Structure refinement of the aldehyde oxidoreductase from Desulfovibrio gigas (MOP) at 1.28 A.
The sulfate-reducing bacterium aldehyde oxidoreductase from Desulfovibrio gigas (MOP) is a member of the xanthine oxidase family of enzymes. It has 907 residues on a single polypeptide chain, a molybdopterin cytosine dinucleotide (MCD) cofactor and two [2Fe-2S] iron-sulfur clusters. Synchrotron data to almost atomic resolution were collected for improved cryo-cooled crystals of this enzyme in the oxidized form. The cell constants of a=b=141.78 A and c=160.87 A are about 2% shorter than those of room temperature data, yielding 233,755 unique reflections in space group P6(1)22, at 1.28 A resolution. Throughout the entire refinement the full gradient least-squares method was used, leading to a final R factor of 14.5 and Rfree factor of 19.3 (4sigma cut-off) with "riding" H-atoms at their calculated positions. The model contains 8146 non-hydrogen atoms described by anisotropic displacement parameters with an observations/parameters ratio of 4.4. It includes alternate conformations for 17 amino acid residues. At 1.28 A resolution, three Cl- and two Mg2+ ions from the crystallization solution were clearly identified. With the exception of one Cl- which is buried and 8 A distant from the Mo atom, the other ions are close to the molecular surface and may contribute to crystal packing. The overall structure has not changed in comparison to the lower resolution model apart from local corrections that included some loop adjustments and alternate side-chain conformations. Based on the estimated errors of bond distances obtained by blocked least-squares matrix inversion, a more detailed analysis of the three redox centres was possible. For the MCD cofactor, the resulting geometric parameters confirmed its reduction state as a tetrahydropterin. At the Mo centre, estimated corrections calculated for the Fourier ripples artefact are very small when compared to the experimental associated errors, supporting the suggestion that the fifth ligand is a water molecule rather than a hydroxide. Concerning the two iron-sulfur centres, asymmetry in the Fe-S distances as well as differences in the pattern of NH.S hydrogen-bonding interactions was observed, which influences the electron distribution upon reduction and causes non-equivalence of the individual Fe atoms in each cluster.
Rebelo
J M
JM
Departamento de Química, CQFB, Faculdade de Ciencias e Tecnologia, Universidade Nova de Lisboa, 2825-114 Monte da Caparica, Portugal.
Dias
J M
JM
Huber
R
R
Moura
J J
JJ
Romão
M J
MJ
D000445:Aldehyde Oxidoreductases / Q000737:chemistry*; D001426:Bacterial Proteins / Q000737:chemistry; D002712:Chlorides / Q000737:chemistry; D003460:Crystallization; D018360:Crystallography, X-Ray; D003901:Desulfovibrio / Q000737:chemistry / Q000201:enzymology*; D007506:Iron-Sulfur Proteins / Q000737:chemistry*; D016018:Least-Squares Analysis; D008274:Magnesium / Q000737:chemistry; D008958:Models, Molecular; D008982:Molybdenum / Q000737:chemistry*; D011487:Protein Conformation; D011622:Pterins / Q000737:chemistry
D001426:Bacterial Proteins; D002712:Chlorides; D007506:Iron-Sulfur Proteins; D011622:Pterins; C011552:tetrahydropterin; D008982:Molybdenum; D000445:Aldehyde Oxidoreductases; D008274:Magnesium