Ntral.com/1472-6807/8/Figure uric acid1 Omit-map showing the electron density
Ntral.com/1472-6807/8/Figure uric acid1 Omit-map showing the electron density corresponding to the two partners of the [UOX/UA/CN] complex, the cyanide and Omit-map showing the electron density corresponding to the two partners of the [UOX/UA/CN] complex, the cyanide and uric acid. The active site is delimited by 1) the conserved residues implicated in the molecular tweezers (Arg 176, Glu 228) that holds the substrate, 2) the Phe 159 closing one end of the cavity below, and 3) the two Asn 254 and Thr 57* forming another tweezers above the mean plane of the ligand that construct a location where efficient electron transfer can take place at a low energy level via the catalytic triad Thr 57* Lys 10* His 256, and the associated water molecules shown in Figure 3). The orientation of cyanide is based on a coherent evolution of the C/N thermal factors during refinements, compared to neighboring atoms (stereo view).oxygen-pressurized [UOX/O2/8-AZA] structures 2zka, 2zkb or 3cks [10], a feature underlining the flexibility of this motif. When the reagent tweezers is void of any ligand, like in 1r56 or 1xy3 structures, the distance increase up to 6 ? In addition, W1 and the O of Thr 57* (building the peroxo hole) are the starting point of a complex proton relay including Lys 10*, His 256, and two water molecules, ending to W2 and to the N9 atom of the ligand, as shown in Figure 2 and 3. However, Thr 57* in [UOX/UA/CN], cannot act as a base because PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/27385778 of the charged cyanide ion. The threonine 57* becomes a proton donor instead and as a result, the extended electron relay through the already mentioned Lys 10* and His 256 residues is interrupted as shown by the increased distance of 3.5 ?between Lys 10* and Thr 57* (Figure 3).All the ligands so far observed in the peroxo hole (water, oxygen, cyanide, chloride ..) are Necrosulfonamide site always tightly bound as illustrated by low thermal B factors, full occupancy factors ?Table 1) and well defined electron densities, including the Lys 10* side-chain.DiscussionAs mentioned above, the catalytic mechanism of urate oxidase is of particular interest because UOX is a cofactorless enzyme that requires no special assistance [12]. Isotopic labeling experiments have proved that the oxygen atoms of hydrogen peroxide derived from dioxygen and that the oxygen atom attached to C5 in the product derived from a water molecule [13]. NMR and spectroscopic studies have shown that UOX transforms the urate anion in a metastable compound identified as 5-hydroxyisourate (5-HIU) [1,14-16] and not allantoin. In addi-Page 3 of(page number not for citation purposes)BMC Structural Biology 2008, 8:http://www.biomedcentral.com/1472-6807/8/HGlnO HN O O H N HN H O N N HThrN O HN O O-OH N C N N OHAspN H O57C O O H H N3 2 NN HNN HOArgH N HO(W2)H2 OO HN O OH H N C N N H O O NH2O (W1)HNO N C N OH O(W2)Figure 2 The reaction pathway from compiled data [10,18,19] The reaction pathway from compiled data[10,18,19]. Although the use of Lewis-type representation is inadequate for strongly mesomeric structures (radicals or anions), this sketches about the different intermediates 1to 4involved in the mechanism leading to 5-HIU, 5.tion, it was suggested from stop-flow kinetics experiments that urate hydro-peroxide would be a primary intermediate, presumably through the addition of a superoxide radical O2-. [16]. Although this intermediate was not firmly established, the structural similarities between UOX, pterin and other reduced flavin proteins reinforces this sugge.