Electrostatic suppression allows tyrosine site-specific recombination in the absence of a conserved catalytic arginine.
The active site of the tyrosine family site-specific recombinase Flp contains a conserved catalytic pentad that includes two arginine residues, Arg-191 and Arg-308. Both arginines are essential for the transesterification steps of strand cleavage and strand joining in DNA substrates containing a phosphate group at the scissile position. During strand cleavage, the active site tyrosine supplies the nucleophile to form a covalent 3'-phosphotyrosyl intermediate. The 5'-hydroxyl group produced by cleavage provides the nucleophile to re-form a 3'-5' phosphodiester bond in a recombinant DNA strand. In previous work we showed that substitution of the scissile phosphate (P) by the charge neutral methylphosphonate (MeP) makes Arg-308 dispensable during the catalytic activation of the MeP diester bond. However, in the Flp(R308A) reaction, water out-competes the tyrosine nucleophile (Tyr-343) to cause direct hydrolysis of the MeP diester bond. We now report that for MeP activation Arg-191 is also not required. In contrast to Flp(R308A), Flp(R191A) primarily mediates normal cleavage by Tyr-343 but also exhibits a weaker direct hydrolytic activity. The cleaved MeP-tyrosyl intermediate formed by Flp(R191A) can be targeted for nucleophilic attack by a 5'-hydroxyl or water and channeled toward strand joining or hydrolysis, respectively. In collaboration with wild type Flp, Flp(R191A) promotes strand exchange between MeP- and P-DNA partners. Loss of a catalytically crucial positively charged side chain can thus be suppressed by a compensatory modification in the DNA substrate that neutralizes the negative charge on the scissile phosphate.
Rowley
Paul A
PA
Section of Molecular Genetics and Microbiology, University of Texas at Austin, Austin, Texas 78712, USA.
Kachroo
Aashiq H
AH
Ma
Chien-Hui
CH
Maciaszek
Anna D
AD
Guga
Piotr
P
Jayaram
Makkuni
M
D001120:Arginine; D001483:Base Sequence; D055162:Biocatalysis; D020134:Catalytic Domain; D017124:Conserved Sequence; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000378:metabolism; D006868:Hydrolysis; D008958:Models, Molecular; D009154:Mutation; D009943:Organophosphorus Compounds / Q000378:metabolism; D045522:Recombinases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011995:Recombination, Genetic; D055672:Static Electricity; D013379:Substrate Specificity; D014443:Tyrosine
D009943:Organophosphorus Compounds; D045522:Recombinases; C032627:methylphosphonic acid; D014443:Tyrosine; D004247:DNA; D001120:Arginine; D004720:Endonucleases
Mol Cell. 2000 Oct;6(4):885-97
11090626
Mol Cell. 2006 Aug 4;23(3):343-54
16885024
Annu Rev Biophys Biomol Struct. 2003;32:135-59
12598365
Annu Rev Biochem. 2006;75:567-605
16756503
EMBO J. 2009 Jun 17;28(12):1745-56
19440204
Nat Biotechnol. 1998 Jul;16(7):657-62
9661200
J Biol Chem. 2003 Jul 4;278(27):24800-7
12716882
Mol Cell Biol. 1992 Sep;12(9):3757-65
1508181
J Biol Chem. 2005 Mar 4;280(9):8290-9
15591069
Proc Natl Acad Sci U S A. 1987 Apr;84(8):2189-93
3104911
Nature. 1997 Sep 4;389(6646):40-6
9288963
Structure. 2005 Apr;13(4):513-20
15837190
Mol Cell. 2003 Jul;12(1):199-208
12887905
J Biol Chem. 1993 Jan 5;268(1):455-63
8416950
J Mol Biol. 2003 Feb 14;326(2):425-34
12559911
Mol Microbiol. 1999 Aug;33(3):449-56
10577069
EMBO J. 1999 Feb 1;18(3):784-91
9927438
J Mol Biol. 2006 Apr 7;357(4):1202-10
16487540
J Mol Biol. 1992 May 20;225(2):313-26
1593623
Mol Cell Biol. 1988 Aug;8(8):3303-10
2974924
J Biol Chem. 1990 Mar 15;265(8):4527-33
2407737
EMBO J. 1986 Feb;5(2):433-40
3011407
Cell. 1998 Mar 20;92(6):841-50
9529259
J Biol Chem. 1998 Nov 13;273(46):30591-8
9804830
PLoS One. 2009 Sep 30;4(9):e7248
19789629
Mol Cell. 1998 Apr;1(5):729-39
9660956