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 - Mota, Cristiano A1 - Esmaeeli Moghaddam Tabalvandani, Mariam A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Wolff, Martin A1 - Foti, Alessandro A1 - Leimkühler, Silke A1 - Romao, Maria Joao T1 - Human aldehyde oxidase (hAOX1) BT - structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms JF - FEBS Open Bio N2 - 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 . KW - human aldehyde oxidase KW - molybdenum cofactor KW - single nucleotide polymorphism KW - xanthine oxidase Y1 - 2019 U6 - https://doi.org/10.1002/2211-5463.12617 SN - 2211-5463 VL - 9 IS - 5 SP - 925 EP - 934 PB - Wiley CY - Hoboken ER - TY - JOUR A1 - Foti, Alessandro A1 - Hartmann, Tobias A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Romao, Maria Joao A1 - Leimkühler, Silke T1 - Optimization of the Expression of Human Aldehyde Oxidase for Investigations of Single-Nucleotide Polymorphisms JF - Drug metabolism and disposition : the biological fate of chemicals N2 - 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. Y1 - 2016 U6 - https://doi.org/10.1124/dmd.115.068395 SN - 0090-9556 SN - 1521-009X VL - 44 SP - 1277 EP - 1285 PB - American Society for Pharmacology and Experimental Therapeutics CY - Bethesda ER - TY - JOUR A1 - Coelho, Catarina A1 - Foti, Alessandro A1 - Hartmann, Tobias A1 - Santos-Silva, Teresa A1 - Leimkühler, Silke A1 - Romao, Maria Joao T1 - Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase JF - Nature chemical biology N2 - 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. Y1 - 2015 U6 - https://doi.org/10.1038/NCHEMBIO.1895 SN - 1552-4450 SN - 1552-4469 VL - 11 IS - 10 SP - 779 EP - + PB - Nature Publ. Group CY - New York ER - TY - JOUR A1 - Mahro, Martin A1 - Coelho, Catarina A1 - Trincao, Jose A1 - Rodrigues, David A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Saggu, Miguel A1 - Lendzian, Friedhelm A1 - Hildebrandt, Peter A1 - Romao, Maria Joao A1 - Leimkühler, Silke T1 - Characterization and crystallization of mouse aldehyde oxidase 3 - from mouse liver to escherichia coli heterologous protein expression JF - Drug metabolism and disposition : the biological fate of chemicals N2 - 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. Y1 - 2011 U6 - https://doi.org/10.1124/dmd.111.040873 SN - 0090-9556 VL - 39 IS - 10 SP - 1939 EP - 1945 PB - American Society for Pharmacology and Experimental Therapeutics CY - Bethesda ER - TY - JOUR A1 - Coelho, Catarina A1 - Mahro, Martin A1 - Trincao, Jose A1 - Carvalho, Alexandra T. P. A1 - Ramos, Maria Joao A1 - Terao, Mineko A1 - Garattini, Enrico A1 - Leimkühler, Silke A1 - Romao, Maria Joao T1 - The first mammalian aldehyde oxidase crystal structure insights into substrate specificity JF - The journal of biological chemistry N2 - 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. Y1 - 2012 U6 - https://doi.org/10.1074/jbc.M112.390419 SN - 0021-9258 VL - 287 IS - 48 SP - 40690 EP - 40702 PB - American Society for Biochemistry and Molecular Biology CY - Bethesda ER - TY - JOUR A1 - Mahro, Martin A1 - Bras, Natercia F. A1 - Cerqueira, Nuno M. F. S. A. A1 - Teutloff, Christian A1 - Coelho, Catarina A1 - Romao, Maria Joao A1 - Leimkühler, Silke T1 - Identification of crucial amino acids in mouse aldehyde oxidase 3 that determine substrate specificity JF - PLoS one N2 - 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. Y1 - 2013 U6 - https://doi.org/10.1371/journal.pone.0082285 SN - 1932-6203 VL - 8 IS - 12 PB - PLoS CY - San Fransisco ER -