Health, Safety & Environment

Aldehyde oxidase

In Vitro and In Silico Analyses of the Inhibition of Human Aldehyde Oxidase by Bazedoxifene, Lasofoxifene, and Structural Analogues

Tamoxifen, raloxifene, and nafoxidine are selective estrogen receptor modulators (SERMs) reported to inhibit the catalytic activity of human aldehyde oxidase 1 (AOX1). How these drugs interact with AOX1 and whether other SERMs inhibit this drug-metabolizing enzyme are not known. Therefore, a detailed in vitro and in silico study involving parent drugs and their analogs was conducted to investigate the effect of specific SERMs, particularly acolbifene, bazedoxifene, and lasofoxifene on AOX1 catalytic activity, as assessed by carbazeran 4-oxidation, an AOX1-selective catalytic marker. The rank order in the potency (based on IC50 values) of AOX1 inhibition by SERMs was raloxifene > bazedoxifene approximately lasofoxifene > tamoxifen > acolbifene. Inhibition of liver cytosolic AOX1 by bazedoxifene, lasofoxifene, and tamoxifen was competitive, whereas that by raloxifene was noncompetitive. Loss of 1-azepanylethyl group increased the inhibitory potency of bazedoxifene, whereas the N-oxide group decreased it. The 7-hydroxy group and the substituted pyrrolidine ring attached to the tetrahydronaphthalene structure contributed to AOX1 inhibition by lasofoxifene. These results are supported by molecular-docking simulations in terms of predicted binding modes, encompassing binding orientation and efficiency, and analysis of key interactions, particularly hydrogen bonds. The extent of AOX1 inhibition by bazedoxifene was increased by estrone sulfate and estrone. In summary, SERMs differentially inhibited human AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contributed to AOX1 inhibition, whereas those of acolbifene rendered it considerably less susceptible to AOX1 inhibition. Overall, our novel biochemical findings and molecular-docking analyses provide new insights into the interaction between SERMs and AOX1. SIGNIFICANCE STATEMENT: Aldehyde oxidase (AOX1) is a molybdo-flavoprotein and has emerged as a drug-metabolizing enzyme of potential therapeutic importance because drugs have been identified as AOX1 substrates. Selective estrogen receptor modulators (SERM), which are drugs used to treat and prevent various conditions, differentially inhibit AOX1 catalytic activity. Structural features of bazedoxifene and lasofoxifene contribute to AOX1 inhibition, whereas those of acolbifene render it considerably less susceptible to AOX1 inhibition. Our novel biochemical findings, together with molecular- docking analyses, provide new insights into the differential inhibitory effect of SERMs on the catalytic activity of human AOX1, how SERMs bind to AOX1, and increase our understanding of the AOX1 pharmacophore in the inhibition of AOX1 by drugs and other chemicals.

S. Chen, K. Austin-Muttitt, L. H. Zhang, J. G. L. Mullins, and A. J. Lau,In Vitro and In Silico Analyses of the Inhibition of Human Aldehyde Oxidase by Bazedoxifene, Lasofoxifene, and Structural Analogues, The Journal of pharmacology and experimental therapeutics, 2019, 371, 75-86.

Aldehyde oxidase

Aldehyde oxidase at the crossroad of metabolism and preclinical screening

Human AOX1 is a member of the mammalian aldehyde oxidase (AOX) family of enzymes and it is an emerging cytosolic enzyme involved in phase I drug-metabolism, bio-transforming a number of therapeutic agents and xenobiotics. The current trend in drug-development is to design molecules which are not recognized and inactivated by CYP450 monooxygenases, the main drug-metabolizing system, to generate novel therapeutic agents characterized by optimal pharmacokinetic and pharmacodynamic properties. Unfortunately, this has resulted in a substantial enrichment in molecules which are recognized and metabolized by AOXs. The observation has raised interest in the generation of tools capable of predicting AOX-dependent drug-metabolism of novel molecules during the early phases of drug development. Such tools are likely to reduce the number of failures occurring at the clinical and late phase of the drug development process. The current review describes different in silico, in vitro and in vivo methods for the prediction of AOX metabolizing ability and focuses on the existing drawbacks and challenges associated with these approaches.

