@article{TeraoGarattiniRomaoetal.2020, author = {Terao, Mineko and Garattini, Enrico and Rom{\~a}o, Maria Jo{\~a}o and Leimk{\"u}hler, Silke}, title = {Evolution, expression, and substrate specificities of aldehyde oxidase enzymes in eukaryotes}, series = {The journal of biological chemistry}, volume = {295}, journal = {The journal of biological chemistry}, number = {16}, publisher = {American Society for Biochemistry and Molecular Biology}, address = {Rockville}, issn = {0021-9258}, doi = {10.1074/jbc.REV119.007741}, pages = {5377 -- 5389}, year = {2020}, abstract = {Aldehyde oxidases (AOXs) are a small group of enzymes belonging to the larger family of molybdo-flavoenzymes, along with the well-characterized xanthine oxidoreductase. The two major types of reactions that are catalyzed by AOXs are the hydroxylation of heterocycles and the oxidation of aldehydes to their corresponding carboxylic acids. Different animal species have different complements of AOX genes. The two extremes are represented in humans and rodents; whereas the human genome contains a single active gene (AOX1), those of rodents, such as mice, are endowed with four genes (Aox1-4), clustering on the same chromosome, each encoding a functionally distinct AOX enzyme. It still remains enigmatic why some species have numerous AOX enzymes, whereas others harbor only one functional enzyme. At present, little is known about the physiological relevance of AOX enzymes in humans and their additional forms in other mammals. These enzymes are expressed in the liver and play an important role in the metabolisms of drugs and other xenobiotics. In this review, we discuss the expression, tissue-specific roles, and substrate specificities of the different mammalian AOX enzymes and highlight insights into their physiological roles.}, 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{KuecuekgoezeTeraoGarattinietal.2017, author = {Kuecuekgoeze, Goekhan and Terao, Mineko and Garattini, Enrico and Leimk{\"u}hler, Silke}, title = {Direct Comparison of the Enzymatic Characteristics and Superoxide Production of the Four Aldehyde Oxidase Enzymes Present in Mouse}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {45}, 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.117.075937}, pages = {947 -- 955}, year = {2017}, abstract = {Aldehyde oxidases (AOXs) are molybdoflavoenzymes with an important role in the metabolism and detoxification of heterocyclic compounds and aliphatic as well as aromatic aldehydes. The enzymes use oxygen as the terminal electron acceptor and produce reduced oxygen species during turnover. Four different enzymes, mAOX1, mAOX3, mAOX4, and mAOX2, which are the products of distinct genes, are present in the mouse. A direct and simultaneous comparison of the enzymatic properties and characteristics of the four enzymes has never been performed. In this report, the four catalytically active mAOX enzymes were purified after heterologous expression in Escherichia coli. The kinetic parameters of the four mouse AOX enzymes were determined and compared with the use of six predicted substrates of physiologic and toxicological interest, i.e., retinaldehyde, N1-methylnicotinamide, pyridoxal, vanillin, 4-(dimethylamino) cinnamaldehyde (p-DMAC), and salicylaldehyde. While retinaldehyde, vanillin, p-DMAC, and salycilaldehyde are efficient substrates for the four mouse AOX enzymes, N1-methylnicotinamide is not a substrate of mAOX1 or mAOX4, and pyridoxal is notmetabolized by any of the purified enzymes. Overall, mAOX1, mAOX2, mAOX3, and mAOX4 are characterized by significantly different KM and kcat values for the active substrates. The four mouse AOXs are also characterized by quantitative differences in their ability to produce superoxide radicals. With respect to this last point, mAOX2 is the enzyme generating the largest rate of superoxide radicals of around 40\% in relation to moles of substrate converted, and mAOX1, the homolog to the human enzyme, produces a rate of approximately 30\% of superoxide radicals with the same substrate.