid: 19659577
references.citation: EMBO J. 2000 Apr 3;19(7):1719-30
references.pmid: 10747039
references.citation: Infect Immun. 2003 Feb;71(2):997-1000
references.pmid: 12540583
references.citation: Front Microbiol. 2011 May 13;2:105
references.pmid: 21734908
references.citation: Acta Crystallogr D Biol Crystallogr. 1994 Sep 1;50(Pt 5):760-3
references.pmid: 15299374
references.citation: J Mol Biol. 2009 Jun 5;389(2):248-63
references.pmid: 19358853
references.citation: Mol Microbiol. 2003 Apr;48(1):77-84
references.pmid: 12657046
references.citation: Biochemistry. 2011 Mar 15;50(10):1590-8
references.pmid: 21226457
references.citation: J Mol Biol. 2005 Jul 22;350(4):657-66
references.pmid: 15964013
references.citation: Chem Res Toxicol. 2011 May 16;24(5):618-39
references.pmid: 21466232
references.citation: J Mol Biol. 1999 Sep 24;292(3):707-16
references.pmid: 10497033
references.citation: Biochemistry. 1999 May 25;38(21):6801-6
references.pmid: 10346901
references.citation: Clin Sci (Lond). 2010 May 25;119(5):187-202
references.pmid: 20522025
references.citation: Nature. 1970 Aug 15;227(5259):680-5
references.pmid: 5432063
references.citation: Mech Ageing Dev. 2012 Apr;133(4):138-46
references.pmid: 21982925
references.citation: Mol Microbiol. 1998 Sep;29(6):1331-9
references.pmid: 9781872
references.citation: Anal Biochem. 1976 May 7;72:248-54
references.pmid: 942051
references.citation: EMBO J. 1994 Apr 1;13(7):1495-501
references.pmid: 8156986
references.citation: Biochemistry. 2005 Nov 22;44(46):15396-405
references.pmid: 16285744
references.citation: DNA Repair (Amst). 2007 Aug 1;6(8):1222-8
references.pmid: 17500045
references.citation: PLoS One. 2008 Feb 06;3(2):e1538
references.pmid: 18253486
references.citation: Nat Med. 2007 Mar;13(3):282-4
references.pmid: 17342138
references.citation: Carcinogenesis. 2002 May;23(5):823-30
references.pmid: 12016156
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Biochem J. 2000 Apr 15;347(Pt 2):519-26
references.pmid: 10749682
references.citation: Nucleic Acids Res. 2000 Jan 15;28(2):393-401
references.pmid: 10606635
references.citation: J Exp Med. 2003 Sep 1;198(5):693-704
references.pmid: 12953091
references.citation: Emerg Infect Dis. 2003 Jul;9(7):838-45
references.pmid: 12890325
references.citation: Proc Natl Acad Sci U S A. 2012 Nov 13;109(46):18755-60
references.pmid: 23112169
references.citation: J Appl Crystallogr. 2007 Aug 1;40(Pt 4):658-674
references.pmid: 19461840
references.citation: Nature. 2009 Jun 11;459(7248):808-13
references.pmid: 19516334
references.citation: Tuberculosis (Edinb). 2007 Nov;87(6):502-8
references.pmid: 17919978
references.citation: Nat Struct Mol Biol. 2004 Aug;11(8):714-20
references.pmid: 15221026
references.citation: Nature. 1977 May 19;267(5608):281-3
references.pmid: 325420
references.citation: Carcinogenesis. 2010 Jan;31(1):59-70
references.pmid: 19875697
references.citation: Protein Expr Purif. 2005 May;41(1):207-34
references.pmid: 15915565
references.citation: J Biol Chem. 2012 Feb 3;287(6):4222-31
references.pmid: 22167184
references.citation: Nat Struct Biol. 1999 May;6(5):458-63
references.pmid: 10331874