N. Cheshmazar, S. Dastmalchi, M. Terao, E. Garattini, and M. Hamzeh-Mivehroud,Aldehyde oxidase at the crossroad of metabolism and preclinical screening, Drug Metabolism Reviews 2019. https://doi.org/10.1080/03602532.2019.1667379

Aldehyde oxidase

Aldehyde oxidase and its role as a drug metabolizing enzyme

Aldehyde oxidase (AO) is a cytosolic enzyme that belongs to the family of structurally related molybdoflavoproteins like xanthine oxidase (XO). The enzyme is characterized by broad substrate specificity and marked species differences. It catalyzes the oxidation of aromatic and aliphatic aldehydes and various heteroaromatic rings as well as reduction of several functional groups. The references to AO and its role in metabolism date back to the 1950s, but the importance of this enzyme in the metabolism of drugs has emerged in the past fifteen years. Several reviews on the role of AO in drug metabolism have been published in the past decade indicative of the growing interest in the enzyme and its influence in drug metabolism. Here, we present a comprehensive monograph of AO as a drug metabolizing enzyme with emphasis on marketed drugs as well as other xenobiotics, as substrates and inhibitors. Although the number of drugs that are primarily metabolized by AO are few, the impact of AO on drug development has been extensive. We also discuss the effect of AO on the systemic exposure and clearance these clinical candidates. The review provides a comprehensive analysis of drug discovery compounds involving AO with the focus on developmental candidates that were reported in the past five years with regards to pharmacokinetics and toxicity. While there is only one known report of AO-mediated clinically relevant drug-drug interaction (DDI), a detailed description of inhibitors and inducers of AO known to date has been presented here and the potential risks associated with DDI. The increasing recognition of the importance of AO has led to significant progress in predicting the site of AO-mediated metabolism using computational methods. Additionally, marked species difference in expression of AO makes it is difficult to predict human clearance with high confidence. The progress made towards developing in vivo, in vitro and in silico approaches for predicting AO metabolism and estimating human clearance of compounds that are metabolized by AO have also been discussed.

D. Dalvie, and L. Di,Aldehyde oxidase and its role as a drug metabolizing enzyme, Pharmacology & therapeutics, 2019, 201, 137-180.

 

Aldehyde Oxidase

Effects of Phenothiazines on Aldehyde Oxidase Activity Towards Aldehydes and N-Heterocycles: an In Vitro and In Silico Study

BACKGROUND: Aldehyde oxidase (AOX) is an important molybdenum-containing enzyme with high similarity with xanthine oxidase (XO). AOX involved in the metabolism of a large array of aldehydes and N-heterocyclic compounds and its activity is highly substrate-dependent. OBJECTIVES: The aim of this work was to study the effect of five important phenothiazine drugs on AOX activity using benzaldehyde and phenanthridine as aldehyde and N-heterocyclic substrates, respectively. METHODS: The effect of trifluperazine, chlorpromazine, perphenazine, thioridazine and promethazine on rat liver AOX was measured spectrophotometrically. To predict the mode of interactions between the studied compounds and AOX, a combination of homology modeling and a molecular docking study was performed. RESULTS: All phenothiazines could inhibit AOX activity measured either by phenanthridine or benzaldehyde with almost no effect on XO activity. In the case of benzaldehyde oxidation, the lowest and highest half-maximal inhibitory concentration (IC50) values were obtained for promethazine (IC50 = 0.9 microM), and trifluoperazine (IC50 = 3.9 microM), respectively; whereas perphenazine (IC50 = 4.3 microM), and trifluoperazine (IC50 = 49.6 microM) showed the strongest and weakest inhibitory activity against AOX-catalyzed phenanthridine oxidation, respectively. The in silico findings revealed that the binding site of thioridazine is near the dimer interference, and that hydrophobic interactions are of great importance in all the tested phenothiazines. CONCLUSION: The five studied phenothiazine drugs showed dual inhibitory effects on AOX activity towards aldehydes and N-heterocycles as two major classes of enzyme substrates. Most of the interactions between the phenothiazine-related drugs and AOX in the binding pocket showed a hydrophobic nature.

F. Deris-Abdolahpour, L. Abdolalipouran-Sadegh, S. Dastmalchi, M. Hamzeh-Mivehroud, O. Zarei, G. Dehgan, and M. R. Rashidi,Effects of Phenothiazines on Aldehyde Oxidase Activity Towards Aldehydes and N-Heterocycles: an In Vitro and In Silico Study, European journal of drug metabolism and pharmacokinetics, 2019, 44, 275-286.

 

               

Aldehyde oxidase

Human aldehyde oxidase (hAOX1): structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms

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. ENZYMES: Aldehyde oxidase (EC1.2.3.1); xanthine dehydrogenase (EC1.17.1.4); xanthine oxidase (EC1.1.3.2). DATABASES: Structural data are available in the Protein Data Bank under the accession number 6Q6Q.

