@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} } @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} } @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{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{FotiDorendorfLeimkuehler2017, author = {Foti, Alessandro and Dorendorf, Frank and Leimk{\"u}hler, Silke}, title = {A single nucleotide polymorphism causes enhanced radical oxygen species production by human aldehyde oxidase}, series = {PLoS one}, volume = {12}, journal = {PLoS one}, publisher = {PLoS}, address = {San Fransisco}, issn = {1932-6203}, doi = {10.1371/journal.pone.0182061}, pages = {18338 -- 18347}, 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. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. The physiological function of mammalian AOX isoenzymes is still unclear, however, human AOX (hAOX1) is an emerging enzyme in phase-I drug metabolism. Indeed, the number of xenobiotics acting as hAOX1 substrates is increasing. Further, numerous single-nucleotide polymorphisms (SNPs) have been identified within the hAOX1 gene. SNPs are a major source of inter-individual variability in the human population, and SNP-based amino acid exchanges in hAOX1 reportedly modulate the catalytic function of the enzyme in either a positive or negative fashion. In this report we selected ten novel SNPs resulting in amino acid exchanges in proximity to the FAD site of hAOX1 and characterized the purified enzymes after heterologous expression in Escherichia coli. The hAOX1 variants were characterized carefully by quantitative differences in their ability to produce superoxide radical. ROS represent prominent key molecules in physiological and pathological conditions in the cell. Our data reveal significant alterations in superoxide anion production among the variants. In particular the SNP-based amino acid exchange L438V in proximity to the isoalloxanzine ring of the FAD cofactor resulted in increased rate of superoxide radical production of 75\%. Considering the high toxicity of the superoxide in the cell, the hAOX1-L438V SNP variant is an eventual candidate for critical or pathological roles of this natural variant within the human population.}, language = {en} }