TY  - JOUR
A1  - De Sousa Mota, Cristiano
A1  - Diniz, Ana
A1  - Coelho, Catarina
A1  - Santos-Silva, Teresa
A1  - Esmaeeli Moghaddam Tabalvandani, Mariam
A1  - Leimkühler, Silke
A1  - Cabrita, Eurico J.
A1  - Marcelo, Filipa
A1  - Romão, Maria João
T1  - Interrogating the inhibition mechanisms of human aldehyde oxidase by X-ray crystallography and NMR spectroscopy
BT  - the raloxifene case
JF  - Journal of medicinal chemistry / American Chemical Society
N2  - Human aldehyde oxidase (hAOX1) is mainly present in the liver and has an emerging role in drug metabolism, since it accepts a wide range of molecules as substrates and inhibitors. Herein, we employed an integrative approach by combining NMR, X-ray crystallography, and enzyme inhibition kinetics to understand the inhibition modes of three hAOX1 inhibitors-thioridazine, benzamidine, and raloxifene. These integrative data indicate that thioridazine is a noncompetitive inhibitor, while benzamidine presents a mixed type of inhibition. Additionally, we describe the first crystal structure of hAOX1 in complex with raloxifene. Raloxifene binds tightly at the entrance of the substrate tunnel, stabilizing the flexible entrance gates and elucidating an unusual substrate-dependent mechanism of inhibition with potential impact on drug-drug interactions. This study can be considered as a proof-of-concept for an efficient experimental screening of prospective substrates and inhibitors of hAOX1 relevant in drug discovery.
Y1  - 2021
U6  - https://doi.org/10.1021/acs.jmedchem.1c01125
SN  - 0022-2623
SN  - 1520-4804
VL  - 64
IS  - 17
SP  - 13025
EP  - 13037
PB  - American Chemical Society
CY  - Washington
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  -