C. Mota, M. Esmaeeli, C. Coelho, T. Santos-Silva, M. Wolff, A. Foti, S. Leimkuhler, and M. J. Romao,Human aldehyde oxidase (hAOX1): structure determination of the Moco-free form of the natural variant G1269R and biophysical studies of single nucleotide polymorphisms, FEBS open bio, 2019, 9, 925-934.

 

Aldehyde Oxidase

In Vitro Metabolism by Aldehyde Oxidase Leads to Poor Pharmacokinetic Profile in Rats for c-Met Inhibitor MET401

BACKGROUND AND OBJECTIVES: MET401 is a potent and selective c-Met inhibitor with a novel triazolopyrimidine scaffold. The aim of this study was to determine the pharmacokinetic profile of MET401 in preclinical species, and to identify the metabolic soft spot and enzyme involved, in order to help medicinal chemists to modify the compound to improve the pharmacokinetic profile. METHODS: A metabolite identification study was performed in different liver fractions from various species. Chemical inhibition with selective cytochrome P450 (CYP) and molybdenum hydroxylase inhibitors was carried out to identify the enzyme involved. The deuterium substitution strategy was adopted to reduce metabolism. Pharmacokinetic studies were performed in rats to confirm the effect. RESULTS: Although M-2 is a minor metabolite in liver microsomal incubations, it became the predominant metabolite in incubations with liver S9, cytosol, hepatocytes and rat pharmacokinetic study. M-2 was synthesized enzymatically and the structure was identified as a mono-oxidation on the triazolopyrimidine moiety. The M-2 formation was ascribed to aldehyde oxidase (AO)-mediated metabolism based on the following evidence-M-2 production was NADPH independent, pan-CYP inhibitor 1-aminobenzotriazole and xanthine oxidase inhibitor allopurinol did not inhibit M-2 formation, and AO inhibitors menadione and raloxifene inhibited M-2 formation. The deuterated analog MET763 demonstrated an improved pharmacokinetic profile with lower clearance, longer terminal half-life and double oral exposure compared with MET401 in rats. CONCLUSIONS: These results indicate that the main metabolic pathway of MET401 is AO-mediated metabolism, which leads to poor in vivo pharmacokinetic profiles in rodents. The deuterium substitution strategy could be used to reduce AO-mediated metabolism liability.

J. W. Zhang, H. B. Deng, C. Y. Zhang, J. Q. Dai, Q. Li, Q. G. Zheng, H. X. Wan, H. P. Yu, F. He, Y. C. Xu, S. Zhao, and J. Y. J. Zhang,In Vitro Metabolism by Aldehyde Oxidase Leads to Poor Pharmacokinetic Profile in Rats for c-Met Inhibitor MET401, European journal of drug metabolism and pharmacokinetics, 2019.

 

 

Adehyde oxidase

New insights about the monomer and homodimer structures of the human AOX1

Human aldehyde oxidase (hAOX1) is a molybdenum dependent enzyme that plays an important role in the metabolism of various compounds either endogenous or xenobiotics. Due to its promiscuity, hAOX1 plays a major role in the pharmacokinetics of many drugs and therefore has gathered a lot of attention from the scientific community and, particularly, from the pharmaceutical industry. In this work, homology modelling, molecular docking and molecular dynamics simulations were used to study the structure of the monomer and dimer of human AOX. The results with the monomer of hAOX1 allowed to shed some light on the role played by thioridazine and two malonate ions that are co-crystalized in the recent X-ray structure of hAOX1. The results show that these molecules endorse several conformational rearrangements in the binding pocket of the enzyme and these changes have an impact in the active site topology as well as in the stability of the substrate (phthalazine). The results show that the presence of both molecules open two gates located at the entrance of the binding pocket, from which results the flooding of the active site. They also endorse several modifications in the shape of the binding pocket (namely the position of Lys893) that, together with the presence of the solvent molecules, favour the release of the substrate to the solvent. Further insights were also obtained with the assembled homodimer of hAOX1. The allosteric inhibitor (THI) binds closely to the region where the dimerization of both monomers occur. These findings suggest that THI can interfere with protein dimerization.

P. Ferreira, N. Cerqueira, C. Coelho, P. A. Fernandes, M. J. Romao, and M. J. Ramos,New insights about the monomer and homodimer structures of the human AOX1, Physical chemistry chemical physics : PCCP, 2019, 21, 13545-13554.

