Structural Basis of Eukaryotic Nitrate Reduction: Crystal Structures of the Nitrate Reductase Active Site
| Autor: | Katrin Fischer, Wilbur H. Campbell, Guillaume G. Barbier, Hans-Juergen Hecht, Guenter Schwarz, Ralf R. Mendel |
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| Jazyk: | angličtina |
| Rok vydání: | 2005 |
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Molecular Stereochemistry Nitrogen assimilation Molecular Sequence Data Plant Science Biology Nitrate reductase Arginine Crystallography X-Ray Cofactor Pichia chemistry.chemical_compound Nitrate Body Water Oxidoreductase Nitrate Reductases Animals Oxidoreductases Acting on Sulfur Group Donors Research Articles chemistry.chemical_classification Flavin adenine dinucleotide Molybdenum Binding Sites Nitrates Sequence Homology Amino Acid Tryptophan Active site Cell Biology Protein Structure Tertiary Eukaryotic Cells chemistry Biochemistry Electron Transport Chain Complex Proteins biology.protein NAD+ kinase Chickens Oxidation-Reduction NADP |
| Popis: | Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 angstroms. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis. |
| Databáze: | OpenAIRE |
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