title: Requirements for catalysis in the Cre recombinase active site.
abstract: Members of the tyrosine recombinase (YR) family of site-specific recombinases catalyze DNA rearrangements using phosphoryl transfer chemistry that is identical to that used by the type IB topoisomerases (TopIBs). To better understand the requirements for YR catalysis and the relationship between the YRs and the TopIBs, we have analyzed the in vivo and in vitro recombination activities of all substitutions of the seven active site residues in Cre recombinase. We have also determined the structure of a vanadate transition state mimic for the Cre-loxP reaction that facilitates interpretation of mutant activities and allows for a comparison with similar structures from the related topoisomerases. We find that active site residues shared by the TopIBs are most sensitive to substitution. Only two, the tyrosine nucleophile and a conserved lysine residue that activates the 5'-hydroxyl leaving group, are strictly required to achieve >5% of wild-type activity. The two conserved arginine residues each tolerate one substitution that results in modest recombination activity and the remaining three active site positions can be substituted with several alternative amino acids while retaining a significant amount of activity. The results are discussed in the context of YR and TopIB structural models and data from related YR systems.
authors.affiliation: Department of Biochemistry and Biophysics and Howard Hughes Medical Institute, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
authors.forename: Bryan
authors.initials: B
authors.lastname: Gibb
authors.forename: Kushol
authors.initials: K
authors.lastname: Gupta
authors.forename: Kaushik
authors.initials: K
authors.lastname: Ghosh
authors.forename: Robert
authors.initials: R
authors.lastname: Sharp
authors.forename: James
authors.initials: J
authors.lastname: Chen
authors.forename: Gregory D
authors.initials: GD
authors.lastname: Van Duyne
mesh_terms: D000595:Amino Acid Sequence; D019943:Amino Acid Substitution; D001120:Arginine / Q000737:chemistry; D055162:Biocatalysis; D020134:Catalytic Domain; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D019426:Integrases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008239:Lysine / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D011995:Recombination, Genetic; D014364:Tryptophan / Q000737:chemistry; D014443:Tyrosine / Q000737:chemistry; D014638:Vanadates / Q000737:chemistry
chemical_list: D018698:Glutamic Acid; D014638:Vanadates; D014443:Tyrosine; D006639:Histidine; D014364:Tryptophan; D001120:Arginine; C045073:Cre recombinase; D019426:Integrases; D008239:Lysine
references.citation: Nature. 1997 Sep 4;389(6646):40-6
references.pmid: 9288963
references.citation: Nucleic Acids Res. 2007 Jan;35(Database issue):D61-5
references.pmid: 17130148
references.citation: Mol Cell. 2000 Jun;5(6):1035-41
references.pmid: 10911997
references.citation: PLoS One. 2009 Sep 30;4(9):e7248
references.pmid: 19789629
references.citation: Annu Rev Biophys Biomol Struct. 2001;30:87-104
references.pmid: 11340053
references.citation: Proc Natl Acad Sci U S A. 1999 Jun 22;96(13):7143-8
references.pmid: 10377382
references.citation: Mol Cell. 2003 Jul;12(1):187-98
references.pmid: 12887904
references.citation: Chem Biol. 2007 Feb;14(2):121-9
references.pmid: 17317566
references.citation: Mol Cell. 2003 Jul;12(1):199-208
references.pmid: 12887905
references.citation: Proc Natl Acad Sci U S A. 1987 Oct;84(19):6840-4
references.pmid: 2821547
references.citation: J Mol Biol. 1992 Aug 5;226(3):661-73
references.pmid: 1324323