 

 

Aldehyde oxidase

Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108

Acetic acid fermentation is widely considered a consequence of ethanol oxidation by two membrane-bound enzymes-alcohol dehydrogenase and aldehyde dehydrogenase (ALDH)-of acetic acid bacteria. Here, we used a markerless gene disruption method to construct a mutant of the Acetobacter pasteurianus strain SKU1108 with a deletion in the aldH gene, which encodes the large catalytic subunit of a heterotrimeric ALDH complex (AldFGH), to examine the role of AldFGH in acetic acid fermentation. The Delta aldH strain grew less on ethanol-containing medium, i.e., acetic acid fermentation conditions, than the wild-type strain and significantly accumulated acetaldehyde in the culture medium. Unexpectedly, acetaldehyde oxidase activity levels of the intact Delta aldH cells and the Delta aldH cell membranes were similar to those of the wild-type strain, which might be attributed to an additional ALDH isozyme (AldSLC). The apparent K-M values of the wild-type and Delta aldH membranes for acetaldehyde were similar to each other, when the cells were cultured in nonfermentation conditions, where Delta aldH cells grow as well as the wild-type cells. However, the membranes of the wild-type cells grown under fermentation conditions showed a 10-fold lower apparent K-M value than those of the cells grown under nonfermentation conditions. Under fermentation conditions, transcriptional levels of a gene for AldSLC were 10-fold lower than those under nonfermentation conditions, whereas aldH transcript levels were not dramatically changed under the two conditions. We suggest that A. pasteurianus SKU1108 has two ALDHs, and the AldFGH complex is indispensable for acetic acid fermentation and is the major enzyme under fermentation conditions.

T. Yakushi, S. Fukunari, T. Kodama, M. Matsutani, S. Nina, N. Kataoka, G. Theeragool, and K. Matsushita,Role of a membrane-bound aldehyde dehydrogenase complex AldFGH in acetic acid fermentation with Acetobacter pasteurianus SKU1108, Applied Microbiology and Biotechnology, 2018, 102, 4549-4561.

Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics

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.

M. J. Romao, C. Coelho, T. Santos-Silva, A. Foti, M. Terao, E. Garattini, and S. Leimkuhler,Structural basis for the role of mammalian aldehyde oxidases in the metabolism of drugs and xenobiotics, Current Opinion in Chemical Biology, 2017, 37, 39-47

The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline

Aldehyde oxidase (AOX) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AOX catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen-containing heterocyclic compounds. AOX is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite and hydroxamic acids. These reductive transformations are not well understood and are generally believed to only occur at low oxygen concentrations. In this study, we used 5-nitroquinoline (5NQ) as a substrate to further understand both the oxidative and the reductive transformations catalyzed by AOX. In vitro reaction of 5NQ with AOX under aerobic conditions generated the oxidized (2-oxo-5-nitroquinoline, 2-oxo-5NQ), the reduced (5-aminoquinoline, 5AQ) and the oxidized/reduced (2-oxo-5-aminoquinoline, 2-oxo-5AQ) metabolites. Interestingly, in human liver cytosol, co-incubation of 5NQ and known AOX oxidative substrates DACA and phthalazine significantly increased the yield of the reduced metabolite, while oxidized metabolites production decreased. These data indicate that 5NQ can be reduced at atmospheric oxygen concentrations and that the reductive transformation occurs at a second site that is kinetically distinct from the oxidative site.

Paragas, E. M., Humphreys, S. C., Min, J., Joswig-Jones, C. A., and Jones, J. P.,The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline, Biochemical pharmacology, 2017, 145, 210-217.

 

ALDEHYDE OXIDASE

Aldehyde oxidase human

Critical overview on the structure and metabolism of human aldehyde oxidase and its role in pharmacokinetics

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. (C) 2018 Elsevier B.V. All rights reserved.

C. Mota, C. Coelho, S. Leimkuhler, E. Garattini, M. Terao, T. Santos-Silva, and M. J. Romao,Critical overview on the structure and metabolism of human aldehyde oxidase and its role in pharmacokinetics, Coordination Chemistry Reviews, 2018, 368, 35-59.

