The 1.5-A structure of XplA-heme, an unusual cytochrome P450 heme domain that catalyzes reductive biotransformation of royal demolition explosive.
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.
Sabbadin
Federico
F
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.
Jackson
Rosamond
R
Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Haider
Kamran
K
York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Tampi
Girish
G
Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Turkenburg
Johan P
JP
York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Hart
Sam
S
York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Bruce
Neil C
NC
Centre for Novel Agricultural Products, Department of Biology, University of York, York YO10 5YW, United Kingdom.
Grogan
Gideon
G
York Structural Biology Laboratory, Department of Biology, University of York, York YO10 5YW, United Kingdom. Electronic address: grogan@ysbl.york.ac.uk.
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*
D053834:Explosive Agents; D007527:Isoenzymes; D014227:Triazines; D006418:Heme; D003577:Cytochrome P-450 Enzyme System; C009160:cyclonite
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