title: Crystal structure of the heme d1 biosynthesis enzyme NirE in complex with its substrate reveals new insights into the catalytic mechanism of S-adenosyl-L-methionine-dependent uroporphyrinogen III methyltransferases.
abstract: During the biosynthesis of heme d(1), the essential cofactor of cytochrome cd(1) nitrite reductase, the NirE protein catalyzes the methylation of uroporphyrinogen III to precorrin-2 using S-adenosyl-L-methionine (SAM) as the methyl group donor. The crystal structure of Pseudomonas aeruginosa NirE in complex with its substrate uroporphyrinogen III and the reaction by-product S-adenosyl-L-homocysteine (SAH) was solved to 2.0 Å resolution. This represents the first enzyme-substrate complex structure for a SAM-dependent uroporphyrinogen III methyltransferase. The large substrate binds on top of the SAH in a "puckered" conformation in which the two pyrrole rings facing each other point into the same direction either upward or downward. Three arginine residues, a histidine, and a methionine are involved in the coordination of uroporphyrinogen III. Through site-directed mutagenesis of the nirE gene and biochemical characterization of the corresponding NirE variants the amino acid residues Arg-111, Glu-114, and Arg-149 were identified to be involved in NirE catalysis. Based on our structural and biochemical findings, we propose a potential catalytic mechanism for NirE in which the methyl transfer reaction is initiated by an arginine catalyzed proton abstraction from the C-20 position of the substrate.
authors.affiliation: Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany.
authors.forename: Sonja
authors.initials: S
authors.lastname: Storbeck
authors.forename: Sayantan
authors.initials: S
authors.lastname: Saha
authors.forename: Joern
authors.initials: J
authors.lastname: Krausze
authors.forename: Björn U
authors.initials: BU
authors.lastname: Klink
authors.forename: Dirk W
authors.initials: DW
authors.lastname: Heinz
authors.forename: Gunhild
authors.initials: G
authors.lastname: Layer
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D002384:Catalysis; D018360:Crystallography, X-Ray; D006418:Heme / Q000031:analogs & derivatives / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism; D008780:Methyltransferases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D020125:Mutation, Missense; D017434:Protein Structure, Tertiary; D011550:Pseudomonas aeruginosa / Q000201:enzymology* / Q000235:genetics; D014577:Uroporphyrinogens / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D014577:Uroporphyrinogens; D006418:Heme; C029400:heme d1; D008780:Methyltransferases