 

Species-Specific Involvement of Aldehyde Oxidase and Xanthine Oxidase in the Metabolism of the Pyrimidine-Containing mGlu5-Negative Allosteric Modulator VU0424238 (Auglurant)

Aldehyde oxidase (AO) and xanthine oxidase (XO) are molybdo-flavoenzymes that catalyze oxidation of aromatic azaheterocycles. Differences in AO activity have been reported among various species, including rats, humans, and monkeys. Herein we report a species difference in the enzymes responsible for the metabolism of the negative allosteric modulator of metabotropic glutamate receptor subtype 5 (mGlu5 NAM) VU0424238 (VU238, auglurant). Hepatic S9 incubations with AO and XO specific inhibitors hydralazine and allopurinol indicated that rats and cynomolgus monkeys both oxidized VU238 to the 6-oxopyrimidine metabolite M1 via an AO-mediated pathway, whereas secondary oxidation to the 2,6-dioxopyrimidine metabolite M2 was mediated predominantly by AO in monkeys and XO in rats. Despite differences in enzymatic pathways, intrinsic clearance (CLint) of M1 was similar between species (cynomolgus and rat CLint = 2.00 +/- 0.040 and 2.19 +/- 0.201 mul/min per milligram of protein, respectively). Inhibitor studies in the S9 of multiple species indicated that oxidation of VU238 to M1 was mediated predominantly by AO in humans, cynomolgus and rhesus monkeys, rats, mice, guinea pigs, and minipigs. Oxidation of M1 to M2 was mediated predominantly by XO in rats and mice and by AO in monkeys and guinea pigs, whereas low turnover prevented enzyme phenotyping in humans and minipigs. Additionally, inhibitor experiments indicated that oxidation at the 2-position of the pyrimidine ring of the known AO substrate, BIBX1382, was mediated by AO in all species, although production of this metabolite was comparatively low in rats and mice. These data may suggest low reactivity of rat AO toward 2-oxidation of pyrimidine-containing compounds and highlight the importance of thoroughly characterizing AO-metabolized drug candidates in multiple preclinical species.

Crouch, R. D., Blobaum, A. L., Felts, A. S., Conn, P. J., and Lindsley, C. W.,Species-Specific Involvement of Aldehyde Oxidase and Xanthine Oxidase in the Metabolism of the Pyrimidine-Containing mGlu5-Negative Allosteric Modulator VU0424238 (Auglurant), Drug metabolism and disposition: the biological fate of chemicals, 2017, 45, 1245-1259.

Aldehyde oxidase

Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp WU-0701

Trans-Aconitic acid is an unsaturated organic acid that is present in some plants such as soybean and wheat; however, it remains unclear how trans-aconitic acid is degraded and/or assimilated by living cells in nature.

From soil, we isolated Pseudomonas sp. WU-0701 assimilating trans-aconitic acid as a sole carbon source.

In the cell-free extract of Pseudomonas sp. WU-0701, aconitate isomerase (AI; EC 5.3.3.7) activity was detected. Therefore, it seems likely that strain Pseudomonas sp. WU-0701 converts trans-aconitic acid to cis-aconitic acid with AI, and assimilates this via the tricarboxylic acid cycle.

For the characterization of AT from Pseudomonas sp. WU-0701, we performed purification, determination of enzymatic properties and gene identification of AI. The molecular mass of AT purified from cell-free extract was estimated to be similar to 25 kDa by both SDS/PAGE and gel filtration analyses, indicating that AT is a monomeric enzyme.

The optimal pH and temperature of purified AT for the reaction were 6.0 degrees C and 37 degrees C, respectively.

The gene ais encoding AT was cloned on the basis of the N-terminal amino acid sequence of the protein, and Southern blot analysis revealed that only one copy of ais is located on the bacterial genome. The gene ais contains an ORF of 786 bp, encoding a polypeptide of 262 amino acids, including the N-terminal 22 amino acids as a putative periplasm-targeting signal peptide. It is noteworthy that the amino acid sequence of AT shows 90% and 74% identity with molybdenum ABC transporter substrate-binding proteins of Pseudomonas psychrotolerans and Xanthomonas albilineans, respectively.

This is the first report on purification to homogeneity, characterization and gene identification of AI.

Yuhara, K., Yonehara, H., Hattori, T., Kobayashi, K., and Kirimura, K.,Enzymatic characterization and gene identification of aconitate isomerase, an enzyme involved in assimilation of trans-aconitic acid, from Pseudomonas sp WU-0701, Febs Journal, 2015, 282, 4257-4267.

