title: Crystal structures of isoorotate decarboxylases reveal a novel catalytic mechanism of 5-carboxyl-uracil decarboxylation and shed light on the search for DNA decarboxylase.
abstract: DNA methylation and demethylation regulate many crucial biological processes in mammals and are linked to many diseases. Active DNA demethylation is believed to be catalyzed by TET proteins and a putative DNA decarboxylase that may share some similarities in sequence, structure and catalytic mechanism with isoorotate decarboxylase (IDCase) that catalyzes decarboxylation of 5caU to U in fungi. We report here the structures of wild-type and mutant IDCases from Cordyceps militaris and Metarhizium anisopliae in apo form or in complexes with 5caU, U, and an inhibitor 5-nitro-uracil. IDCases adopt a typical (β/α)8 barrel fold of the amidohydrolase superfamily and function as dimers. A Zn(2+) is bound at the active site and coordinated by four strictly conserved residues, one Asp and three His. The substrate is recognized by several strictly conserved residues. The functional roles of the key residues at the active site are validated by mutagenesis and biochemical studies. Based on the structural and biochemical data, we present for the first time a novel catalytic mechanism of decarboxylation for IDCases, which might also apply to other members of the amidohydrolase superfamily. In addition, our biochemical data show that IDCases can catalyze decarboxylation of 5caC to C albeit with weak activity, which is the first in vitro evidence for direct decarboxylation of 5caC to C by an enzyme. These findings are valuable in the identification of potential DNA decarboxylase in mammals.
authors.affiliation: 1] State Key Laboratory of Molecular Biology, Institute of Biochemistry and Cell Biology, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China [2] Graduate School of Chinese Academy of Sciences, 320 Yue-Yang Road, Shanghai 200031, China.
authors.forename: Shutong
authors.initials: S
authors.lastname: Xu
authors.forename: Wenjing
authors.initials: W
authors.lastname: Li
authors.forename: Junjun
authors.initials: J
authors.lastname: Zhu
authors.forename: Rong
authors.initials: R
authors.lastname: Wang
authors.forename: Zheng
authors.initials: Z
authors.lastname: Li
authors.forename: Guo-Liang
authors.initials: GL
authors.lastname: Xu
authors.forename: Jianping
authors.initials: J
authors.lastname: Ding
mesh_terms: D055162:Biocatalysis; D002262:Carboxy-Lyases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D032661:Cordyceps / Q000201:enzymology; D018360:Crystallography, X-Ray; D004247:DNA / Q000378:metabolism*; D003653:Decarboxylation; D052981:Metarhizium / Q000201:enzymology; D008958:Models, Molecular; D009154:Mutation; D014498:Uracil / Q000031:analogs & derivatives* / Q000737:chemistry / Q000378:metabolism*
chemical_list: D014498:Uracil; D004247:DNA; D002262:Carboxy-Lyases; C117582:isoorotate decarboxylase
references.citation: Nat Rev Genet. 2005 Aug;6(8):597-610
references.pmid: 16136652
references.citation: Biochim Biophys Acta. 2005 May 25;1723(1-3):256-64
references.pmid: 15794921
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Trends Genet. 2005 Aug;21(8):457-65
references.pmid: 15990197
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Curr Med Chem. 2005;12(19):2241-58
references.pmid: 16178783
references.citation: Biochemistry. 2005 Aug 9;44(31):10562-9
references.pmid: 16060665
references.citation: Science. 2009 May 15;324(5929):930-5
references.pmid: 19372391
references.citation: Angew Chem Int Ed Engl. 2012 Jun 25;51(26):6516-20
references.pmid: 22644704
references.citation: J Biol Chem. 1962 Jul;237:2289-90
references.pmid: 13892978
references.citation: FEBS J. 2009 Nov;276(22):6615-23
references.pmid: 19843166
references.citation: Nat Rev Mol Cell Biol. 2010 Sep;11(9):607-20
references.pmid: 20683471
references.citation: Annu Rev Plant Biol. 2006;57:805-36
references.pmid: 16669783
references.citation: Biochemistry. 1972 May 23;11(11):2172-6
references.pmid: 4260374
references.citation: J Chem Theory Comput. 2011 Feb 8;7(2):525-37
references.pmid: 26596171
references.citation: Adv Exp Med Biol. 2008;625:141-54
references.pmid: 18365665
references.citation: Biochim Biophys Acta. 1967 Mar 15;132(2):525-8
references.pmid: 6031142
references.citation: Acta Crystallogr D Biol Crystallogr. 2011 Apr;67(Pt 4):235-42
references.pmid: 21460441
references.citation: J Cell Physiol. 2007 Nov;213(2):384-90
references.pmid: 17708532
references.citation: J Biol Chem. 1971 May 25;246(10):3314-9
references.pmid: 5574400
references.citation: Annu Rev Biochem. 1995;64:721-62
references.pmid: 7574499
references.citation: Science. 2011 Sep 2;333(6047):1303-7
references.pmid: 21817016
references.citation: Angew Chem Int Ed Engl. 2011 Jul 25;50(31):7008-12
references.pmid: 21721093
references.citation: Biochemistry. 2005 May 3;44(17):6383-91
references.pmid: 15850372
references.citation: J Biol Chem. 1993 Nov 15;268(32):24005-11
references.pmid: 8226944
references.citation: Nature. 2010 Aug 26;466(7310):1129-33
references.pmid: 20639862
references.citation: J Biol Chem. 1970 Dec 25;245(24):6706-10
references.pmid: 5482775
references.citation: J Am Chem Soc. 2003 Feb 5;125(5):1244-52
references.pmid: 12553826
references.citation: J Biol Chem. 1995 Aug 4;270(31):18309-12
references.pmid: 7629151
references.citation: Annu Rev Med. 2008;59:267-80
references.pmid: 17937590
references.citation: Biochemistry. 2006 Sep 5;45(35):10412-21
references.pmid: 16939194
references.citation: Anal Biochem. 1999 Jan 1;266(1):85-92
references.pmid: 9887216
references.citation: J Mol Biol. 2007 Sep 21;372(3):774-97
references.pmid: 17681537
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Nat Chem Biol. 2012 Feb 12;8(4):328-30
references.pmid: 22327402
references.citation: J Mol Biol. 2002 Jan 25;315(4):687-97
references.pmid: 11812140
references.citation: Annu Rev Biochem. 1980;49:253-79
references.pmid: 6105839
references.citation: J Biol Chem. 1970 Apr 25;245(8):2023-6
references.pmid: 5440840
references.citation: Acta Crystallogr D Biol Crystallogr. 2010 Feb;66(Pt 2):213-21
references.pmid: 20124702
references.citation: Arch Biochem Biophys. 1973 Nov;159(1):180-7
references.pmid: 4274083
references.citation: Nat Rev Genet. 2008 Jun;9(6):465-76
references.pmid: 18463664
references.citation: Science. 1991 May 31;252(5010):1278-84
references.pmid: 1925539
references.citation: Science. 2011 Sep 2;333(6047):1300-3
references.pmid: 21778364
references.citation: Annu Rev Genomics Hum Genet. 2005;6:69-92
references.pmid: 16124854
references.citation: Cell. 2008 Jun 27;133(7):1145-8
references.pmid: 18585349