@article{OtreloCardosoSchwuchowRodriguesetal.2014,
  author    = {Otrelo-Cardoso, Ana Rita and Schwuchow, Viola and Rodrigues, David and Cabrita, Eurico J. and Leimk{\"u}hler, Silke and Romao, Maria Joao and Santos-Silva, Teresa},
  title     = {Biochemical, stabilization and crystallization studies on a molecular chaperone (PaoD) involved in the maturation of molybdoenzymes},
  series = {PLoS one},
  volume    = {9},
  journal   = {PLoS one},
  number    = {1},
  publisher = {PLoS},
  address   = {San Fransisco},
  issn      = {1932-6203},
  doi       = {10.1371/journal.pone.0087295},
  pages     = {9},
  year      = {2014},
  abstract  = {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.},
  language  = {en}
}
@article{MahroCoelhoTrincaoetal.2011,
  author    = {Mahro, Martin and Coelho, Catarina and Trincao, Jose and Rodrigues, David and Terao, Mineko and Garattini, Enrico and Saggu, Miguel and Lendzian, Friedhelm and Hildebrandt, Peter and Romao, Maria Joao and Leimk{\"u}hler, Silke},
  title     = {Characterization and crystallization of mouse aldehyde oxidase 3 - from mouse liver to escherichia coli heterologous protein expression},
  series = {Drug metabolism and disposition : the biological fate of chemicals},
  volume    = {39},
  journal   = {Drug metabolism and disposition : the biological fate of chemicals},
  number    = {10},
  publisher = {American Society for Pharmacology and Experimental Therapeutics},
  address   = {Bethesda},
  issn      = {0090-9556},
  doi       = {10.1124/dmd.111.040873},
  pages     = {1939 -- 1945},
  year      = {2011},
  abstract  = {Aldehyde oxidase (AOX) is characterized by a broad substrate specificity, oxidizing aromatic azaheterocycles, such as N(1)-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. In the past decade, AOX has been recognized increasingly to play an important role in the metabolism of drugs through its complex cofactor content, tissue distribution, and substrate recognition. In humans, only one AOX gene (AOX1) is present, but in mouse and other mammals different AOX homologs were identified. The multiple AOX isoforms are expressed tissue-specifically in different organisms, and it is believed that they recognize distinct substrates and carry out different physiological tasks. AOX is a dimer with a molecular mass of approximately 300 kDa, and each subunit of the homodimeric enzyme contains four different cofactors: the molybdenum cofactor, two distinct [2Fe-2S] clusters, and one FAD. We purified the AOX homolog from mouse liver (mAOX3) and established a system for the heterologous expression of mAOX3 in Escherichia coli. The purified enzymes were compared. Both proteins show the same characteristics and catalytic properties, with the difference that the recombinant protein was expressed and purified in a 30\% active form, whereas the native protein is 100\% active. Spectroscopic characterization showed that FeSII is not assembled completely in mAOX3. In addition, both proteins were crystallized. The best crystals were from native mAOX3 and diffracted beyond 2.9 angstrom. The crystals belong to space group P1, and two dimers are present in the unit cell.},
  language  = {en}
}