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 |
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Authors: | Storbeck Sonja Saha Sayantan Krausze Joern Klink Björn U Heinz Dirk W Layer Gunhild |
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Institution: | From the ?Institute of Microbiology, Technische Universität Braunschweig, 38106 Braunschweig, Germany.;the §Department of Molecular Structural Biology, Helmholtz Centre for Infection Research, 38124 Braunschweig, Germany, and ;the ¶Duke University Medical Center, Durham, North Carolina 27705 |
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Abstract: | During the biosynthesis of heme d1, the essential cofactor of cytochrome cd1 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. |
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Keywords: | Enzyme Catalysis Enzyme Mechanisms Enzyme Structure S-Adenosylmethionine (SAM) Site-directed Mutagenesis Pseudomonas aeruginosa SUMT Heme d1 Tetrapyrrole Uroporphyrinogen III |
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