@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{OtreloCardosodaSilvaCorreiaSchwuchowetal.2014, author = {Otrelo-Cardoso, Ana Rita and da Silva Correia, Marcia Alexandra and Schwuchow, Viola and Svergun, Dmitri I. and Romao, Maria Joao and Leimk{\"u}hler, Silke and Santos-Silva, Teresa}, title = {Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis}, series = {International journal of molecular sciences}, volume = {15}, journal = {International journal of molecular sciences}, number = {2}, publisher = {MDPI}, address = {Basel}, issn = {1422-0067}, doi = {10.3390/ijms15022223}, pages = {2223 -- 2236}, year = {2014}, abstract = {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.}, language = {en} } @article{MotaEsmaeeliMoghaddamTabalvandaniCoelhoetal.2019, author = {Mota, Cristiano and Esmaeeli Moghaddam Tabalvandani, Mariam and Coelho, Catarina and Santos-Silva, Teresa and Wolff, Martin and Foti, Alessandro and Leimk{\"u}hler, Silke and Romao, Maria Joao}, title = {Human aldehyde oxidase (hAOX1)}, series = {FEBS Open Bio}, volume = {9}, journal = {FEBS Open Bio}, number = {5}, publisher = {Wiley}, address = {Hoboken}, issn = {2211-5463}, doi = {10.1002/2211-5463.12617}, pages = {925 -- 934}, year = {2019}, abstract = {Human aldehyde oxidase (hAOX1) is a molybdenum enzyme with high toxicological importance, but its physiological role is still unknown. hAOX1 metabolizes different classes of xenobiotics and is one of the main drug-metabolizing enzymes in the liver, along with cytochrome P450. hAOX1 oxidizes and inactivates a large number of drug molecules and has been responsible for the failure of several phase I clinical trials. The interindividual variability of drug-metabolizing enzymes caused by single nucleotide polymorphisms (SNPs) is highly relevant in pharmaceutical treatments. In this study, we present the crystal structure of the inactive variant G1269R, revealing the first structure of a molybdenum cofactor (Moco)-free form of hAOX1. These data allowed to model, for the first time, the flexible Gate 1 that controls access to the active site. Furthermore, we inspected the thermostability of wild-type hAOX1 and hAOX1 with various SNPs (L438V, R1231H, G1269R or S1271L) by CD spectroscopy and ThermoFAD, revealing that amino acid exchanges close to the Moco site can impact protein stability up to 10 degrees C. These results correlated with biochemical and structural data and enhance our understanding of hAOX1 and the effect of SNPs in the gene encoding this enzyme in the human population. EnzymesAldehyde oxidase (); xanthine dehydrogenase (); xanthine oxidase (). DatabasesStructural data are available in the Protein Data Bank under the accession number .}, 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} } @article{MahroBrasCerqueiraetal.2013, author = {Mahro, Martin and Bras, Natercia F. and Cerqueira, Nuno M. F. S. A. and Teutloff, Christian and Coelho, Catarina and Romao, Maria Joao and Leimk{\"u}hler, Silke}, title = {Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity}, series = {PLoS one}, volume = {8}, journal = {PLoS one}, number = {12}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0082285}, pages = {12}, year = {2013}, abstract = {In order to elucidate factors that determine substrate specificity and activity of mammalian molybdo-flavoproteins we performed site directed mutagenesis of mouse aldehyde oxidase 3 (mAOX3). The sequence alignment of different aldehyde oxidase (AOX) isoforms identified variations in the active site of mAOX3 in comparison to other AOX proteins and xanthine oxidoreductases (XOR). Based on the structural alignment of mAOX3 and bovine XOR, differences in amino acid residues involved in substrate binding in XORs in comparison to AOXs were identified. We exchanged several residues in the active site to the ones found in other AOX homologues in mouse or to residues present in bovine XOR in order to examine their influence on substrate selectivity and catalytic activity. Additionally we analyzed the influence of the [2Fe-2S] domains of mAOX3 on its kinetic properties and cofactor saturation. We applied UV-VIS and EPR monitored redox-titrations to determine the redox potentials of wild type mAOX3 and mAOX3 variants containing the iron-sulfur centers of mAOX1. In addition, a combination of molecular docking and molecular dynamic simulations (MD) was used to investigate factors that modulate the substrate specificity and activity of wild type and AOX variants. The successful conversion of an AOX enzyme to an XOR enzyme was achieved exchanging eight residues in the active site of mAOX3. It was observed that the absence of the K889H exchange substantially decreased the activity of the enzyme towards all substrates analyzed, revealing that this residue has an important role in catalysis.}, language = {en} } @article{FotiHartmannCoelhoetal.2016, author = {Foti, Alessandro and Hartmann, Tobias and Coelho, Catarina and Santos-Silva, Teresa and Romao, Maria Joao and Leimk{\"u}hler, Silke}, title = {Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {44}, journal = {Drug metabolism and disposition : the biological fate of chemicals}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, address = {Bethesda}, issn = {0090-9556}, doi = {10.1124/dmd.115.068395}, pages = {1277 -- 1285}, year = {2016}, abstract = {Aldehyde oxidase (AOX1) is an enzyme with broad substrate specificity, catalyzing the oxidation of a wide range of endogenous and exogenous aldehydes as well as N-heterocyclic aromatic compounds. In humans, the enzyme's role in phase I drug metabolism has been established and its importance is now emerging. However, the true physiologic function of AOX1 in mammals is still unknown. