The crystal structure of E. coli copper amine oxidase reveals a number of unexpected features and provides a basis for investigating the intriguing similarities and differences in catalytic mechanism of members of this enzyme family. In addition to the three conserved histidines that bind the copper, our studies identify a number of other conserved residues close to the active site, including a candidate for the catalytic base and a fourth conserved histidine which is involved in an interesting intersubunit interaction.

The crystal structures of Hydroxyquinol 1,2-dioxygenase (1,2-HQD) from Nocardioides simplex 3E [1] and of 3-[2] and 4-chlorocatechol 1,2-dioxygenases [3,4] from the Gram-positive bacterium Rhodococcus opacus 1CP, three Fe(III) ion containing enzymes involved in the aerobic biodegradation of different chloroaromatic compounds, have been recently solved.The analysis of the structures and their comparison with the catechol 1,2-dioxygenase from Acinetobacter calcoaceticus ADP1 (1,2-CTD), highlights significant differences between these enzymes. The active site cavities present several dissimilarities, with respect to the known catechol cleaving enzyme, suggesting the key-role of specific amino-acidic residues in substrate selection. A co-crystallized benzoate-or hydroxamate-like ions, were found bound to the metal center of the three enzymes and revealed details on novel modes of inhibitors binding. The 1,2-HQD structure show one of the most distinctive characteristics among all intradiol dioxygenases; two extensive openings and the consequent exposure to solvent of the upper part of the catalytic cavity are arranged to favor the binding of hydroxyquinols but not catechols. Among the amino acid residues expected to interact with substrates that are different from the corresponding analogues of 1,2-CTD, a few were selected as responsible for the observed substrate selectivity differences in the three distinctive enzymes.[1] Ferraroni M., Seifert J., Travkin V.M., Thiel M., Kaschabek S., Scozzafava A., Golovleva L., Schlöman M., Briganti F., J. Biol. Chem., 2005, in press Superoxide dismutase scavenges the superoxide radical (O 2 -) to form molecular oxygen (O 2 ) and hydrogen peroxide (H 2 O 2 ) and forms part of the defense mechanism of cells against free radical oxidative damages. We identified the iron superoxide dismutase from obligate anaerobic bacterium Desulfovibrio vulgaris Miyazaki F and constructed an expression system in Escherichia coli [1]. Crystallization was carried out using hanging drop vapor diffusion method with PEG6000 (space group P2 1 ; a=51.96 Å, b=83.07 Å, c=61.16 Å, â=114.5 o ). The crystal structure has been determined by molecular replacement and refined to 1.0 Å resolution. The crystallographic R and free R are 17.9% and 19.2%, respectively. There are two identical monomers in the asymmetric unit. The monomer has a molecular weight of 22 kDa and consists of 205 amino acid residues of which 201 are visible in the electron density map. The overall fold of the monomer of D. vulgaris Fe-SOD is similar to that of other known Fe/Mn-SODs. The active site is composed of one iron, four metal ligand residues (His34, His84, Asp170 and His174) and one water molecule. The interaction of the dimer interface is also similar to that of other Fe/Mn-SODs. The structure differences compared with other Fe/Mn-SODs are at the loop regions on the surface of the molecule (Asp68-Ala72 and Gly143-Asp145).[1] Nakanishi T., et. Alzheimer's disease is a debilitating neurodegenerative disorder. Soluble oligomers of A...

Three hemoglobin molecules (Gower I. Gower II and Portland) are synthesised by the human embryo between weeks two and twelve of gestation. These hemoglobins appear to function as scavengers of 02 from the mother's interstitial fluid before the placenta has developed. Functional Gower II hemoglobin is a telramer comprising the adult u. chain and the embryomc £, which has 79% sequence identity to the adult ~-Binding studies have indicated that Gower II hemoglobin binds Oz cooperatively, with a higher affinity than the adult molecule, and displays simihu-al-_ losteric behaviour towards H+. Cl-and 2,3 DPG.Gower II hemoglobin with carbon monoxide bound was crystallised. The crystals proved to be tetragonal (spacegroup P432I2) a=b=62.8, c=320.8A with one U.2£2 tetramer in the asymmetric unit. The 3D structure has been solved at 2.9A resolution by molecular replacement and refined to a crystallographic R-factor of 0.204 (Rfree of 0.279) with good geometry.The quatemary structure is very, similar to that of the adult molecule. Within the £ subunit the main difference from the adult ~ is a small shift of theN terminal helix over the central cavity of the tetramer. The environment of the heme pocket is like that of the adult with the major variation being a closer packing of Ser 70 (Ala in ~). Clear density is visible for the bound CO ligand. We present the crystal structure and structural analysis of a bacterial copper-containing amine oxidase (AO). AOs are homodimeric proteins with molecular weights of between 70-90 kDa per subunit. Their function is the oxidative metabolism of amines in the presence of molecular dioxygen. They are involved in many fundamental cellular processes including: tissue differentiation, tissue development, wound healing, cancer and possibly programmed cell death. AOs are of particular importance in grampositive methylotrophs, including Artlzrobacter spp., as these organisms are able to utilise methylamine as their sole carbon and energy source. PS04.02.46 CRYSTAL STRUCTURE OF A BACTERIAL COPPER-CONTAINING AMINE OXIDASE FROMA. globofonnis AO crystallises in a number of fom1s with and without the presence of ammonium salts that are known to inactivate the enzyme. The structure of the crystal form known as type II is reported here. The crystals were grown from LiS04. The space group is C2 (a=l57.6, b=64.3, c=92.6 A. ~=112.6°) with one subunit per asymmetric unit. Molecular replacement was used to detem1ine an initial phase set. Both the pea seedling and E. coli AO [Parsons, M.R. et al. (1995). Structure, 3, 1171-1184] structures were successfully used as search models. The structure has been refined at 2.8A resolution. The structure of A. globofonnis AO is compmed with both the E. coli AO and pea seedling AO structures with pmticulm reference to the active site. The crystal structure of the complex between the copper amine oxidase from Escherichia coli (ECAO) and a covalently bound inhibitor, 2-hydrazinopyridine, has been determined to a resolution of 2.5A. The enzyme contains a cofactor, 2,4,5-t...

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