@misc{RomaoCoelhoSantosSilvaetal.2017, author = {Romao, Maria Joao and Coelho, Catarina and Santos-Silva, Teresa and Foti, Alessandro and Terao, Mineko and Garattini, Enrico and Leimk{\"u}hler, Silke}, title = {Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics}, series = {Current Opinion in Chemical Biology}, volume = {37}, journal = {Current Opinion in Chemical Biology}, publisher = {Elsevier}, address = {Oxford}, issn = {1367-5931}, doi = {10.1016/j.cbpa.2017.01.005}, pages = {39 -- 47}, year = {2017}, abstract = {Aldehyde oxidases (AOXs) are molybdo-flavoenzymes characterized by broad substrate specificity, oxidizing aromatic/aliphatic aldehydes into the corresponding carboxylic acids and hydroxylating various heteroaromatic rings. Mammals are characterized by a complement of species specific AOX isoenzymes, that varies from one in humans (AOX1) to four in rodents (AOX1, AOX2, AOX3 and AOX4). The physiological function of mammalian AOX isoenzymes is unknown, although human AOX1 is an emerging enzyme in phase-I drug metabolism. Indeed, the number of therapeutic molecules under development which act as AOX substrates is increasing. The recent crystallization and structure determination of human AOX1 as well as mouse AOX3 has brought new insights into the mechanisms underlying substrate/inhibitor binding as well as the catalytic activity of this class of enzymes.}, language = {en} } @misc{MotaCoelhoLeimkuehleretal.2018, author = {Mota, Cristiano and Coelho, Catarina and Leimk{\"u}hler, Silke and Garattini, Enrico and Terao, Mineko and Santos-Silva, Teresa and Romao, Maria Joao}, title = {Critical overview on the structure and metabolism of human aldehyde oxidase and its role in pharmacokinetics}, series = {Coordination chemistry reviews}, volume = {368}, journal = {Coordination chemistry reviews}, publisher = {Elsevier}, address = {Lausanne}, issn = {0010-8545}, doi = {10.1016/j.ccr.2018.04.006}, pages = {35 -- 59}, year = {2018}, abstract = {Aldehyde oxidases are molybdenum and flavin dependent enzymes characterized by a very wide substrate specificity and performing diverse reactions that include oxidations (e.g., aldehydes and azaheterocycles), hydrolysis of amide bonds, and reductions (e.g., nitro, S-oxides and N-oxides). Oxidation reactions and amide hydrolysis occur at the molybdenum site while the reductions are proposed to occur at the flavin site. AOX activity affects the metabolism of different drugs and xenobiotics, some of which designed to resist other liver metabolizing enzymes (e.g., cytochrome P450 monooxygenase isoenzymes), raising its importance in drug development. This work consists of a comprehensive overview on aldehyde oxidases, concerning the genetic evolution of AOX, its diversity among the human population, the crystal structures available, the known catalytic reactions and the consequences in pre-clinical pharmacokinetic and pharmacodynamic studies. Analysis of the different animal models generally used for pre-clinical trials and comparison between the human (hAOX1), mouse homologs as well as the related xanthine oxidase (XOR) are extensively considered. The data reviewed also include a systematic analysis of representative classes of molecules that are hAOX1 substrates as well as of typical and well characterized hAOX1 inhibitors. The considerations made on the basis of a structural and functional analysis are correlated with reported kinetic and metabolic data for typical classes of drugs, searching for potential structural determinants that may dictate substrate and/or inhibitor specificities.}, 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} } @misc{TeraoRomaoLeimkuehleretal.2016, author = {Terao, Mineko and Romao, Maria Joao and Leimk{\"u}hler, Silke and Bolis, Marco and Fratelli, Maddalena and Coelho, Catarina and Santos-Silva, Teresa and Garattini, Enrico}, title = {Structure and function of mammalian aldehyde oxidases}, series = {Archives of toxicology : official journal of EUROTOX}, volume = {90}, journal = {Archives of toxicology : official journal of EUROTOX}, publisher = {Springer}, address = {Heidelberg}, issn = {0340-5761}, doi = {10.1007/s00204-016-1683-1}, pages = {753 -- 780}, year = {2016}, abstract = {Mammalian aldehyde oxidases (AOXs; EC1.2.3.1) are a group of conserved proteins belonging to the family of molybdo-flavoenzymes along with the structurally related xanthine dehydrogenase enzyme. AOXs are characterized by broad substrate specificity, oxidizing not only aromatic and aliphatic aldehydes into the corresponding carboxylic acids, but also hydroxylating a series of heteroaromatic rings. The number of AOX isoenzymes expressed in different vertebrate species is variable. The two extremes are represented by humans, which express a single enzyme (AOX1) in many organs and mice or rats which are characterized by tissue-specific expression of four isoforms (AOX1, AOX2, AOX3, and AOX4). In vertebrates each AOX isoenzyme is the product of a distinct gene consisting of 35 highly conserved exons. The extant species-specific complement of AOX isoenzymes is the result of a complex evolutionary process consisting of a first phase characterized by a series of asynchronous gene duplications and a second phase where the pseudogenization and gene deletion events prevail. In the last few years remarkable advances in the elucidation of the structural characteristics and the catalytic mechanisms of mammalian AOXs have been made thanks to the successful crystallization of human AOX1 and mouse AOX3. Much less is known about the physiological function and physiological substrates of human AOX1 and other mammalian AOX isoenzymes, although the importance of these proteins in xenobiotic metabolism is fairly well established and their relevance in drug development is increasing. This review article provides an overview and a discussion of the current knowledge on mammalian AOX.}, 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{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} } @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{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} }