title: The 1.5-A structure of XplA-heme, an unusual cytochrome P450 heme domain that catalyzes reductive biotransformation of royal demolition explosive.
abstract: XplA is a cytochrome P450 of unique structural organization, consisting of a heme-domain that is C-terminally fused to its native flavodoxin redox partner. XplA, along with flavodoxin reductase XplB, has been shown to catalyze the breakdown of the nitramine explosive and pollutant hexahydro-1,3,5-trinitro-1,3,5-triazine (royal demolition explosive) by reductive denitration. The structure of the heme domain of XplA (XplA-heme) has been solved in two crystal forms: as a dimer in space group P2(1) to a resolution of 1.9 A and as a monomer in space group P2(1)2(1)2 to a resolution of 1.5 A, with the ligand imidazole bound at the heme iron. Although it shares the overall fold of cytochromes P450 of known structure, XplA-heme is unusual in that the kinked I-helix that traverses the distal face of the heme is broken by Met-394 and Ala-395 in place of the well conserved Asp/Glu plus Thr/Ser, important in oxidative P450s for the scission of the dioxygen bond prior to substrate oxygenation. The heme environment of XplA-heme is hydrophobic, featuring a cluster of three methionines above the heme, including Met-394. Imidazole was observed bound to the heme iron and is in close proximity to the side chain of Gln-438, which is situated over the distal face of the heme. Imidazole is also hydrogen-bonded to a water molecule that sits in place of the threonine side-chain hydroxyl exemplified by Thr-252 in Cyt-P450cam. Both Gln-438 --> Ala and Ala-395 --> Thr mutants of XplA-heme displayed markedly reduced activity compared with the wild type for royal demolition explosive degradation when combined with surrogate electron donors.
authors.affiliation: York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom; Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Federico
authors.initials: F
authors.lastname: Sabbadin
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Rosamond
authors.initials: R
authors.lastname: Jackson
authors.affiliation: York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Kamran
authors.initials: K
authors.lastname: Haider
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Girish
authors.initials: G
authors.lastname: Tampi
authors.affiliation: York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Johan P
authors.initials: JP
authors.lastname: Turkenburg
authors.affiliation: York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Sam
authors.initials: S
authors.lastname: Hart
authors.affiliation: Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
authors.forename: Neil C
authors.initials: NC
authors.lastname: Bruce
authors.affiliation: York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom. Electronic address: grogan@ysbl.york.ac.uk.
authors.forename: Gideon
authors.initials: G
authors.lastname: Grogan
mesh_terms: D000595:Amino Acid Sequence; D001665:Binding Sites; D001711:Biotransformation; D018360:Crystallography, X-Ray; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D053834:Explosive Agents / Q000737:chemistry / Q000378:metabolism*; D006418:Heme / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007527:Isoenzymes / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D015394:Molecular Structure; D010084:Oxidation-Reduction; D017434:Protein Structure, Tertiary; D016415:Sequence Alignment; D014227:Triazines / Q000737:chemistry / Q000378:metabolism*
chemical_list: D053834:Explosive Agents; D007527:Isoenzymes; D014227:Triazines; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C009160:cyclonite
references.citation: Antimicrob Agents Chemother. 2004 Jul;48(7):2747-50
references.pmid: 15215142