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified in human AOX1. SNPs are a major source of interindividual variability in the human population, and SNP-based amino acid exchanges in AOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. For the reliable analysis of the effect of amino acid exchanges in human proteins, the existence of reproducible expression systems for the production of active protein in ample amounts for kinetic, spectroscopic, and crystallographic studies is required. In our study we report an optimized expression system for hAOX1 in Escherichia coli using a codon-optimized construct. The codon-optimization resulted in an up to 15-fold increase of protein production and a simplified purification procedure. The optimized expression system was used to study three SNPs that result in amino acid changes C44W, G1269R, and S1271L. In addition, the crystal structure of the S1271L SNP was solved. We demonstrate that the recombinant enzyme can be used for future studies to exploit the role of AOX in drug metabolism, and for the identification and synthesis of new drugs targeting AOX when combined with crystallographic and modeling studies.}, language = {en} } @article{CorreiaOtreloCardosoSchwuchowetal.2016, author = {Correia, Marcia A. S. and Otrelo-Cardoso, Ana Rita and Schwuchow, Viola and Clauss, Kajsa G. V. Sigfridsson and Haumann, Michael and Romao, Maria Joao and Leimk{\"u}hler, Silke and Santos-Silva, Teresa}, title = {The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes}, series = {ACS chemical biology}, volume = {11}, journal = {ACS chemical biology}, publisher = {American Chemical Society}, address = {Washington}, issn = {1554-8929}, doi = {10.1021/acschembio.6b00572}, pages = {2923 -- 2935}, year = {2016}, abstract = {The xanthine oxidase (XO) family comprises molybdenum-dependent enzymes that usually form homodimers (or dimers of heterodimers/trimers) organized in three domains that harbor two [2Fe-2S] clusters, one FAD, and a Mo cofactor. In this work, we crystallized an unusual member of the family, the periplasmic aldehyde oxidoreductase PaoABC from Escherichia coli. This is the first example of an E. coli protein containing a molybdopterin-cytosine-dinucleotide cofactor and is the only heterotrimer of the XO family so far structurally characterized. The crystal structure revealed the presence of an unexpected [4Fe-4S] cluster, anchored to an additional 40 residues subdomain. According to phylogenetic analysis, proteins containing this cluster are widely spread in many bacteria phyla, putatively through repeated gene transfer events. The active site of PaoABC is highly exposed to the surface with no aromatic residues and an arginine (PaoC-R440) making a direct interaction with PaoC-E692, which acts as a base catalyst. In order to understand the importance of R440, kinetic assays were carried out, and the crystal structure of the PaoC-R440H variant was also determined.}, language = {en} } @article{CoelhoMahroTrincaoetal.2012, author = {Coelho, Catarina and Mahro, Martin and Trincao, Jose and Carvalho, Alexandra T. P. and Ramos, Maria Joao and Terao, Mineko and Garattini, Enrico and Leimk{\"u}hler, Silke and Romao, Maria Joao}, title = {The first mammalian aldehyde oxidase crystal structure insights into substrate specificity}, series = {The journal of biological chemistry}, volume = {287}, journal = {The journal of biological chemistry}, number = {48}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Bethesda}, issn = {0021-9258}, doi = {10.1074/jbc.M112.390419}, pages = {40690 -- 40702}, year = {2012}, abstract = {Aldehyde oxidases (AOXs) are homodimeric proteins belonging to the xanthine oxidase family of molybdenum-containing enzymes. Each 150-kDa monomer contains a FAD redox cofactor, two spectroscopically distinct [2Fe-2S] clusters, and a molybdenum cofactor located within the protein active site. AOXs are characterized by broad range substrate specificity, oxidizing different aldehydes and aromatic N-heterocycles. Despite increasing recognition of its role in the metabolism of drugs and xenobiotics, the physiological function of the protein is still largely unknown. We have crystallized and solved the crystal structure of mouse liver aldehyde oxidase 3 to 2.9 angstrom. This is the first mammalian AOX whose structure has been solved. The structure provides important insights into the protein active center and further evidence on the catalytic differences characterizing AOX and xanthine oxidoreductase. The mouse liver aldehyde oxidase 3 three-dimensional structure combined with kinetic, mutagenesis data, molecular docking, and molecular dynamics studies make a decisive contribution to understand the molecular basis of its rather broad substrate specificity.}, language = {en} } @article{CoelhoFotiHartmannetal.2015, author = {Coelho, Catarina and Foti, Alessandro and Hartmann, Tobias and Santos-Silva, Teresa and Leimk{\"u}hler, Silke and Romao, Maria Joao}, title = {Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase}, series = {Nature chemical biology}, volume = {11}, journal = {Nature chemical biology}, number = {10}, publisher = {Nature Publ. Group}, address = {New York}, issn = {1552-4450}, doi = {10.1038/NCHEMBIO.1895}, pages = {779 -- +}, year = {2015}, abstract = {Aldehyde oxidase (AOX) is a xanthine oxidase (XO)-related enzyme with emerging importance due to its role in the metabolism of drugs and xenobiotics. We report the first crystal structures of human AOX1, substrate free (2.6-angstrom resolution) and in complex with the substrate phthalazine and the inhibitor thioridazine (2.7-angstrom resolution). Analysis of the protein active site combined with steady-state kinetic studies highlight the unique features, including binding and substrate orientation at the active site, that characterize human AOX1 as an important drug-metabolizing enzyme. Structural analysis of the complex with the noncompetitive inhibitor thioridazine revealed a new, unexpected and fully occupied inhibitor-binding site that is structurally conserved among mammalian AOXs and XO. The new structural insights into the catalytic and inhibition mechanisms of human AOX that we now report will be of great value for the rational analysis of clinical drug interactions involving inhibition of AOX1 and for the prediction and design of AOX-stable putative drugs.}, language = {en} }