references.citation: J Mol Biol. 2007 Apr 20;368(1):183-96
references.pmid: 17367810
references.citation: J Biol Chem. 1997 Mar 28;272(13):8263-9
references.pmid: 9079646
references.citation: J Mol Biol. 1999 Jun 11;289(3):517-27
references.pmid: 10356326
references.citation: J Biol Chem. 2003 Sep 19;278(38):36905-15
references.pmid: 12851389
references.citation: J Mol Biol. 2006 Apr 7;357(4):1202-10
references.pmid: 16487540
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Plasmid. 1999 Jan;41(1):78-81
references.pmid: 9887310
references.citation: Acta Crystallogr D Biol Crystallogr. 1998 Sep 1;54(Pt 5):905-21
references.pmid: 9757107
references.citation: Philos Trans R Soc Lond B Biol Sci. 2004 Jan 29;359(1441):61-9
references.pmid: 15065657
references.citation: Mol Cell. 2000 Oct;6(4):885-97
references.pmid: 11090626
references.citation: J Mol Biol. 1992 May 20;225(2):313-26
references.pmid: 1593623
references.citation: J Biol Chem. 1984 Feb 10;259(3):1509-14
references.pmid: 6319400
references.citation: Mol Cell Biol. 1988 Aug;8(8):3303-10
references.pmid: 2974924
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Biochemistry. 2000 May 9;39(18):5561-72
references.pmid: 10820030
references.citation: Mol Cell. 2006 Aug 4;23(3):343-54
references.pmid: 16885024
references.citation: EMBO J. 1998 Jul 15;17(14):4175-87
references.pmid: 9670032
references.citation: Nucleic Acids Res. 1998 Aug 15;26(16):3700-6
references.pmid: 9685485
references.citation: J Biol Chem. 2008 Jun 6;283(23):16093-103
references.pmid: 18367446
references.citation: Methods Enzymol. 1991;201:477-82
references.pmid: 1943774
references.citation: Annu Rev Biophys Biomol Struct. 2003;32:135-59
references.pmid: 12598365
references.citation: Methods. 2002 Nov;28(3):374-83
references.pmid: 12431441
references.citation: Annu Rev Biochem. 2006;75:567-605
references.pmid: 16756503
references.citation: EMBO J. 2009 Jun 17;28(12):1745-56
references.pmid: 19440204
references.citation: Science. 1998 Mar 6;279(5356):1504-13
references.pmid: 9488644
references.citation: Nature. 2005 Jun 23;435(7045):1059-66
references.pmid: 15973401
references.citation: J Biol Chem. 2007 Aug 17;282(33):24004-16
references.pmid: 17573343
references.citation: Mol Gen Genet. 1998 Jan;257(2):205-12
references.pmid: 9491079
references.citation: Cell. 1998 Mar 20;92(6):841-50
references.pmid: 9529259
references.citation: Gene. 1984 Feb;27(2):161-72
references.pmid: 6202587
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Structure. 2010 Jan 13;18(1):127-37
references.pmid: 20152159
references.citation: Nucleic Acids Res. 1997 Sep 1;25(17):3389-402
references.pmid: 9254694
references.citation: J Biol Chem. 1997 Feb 14;272(7):3891-6
references.pmid: 9020090
references.citation: Cell. 1998 Apr 17;93(2):149-52
references.pmid: 9568707
references.citation: Nucleic Acids Res. 1998 Jan 15;26(2):391-406
references.pmid: 9421491
references.citation: J Mol Biol. 2005 Nov 25;354(2):233-45
references.pmid: 16242714
references.citation: J Biol Chem. 2003 Jul 4;278(27):24800-7
references.pmid: 12716882
references.citation: Biochim Biophys Acta. 1998 Oct 1;1400(1-3):321-37
references.pmid: 9748643
references.citation: Nat Chem Biol. 2005 Oct;1(5):275-82
references.pmid: 16408057
references.citation: Mol Cell Biol. 1992 Sep;12(9):3757-65
references.pmid: 1508181
references.citation: J Mol Biol. 2000 Jun 2;299(2):391-403
references.pmid: 10860747
references.citation: J Biol Chem. 2002 Feb 22;277(8):5711-4
references.pmid: 11756402
references.citation: J Am Chem Soc. 2005 Mar 16;127(10):3321-31
references.pmid: 15755148
references.citation: Cell. 1992 May 15;69(4):647-58
references.pmid: 1586945