The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline

Aldehyde oxidase (AOX) is a cytosolic enzyme responsible for the metabolism of some drugs and drug candidates. AOX catalyzes the oxidative hydroxylation of substrates including several aliphatic and aromatic aldehydes, and nitrogen-containing heterocyclic compounds. AOX is also reported to catalyze the reductive metabolism of nitro-compounds, N-oxides, sulfoxides, isoxazoles, isothiazoles, nitrite and hydroxamic acids. These reductive transformations are not well understood and are generally believed to only occur at low oxygen concentrations. In this study, we used 5-nitroquinoline (5NQ) as a substrate to further understand both the oxidative and the reductive transformations catalyzed by AOX. In vitro reaction of 5NQ with AOX under aerobic conditions generated the oxidized (2-oxo-5-nitroquinoline, 2-oxo-5NQ), the reduced (5-aminoquinoline, 5AQ) and the oxidized/reduced (2-oxo-5-aminoquinoline, 2-oxo-5AQ) metabolites. Interestingly, in human liver cytosol, co-incubation of 5NQ and known AOX oxidative substrates DACA and phthalazine significantly increased the yield of the reduced metabolite, while oxidized metabolites production decreased. These data indicate that 5NQ can be reduced at atmospheric oxygen concentrations and that the reductive transformation occurs at a second site that is kinetically distinct from the oxidative site.

Paragas, E. M., Humphreys, S. C., Min, J., Joswig-Jones, C. A., and Jones, J. P.,The two faces of aldehyde oxidase: Oxidative and reductive transformations of 5-nitroquinoline, Biochemical pharmacology, 2017, 145, 210-217.

Aldehyde oxidase-dependent species difference in hepatic metabolism of fasudil to hydroxyfasudil

1. An investigation on the metabolic mechanism of fasudil to hydroxyfasudil was conducted in vitro using liver subcellular fractions of different species. Hydroxyfasudil was generated in large amounts by rat liver S9 and to a similar extent by human liver S9 but was not detected in dog liver S9 incubations.

2. Studies with various molybdenum hydroxylase inhibitors demonstrated that aldehyde oxidase (AO), but not xanthine oxidase (XO), selectively catalyzed fasudil to hydroxyfasudil in both rat and human liver cytosol. In addition, the oxygen atom incorporated into hydroxyfasudil was derived from water rather than atmospheric oxygen, which further corroborated AO involvement.

3. Enzyme kinetics experiments revealed that fasudil had a higher affinity to human hepatic AO than to rat hepatic AO. Besides, significantly different in vivo pharmacokinetic parameters observed between male and female rats indicated that the AO activity in rats was gender-dependent.

4. The present study provided first evidences that AO causes differences in fasudil metabolism between species.

Mao, Z., Wu, Y., Li, Q., Wang, X., Liu, Y., and Di, X.,Aldehyde oxidase-dependent species difference in hepatic metabolism of fasudil to hydroxyfasudil, Xenobiotica; the fate of foreign compounds in biological systems, 2017, 1-8.

 

[Fasudil: potent Rho-kinase inhibitor and vasodilator; treatment of cerebral vasospasm due to subarachnoid hemorrhage; improve cognitive decline seen in stroke victims; treatment of pulmonary hypertension; improve memory in normal mice;possible treatment for age related or neurodegenerative memory loss. https://en.wikipedia.org/wiki/Fasudil.]

Reaction mechanism and prediction of site of metabolism

Aldehyde oxidase (AO) is a molybdenum containing enzyme involved in the clearance of drug compounds containing aldehydes and N-containing heterocyclic fragments. AO has gained considerable interest in recent years because of examples of too fast clearance of drug compounds in development. Thus, it is important to be able to predict AO-mediated drug metabolism. Therefore, we have characterized the structural and energetic aspects of different mechanisms with density functional theory using the molybdenum cofactor as a model for the reactive part of the enzyme. For a series of 6-substituted 4-quinazolinones, the trend in activation energies is the same for three tested reaction mechanisms. Using the concerted mechanism as a model for the enzymatic reaction, the transition states (TSs) for the formation of all possible metabolites for a series of known AO substrates were determined. The lowest activation energies correspond in all cases to the experimentally observed sites of metabolism (SOMs). Various molecular properties were calculated and investigated as more easily determinable markers for reactivity. The stabilities of both intermediates and products correlate to some extent with the TS energies and may be used to predict the SOM. The electrostatic-potential-derived charges are also good markers for the prediction of the experimental SOM for this set of compounds and may pave the way for the development of fast methods for the prediction of SOM for AO substrates.

Montefiori, M., Jorgensen, F. S., and Olsen, L.,Aldehyde Oxidase: Reaction Mechanism and Prediction of Site of Metabolism, Acs Omega, 2017, 2, 4237-4244.

