title: Crystal structure of the first dissimilatory nitrate reductase at 1.9 A solved by MAD methods.
abstract: BACKGROUND
  The periplasmic nitrate reductase (NAP) from the sulphate reducing bacterium Desulfovibrio desulfuricans ATCC 27774 is induced by growth on nitrate and catalyses the reduction of nitrate to nitrite for respiration. NAP is a molybdenum-containing enzyme with one bis-molybdopterin guanine dinucleotide (MGD) cofactor and one [4Fe-4S] cluster in a single polypeptide chain of 723 amino acid residues. To date, there is no crystal structure of a nitrate reductase.
  
  
  RESULTS
  The first crystal structure of a dissimilatory (respiratory) nitrate reductase was determined at 1.9 A resolution by multiwavelength anomalous diffraction (MAD) methods. The structure is folded into four domains with an alpha/beta-type topology and all four domains are involved in cofactor binding. The [4Fe-4S] centre is located near the periphery of the molecule, whereas the MGD cofactor extends across the interior of the molecule interacting with residues from all four domains. The molybdenum atom is located at the bottom of a 15 A deep crevice, and is positioned 12 A from the [4Fe-4S] cluster. The structure of NAP reveals the details of the catalytic molybdenum site, which is coordinated to two MGD cofactors, Cys140, and a water/hydroxo ligand. A facile electron-transfer pathway through bonds connects the molybdenum and the [4Fe-4S] cluster.
  
  
  CONCLUSIONS
  The polypeptide fold of NAP and the arrangement of the cofactors is related to that of Escherichia coli formate dehydrogenase (FDH) and distantly resembles dimethylsulphoxide reductase. The close structural homology of NAP and FDH shows how small changes in the vicinity of the molybdenum catalytic site are sufficient for the substrate specificity.
authors.affiliation: Departamento de Quimica, CQFB, FCT, Universidade Nova de Lisboa, Caparica, Portugal.
authors.forename: J M
authors.initials: JM
authors.lastname: Dias
authors.forename: M E
authors.initials: ME
authors.lastname: Than
authors.forename: A
authors.initials: A
authors.lastname: Humm
authors.forename: R
authors.initials: R
authors.lastname: Huber
authors.forename: G P
authors.initials: GP
authors.lastname: Bourenkov
authors.forename: H D
authors.initials: HD
authors.lastname: Bartunik
authors.forename: S
authors.initials: S
authors.lastname: Bursakov
authors.forename: J
authors.initials: J
authors.lastname: Calvete
authors.forename: J
authors.initials: J
authors.lastname: Caldeira
authors.forename: C
authors.initials: C
authors.lastname: Carneiro
authors.forename: J J
authors.initials: JJ
authors.lastname: Moura
authors.forename: I
authors.initials: I
authors.lastname: Moura
authors.forename: M J
authors.initials: MJ
authors.lastname: Romão
mesh_terms: D000595:Amino Acid Sequence; D003001:Cloning, Molecular; D018360:Crystallography, X-Ray; D003901:Desulfovibrio / Q000201:enzymology*; D006150:Guanine Nucleotides / Q000737:chemistry / Q000378:metabolism; D007506:Iron-Sulfur Proteins / Q000737:chemistry / Q000378:metabolism; D008958:Models, Molecular; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000378:metabolism; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism; D009942:Organometallic Compounds / Q000737:chemistry / Q000378:metabolism; D016133:Polymerase Chain Reaction; D011487:Protein Conformation; D017433:Protein Structure, Secondary; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid; D014961:X-Ray Diffraction
chemical_list: D006150:Guanine Nucleotides; D007506:Iron-Sulfur Proteins; D009942:Organometallic Compounds; D011994:Recombinant Proteins; C106961:bis(molybdopterin guanine dinucleotide)molybdenum; D008982:Molybdenum; D009565:Nitrate Reductases; D050901:Nitrate Reductase