references.citation: J Biol Chem. 2005 Jun 10;280(23):22102-7
references.pmid: 15824115
references.citation: Acta Crystallogr D Biol Crystallogr. 1997 May 1;53(Pt 3):240-55
references.pmid: 15299926
references.citation: Appl Environ Microbiol. 1995 Apr;61(4):1318-22
references.pmid: 7747953
references.citation: Appl Environ Microbiol. 2003 Mar;69(3):1347-51
references.pmid: 12620815
references.citation: Nucleic Acids Res. 1996 Jan 1;24(1):21-5
references.pmid: 8594581
references.citation: Acta Crystallogr D Biol Crystallogr. 2006 Sep;62(Pt 9):1002-11
references.pmid: 16929101
references.citation: J Biol Chem. 1985 Dec 25;260(30):16122-30
references.pmid: 4066706
references.citation: Proc Natl Acad Sci U S A. 2002 Apr 16;99(8):5361-6
references.pmid: 11959989
references.citation: J Biol Chem. 2000 Nov 17;275(46):35999-6006
references.pmid: 10956654
references.citation: Biochemistry. 2008 Mar 25;47(12):3789-99
references.pmid: 18311941
references.citation: Biochem Biophys Res Commun. 1989 Sep 29;163(3):1210-6
references.pmid: 2506859
references.citation: Curr Microbiol. 1998 Aug;37(2):127-31
references.pmid: 9662613
references.citation: Biochemistry. 1998 Jun 30;37(26):9211-9
references.pmid: 9649301
references.citation: J Physiol. 2006 Mar 1;571(Pt 2):329-48
references.pmid: 16396928
references.citation: J Biol Chem. 2003 Feb 14;278(7):5141-7
references.pmid: 12435731
references.citation: Appl Environ Microbiol. 2008 Jul;74(14):4550-2
references.pmid: 18487400
references.citation: Structure. 1998 Sep 15;6(9):1079-85
references.pmid: 9753700
references.citation: J Biol Chem. 1994 Jun 3;269(22):15546-52
references.pmid: 8195199
references.citation: Biochim Biophys Acta. 2007 Mar;1770(3):390-401
references.pmid: 16920266
references.citation: Methods Enzymol. 1997;276:307-26
references.pmid: 27754618
references.citation: Chem Res Toxicol. 2001 Jun;14(6):611-50
references.pmid: 11409933
references.citation: J Biol Chem. 2009 Apr 24;284(17):11676-89
references.pmid: 19181667
references.citation: J Mol Biol. 2008 Dec 31;384(5):1218-31
references.pmid: 18951902
references.citation: Proc Natl Acad Sci U S A. 2007 Oct 23;104(43):16822-7
references.pmid: 17940033
references.citation: Proc Natl Acad Sci U S A. 2008 Oct 14;105(41):15696-701
references.pmid: 18838690
references.citation: Proc Natl Acad Sci U S A. 2007 Jul 10;104(28):11591-6
references.pmid: 17606921
references.citation: J Biol Chem. 2008 Apr 18;283(16):10804-12
references.pmid: 18270198
references.citation: Chem Biol Interact. 1996 Mar 25;100(2):113-24
references.pmid: 8646785
references.citation: Acta Crystallogr D Biol Crystallogr. 2008 Jan;64(Pt 1):125-32
references.pmid: 18094476
references.citation: J Mol Biol. 2001 Aug 3;311(1):101-10
references.pmid: 11469860
references.citation: J Inorg Biochem. 2000 Nov;82(1-4):103-11
references.pmid: 11132616
references.citation: Acta Crystallogr D Biol Crystallogr. 2004 Dec;60(Pt 12 Pt 1):2126-32
references.pmid: 15572765
references.citation: Nat Biotechnol. 2006 Feb;24(2):216-9
references.pmid: 16429147
references.citation: Nat Prod Rep. 2007 Jun;24(3):585-609
references.pmid: 17534532
references.citation: Biochem Biophys Res Commun. 1980 Nov 17;97(1):294-300
references.pmid: 7458936
references.citation: Appl Environ Microbiol. 2002 Oct;68(10):4764-71
references.pmid: 12324318
references.citation: J Biol Chem. 2005 Sep 9;280(36):31659-63
references.pmid: 15994329
references.citation: Appl Environ Microbiol. 2009 May;75(10):3258-62
references.pmid: 19270122
references.citation: Org Biomol Chem. 2006 Apr 7;4(7):1252-60
references.pmid: 16557313
references.citation: Nat Struct Biol. 1999 May;6(5):458-63
references.pmid: 10331874
references.citation: FEMS Microbiol Ecol. 2004 Sep 1;49(3):349-57
references.pmid: 19712285