TY - JOUR A1 - Terao, Mineko A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Bolis, Marco A1 - Fratelli, Maddalena A1 - Coelho, Catarina A1 - Santos-Silva, Teresa A1 - Garattini, Enrico T1 - Structure and function of mammalian aldehyde oxidases JF - Archives of toxicology : official journal of EUROTOX N2 - 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. KW - Aldehyde oxidase KW - Molybdo-flavoenzymes KW - Xanthine oxidoreductase KW - Drug metabolism Y1 - 2016 U6 - https://doi.org/10.1007/s00204-016-1683-1 SN - 0340-5761 SN - 1432-0738 VL - 90 SP - 753 EP - 780 PB - Springer CY - Heidelberg 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 - Correia, Marcia A. S. A1 - Otrelo-Cardoso, Ana Rita A1 - Schwuchow, Viola A1 - Clauss, Kajsa G. V. Sigfridsson A1 - Haumann, Michael A1 - Romao, Maria Joao A1 - Leimkühler, Silke A1 - Santos-Silva, Teresa T1 - The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes JF - ACS chemical biology N2 - 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. Y1 - 2016 U6 - https://doi.org/10.1021/acschembio.6b00572 SN - 1554-8929 SN - 1554-8937 VL - 11 SP - 2923 EP - 2935 PB - American Chemical Society CY - Washington ER -