The escherichia coli periplasmic aldehyde oxidoreductase Is an exceptional member of the xanthine oxidase family of molybdoenzymes

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.

Correia, M. A., Otrelo-Cardoso, A. R., Schwuchow, V., Sigfridsson Clauss, K. G., Haumann, M., Romao, M. J., Leimkuhler, S., and Santos-Silva, T.,The Escherichia coli Periplasmic Aldehyde Oxidoreductase Is an Exceptional Member of the Xanthine Oxidase Family of Molybdoenzymes, ACS chemical biology, 2016

Structure and function of mammalian aldehyde oxidases

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.

Terao, M., Romao, M. J., Leimkuhler, S., Bolis, M., Fratelli, M., Coelho, C., Santos-Silva, T., and Garattini, E.,Structure and function of mammalian aldehyde oxidases, Archives of Toxicology, 2016, 90, 753-780.

Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase

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.

Coelho, C., Foti, A., Hartmann, T., Santos-Silva, T., Leimkuhler, S., and Romao, M. J.,Structural insights into xenobiotic and inhibitor binding to human aldehyde oxidase, Nature Chemical Biology, 2015, 11, 779-+.

 [Xenobiotic: a foreign chemical substance found within an organism that is not normally naturally produced by or expected to be present within that organism. Wikipedia.]

The aldehyde oxidoreductase, isolated from the sulfate reducer Desulfovibrio desulfuricans (ATCC 27774), and the homologous enzyme from Desulfovibrio gigas are members of the xanthine oxidase family of molybdenum-containing enzymes. They have similar substrate specificity. The primary sequences from both enzymes show 68 % identity. Both enzymes are very closely related in their sequences and 3D structures. The comparison allowed confirmation and establishment of features that are essential for their function: conserved residues in the active site, catalytically relevant water molecules and recognition of the physiological electron acceptor docking site.

Rebelo, J., Macieira, S., Dias, J. M., Huber, R., Ascenso, C. S., Rusnak, F., Moura, J. J. G., Moura, I., and Romao, M. J., Gene sequence and crystal structure of the aldehyde oxidoreductase from Desulfovibrio desulfuricans ATCC 27774, Journal of Molecular Biology, 2000, 297, 135-146.

The current knowledge of genetics, evolution, structure, enzymology, tissue distribution and regulation of mammalian aldehyde oxidases is reviewed.

Garattini, E., Fratelli, M., and Terao, M., Mammalian aldehyde oxidases: genetics, evolution and biochemistry, Cellular and Molecular Life Sciences, 2008, 65, 1019-1048.

Oxidative removal of glutaraldehyde

Maeda, Y., Yagyu, A., Sakurai, A., Fujii, Y., and Uchida, H., Characterization of aldehyde oxidase from Brevibacillus sp MEY43 and its application to oxidative removal of glutaraldehyde, World Journal of Microbiology & Biotechnology, 2008, 24, 797-804.

Molybdenum iron-sulfur flavin hydroxylases in the pathogenesis of liver injuries injuries

The role of molybdenum iron-sulfur flavin hydroxylases in the pathogenesis of liver injuries injuries in rats induced

(a) by carbon tetrachloride (CCl4), thioacetamide (TAA) and chloroform (CHCl3), which produce free radicals were associated with elevated activity levels of hepatic Mo-Fe-S flavin hydroxylases. Inhibition of these hydroxylases by sodium tungstate suppressed biochemical and oxidative stress markers of hepatic tissue damage.

(b) by acetaminophen (AAP) and bromobenzene (BB), which cause severe glutathione depletion. Mo-Fe-S flavin hydroxylases did not with these toxicants show any change. Mo-Fe-S hydroxylases contribute to the hepatic injury inflicted by free radical generating agents. and does not play any role in hepatic injury produced by glutathione depleting agents. The study has implication for understanding human liver diseases caused by liver toxicants and for inhibitors of Mo-Fe-S flavin hydroxylases as potential therapeutic agents.

Ali, S., Pawa, S., Naime, M., Prasad, R., Ahmad, T., Farooqui, H., and Zafar, H., Role of mammalian cytosolic molybdenum Fe-S flavin hydroxylases in hepatic injury, Life Sciences, 2008, 82, 780-788.