}, 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{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{HartmannTeraoGarattinietal.2012, author = {Hartmann, Tobias and Terao, Mineko and Garattini, Enrico and Teutloff, Christian and Alfaro, Joshua F. and Jones, Jeffrey P. and Leimk{\"u}hler, Silke}, title = {The impact of single nucleotide polymorphisms on human aldehyde oxidase}, series = {Drug metabolism and disposition : the biological fate of chemicals}, volume = {40}, journal = {Drug metabolism and disposition : the biological fate of chemicals}, number = {5}, publisher = {American Society for Pharmacology and Experimental Therapeutics}, address = {Bethesda}, issn = {0090-9556}, doi = {10.1124/dmd.111.043828}, pages = {856 -- 864}, year = {2012}, abstract = {Aldehyde oxidase (AO) is a complex molybdo-flavoprotein that belongs to the xanthine oxidase family. AO is active as a homodimer, and each 150-kDa monomer binds two distinct [2Fe2S] clusters, FAD, and the molybdenum cofactor. AO has an important role in the metabolism of drugs based on its broad substrate specificity oxidizing aromatic aza-heterocycles, for example, N-1-methylnicotinamide and N-methylphthalazinium, or aldehydes, such as benzaldehyde, retinal, and vanillin. Sequencing the 35 coding exons of the human AOX1 gene in a sample of 180 Italian individuals led to the identification of relatively frequent, synonymous, missense and nonsense single-nucleotide polymorphisms (SNPs). Human aldehyde oxidase (hAOX1) was purified after heterologous expression in Escherichia coli. The recombinant protein was obtained with a purity of 95\% and a yield of 50 mu g/l E. coli culture. Site-directed mutagenesis of the hAOX1 cDNA allowed the purification of protein variants bearing the amino acid changes R802C, R921H, N1135S, and H1297R, which correspond to some of the identified SNPs. The hAOX1 variants were purified and compared with the wild-type protein relative to activity, oligomerization state, and metal content. Our data show that the mutation of each amino acid residue has a variable impact on the ability of hAOX1 to metabolize selected substrates. Thus, the human population is characterized by the presence of functionally inactive hAOX1 allelic variants as well as variants encoding enzymes with different catalytic activities. Our results indicate that the presence of these allelic variants should be considered for the design of future drugs.}, 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} } @misc{SchumannTeraoGarattinietal.2009, author = {Schumann, Silvia and Terao, Mineko and Garattini, Enrico and Saggu, Miguel and Lendzian, Friedhelm and Hildebrandt, Peter and Leimk{\"u}hler, Silke}, title = {Site directed mutagenesis of amino acid residues at the active site of mouse aldehyde oxidase AOX1}, url = {http://nbn-resolving.de/urn:nbn:de:kobv:517-opus-45030}, year = {2009}, abstract = {Mouse aldehyde oxidase (mAOX1) forms a homodimer and belongs to the xanthine oxidase family of molybdoenzymes which are characterized by an essential equatorial sulfur ligand coordinated to the molybdenum atom. In general, mammalian AOs are characterized by broad substrate specificity and an yet obscure physiological function. To define the physiological substrates and the enzymatic characteristics of mAOX1, we established a system for the heterologous expression of the enzyme in Eschericia coli. The recombinant protein showed spectral features and a range of substrate specificity similar to the native protein purified from mouse liver. The EPR data of recombinant mAOX1 were similar to those of AO from rabbit liver, but differed from the homologous xanthine oxidoreductase enzymes. Site-directed mutagenesis of amino acids Val806, Met884 and Glu1265 at the active site resulted in a drastic decrease in the oxidation of aldehydes with no increase in the oxidation of purine substrates. The double mutant V806E/M884R and the single mutant E1265Q were catalytically inactive enzymes regardless of the aldehyde or purine substrates tested. Our results show that only Glu1265 is essential for the catalytic activity by initiating the base-catalyzed mechanism of substrate oxidation. In addition, it is concluded that the substrate specificity of molybdo-flavoenzymes is more complex and not only defined by the three characterized amino acids in the active site.}, language = {en} }