TY  - JOUR
A1  - Otrelo-Cardoso, Ana Rita
A1  - da Silva Correia, Marcia Alexandra
A1  - Schwuchow, Viola
A1  - Svergun, Dmitri I.
A1  - Romao, Maria Joao
A1  - Leimkühler, Silke
A1  - Santos-Silva, Teresa
T1  - Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis
JF  - International journal of molecular sciences
N2  - The periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli is a molybdenum enzyme involved in detoxification of aldehydes in the cell. It is an example of an heterotrimeric enzyme of the xanthine oxidase family of enzymes which does not dimerize via its molybdenum cofactor binding domain. In order to structurally characterize PaoABC, X-ray crystallography and small angle X-ray scattering (SAXS) have been carried out. The protein crystallizes in the presence of 20% (w/v) polyethylene glycol 3350 using the hanging-drop vapour diffusion method. Although crystals were initially twinned, several experiments were done to overcome twinning and lowering the crystallization temperature (293 K to 277 K) was the solution to the problem. The non-twinned crystals used to solve the structure diffract X-rays to beyond 1.80 angstrom and belong to the C2 space group, with cell parameters a = 109.42 angstrom, b = 78.08 angstrom, c = 151.77 angstrom, = 99.77 degrees, and one molecule in the asymmetric unit. A molecular replacement solution was found for each subunit separately, using several proteins as search models. SAXS data of PaoABC were also collected showing that, in solution, the protein is also an heterotrimer.
KW  - periplasmic aldehyde oxidoreductase
KW  - X-ray crystallography
KW  - small angle X-ray scattering
KW  - crystal twinning
Y1  - 2014
U6  - https://doi.org/10.3390/ijms15022223
SN  - 1422-0067
VL  - 15
IS  - 2
SP  - 2223
EP  - 2236
PB  - MDPI
CY  - Basel
ER  - 
TY  - JOUR
A1  - Otrelo-Cardoso, Ana Rita
A1  - Schwuchow, Viola
A1  - Rodrigues, David
A1  - Cabrita, Eurico J.
A1  - Leimkühler, Silke
A1  - Romao, Maria Joao
A1  - Santos-Silva, Teresa
T1  - Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes
JF  - PLoS one
N2  - Molybdenum and tungsten enzymes require specific chaperones for folding and cofactor insertion. PaoD is the chaperone of the periplasmic aldehyde oxidoreductase PaoABC. It is the last gene in the paoABCD operon in Escherichia coli and its presence is crucial for obtaining mature enzyme. PaoD is an unstable, 35 kDa, protein. Our biochemical studies showed that it is a dimer in solution with a tendency to form large aggregates, especially after freezing/thawing cycles. In order to improve stability, PaoD was thawed in the presence of two ionic liquids [C(4)mim]Cl and [C(2)OHmim]PF6 and no protein precipitation was observed. This allowed protein concentration and crystallization using polyethylene glycol or ammonium sulfate as precipitating agents. Saturation transfer difference - nuclear magnetic resonance (STD-NMR) experiments have also been performed in order to investigate the effect of the ionic liquids in the stabilization process, showing a clear interaction between the acidic ring protons of the cation and, most likely, negatively charged residues at the protein surface. DLS assays also show a reduction of the overall size of the protein aggregates in presence of ionic liquids. Furthermore, cofactor binding studies on PaoD showed that the protein is able to discriminate between molybdenum and tungsten bound to the molybdenum cofactor, since only a Mo-MPT form of the cofactor remained bound to PaoD.
Y1  - 2014
U6  - https://doi.org/10.1371/journal.pone.0087295
SN  - 1932-6203
VL  - 9
IS  - 1
PB  - PLoS
CY  - San Fransisco
ER  -