Cytosol = the non-particulate components of the cytoplasm
Cytoplasm = that part of the cell outside the nucleus but inside the cell wall if it exists

Aldehyde oxidase functions as a superoxide generating NADH oxidase: An important redox regulated pathway of cellular oxygen radical formation

The enzyme aldehyde oxidase (AO) is a member of the molybdenum hydroxylase family that includes xanthine oxidoreductase (XOR); however, its physiological substrates and functions remain unclear. Moreover, little is known about its role in cellular redox stress.

Utilizing electron paramagnetic resonance spin trapping, we measured the role of AO in the generation of reactive oxygen species (ROS) through the oxidation of NADH and the effects of inhibitors of AO on NADH-mediated superoxide (O2)generation.

NADH was found to be a good substrate for AO with apparent K-m and V-max values of 29 mu M and 12 nmol min-1 mg-1, respectively.

From O2 generation measurements by cytochrome c reduction the apparent K-m and V-max values of NADH for AO were 11 mu M and 15 nmol min-1 mg-1, respectively. With NADH oxidation by AO, >= 65% of the total electron flux led to O2 generation. Diphenyleneiodonium completely inhibited AO-mediated O2 production, confirming that this occurs at the FAD site.

Inhibitors of this NADH-derived O2generation were studied with amidone the most potent exerting complete inhibition at 100 mu M concentration, while 150 mu M menadione, raloxifene, or beta-estradiol led to 81%, 46%, or 26% inhibition, respectively.

From the kinetic data, and the levels of AO and NADH, O2 production was estimated to be 89 and 4 nM/s in liver and heart, respectively, much higher than that estimated for XOR under similar conditions.

Owing to the ubiquitous distribution of NADH, aldehydes, and other endogenous AO substrates, AO is predicted to have an important role in cellular redox stress and related disease pathogenesis

Kundu, Tapan K., Velayutham, Murugesan, and Zweier, Jay L., Aldehyde Oxidase Functions as a Superoxide Generating NADH Oxidase: An Important Redox Regulated Pathway of Cellular Oxygen Radical Formation, Biochemistry, 2012, 51, 2930-2939.

Molybdenum hydroxylases drug-metabolizing ability

Drug metabolizing ability of molybdenum hydroxylases, which include aldehyde oxidase and xanthine oxidoreductase, and the variation of the activity amongst humans, with the highest activity, rats and mice and dogs are described in this review. Molybdenum hydroxylases, are involved in the metabolism of some medicines in humans. Interindividual variation of aldehyde oxidase activity is present in humans. Drug-drug interactions associated with aldehyde oxidase and xanthine oxidoreductase are of potential clinical significance.

Kitamura, S., Sugihara, K., and Ohta, S., Drug-metabolizing ability of molybdenum hydroxylases, Drug Metabolism and Pharmacokinetics, 2006, 21, 83-98.

Reduction of N-hydroxylated prodrugs by a molybdenum enzyme

The recently discovered mammalian molybdoprotein mARC1 is capable of reducing N-hydroxylated compounds. Upon reconstitution with cytochrome b(5) and b5 reductase, benzamidoxime, pentamidine, and diminazene amidoximes, N-hydroxymelagatran, guanoxabenz, and N-hydroxydebrisoquine are efficiently reduced. These substances are amidoxime/N-hydroxyguanidine prodrugs, leading to improved bioavailability compared to the active amidines/guanidines. Thus, the recombinant enzyme allows prediction about in vivo reduction of N-hydroxylated prodrugs. Furthermore, the prodrug principle is not dependent on cytochrome P450 enzymes

Gruenewald, S., Wahl, B., Bittner, F., Hungeling, H., Kanzow, S., Kotthaus, J., Schwering, U., Mendel, R. R., and Clement, B., The Fourth Molybdenum Containing Enzyme mARC: Cloning and Involvement in the Activation of N-Hydroxylated Prodrugs, Journal of Medicinal Chemistry, 2008, 51, 8173-8177.

Selenium-dependent molybdenum hydroxylases

Haft, D.H. and Self, W. T., Orphan SeID proteins and selenium-dependent molybdenum hydroxylases, Biology Direct, 2008, 3

Functional analysis aldehyde oxidase

Aldehyde oxidase (AO) is a homodimer with a subunit molecular mass of approximately 150 kDa. Each subunit consists of about 20 kDa 2Fe-2S cluster domain storing reducing equivalents, about 40 kDa flavine adenine dinucleotide (FAD) domain and about 85 kDa molybdenum cofactor (MoCo) domain containing a substrate binding site. In order to clarify the properties of each domain, especially substrate binding domain, chimeric cDNAs were constructed by mutual exchange of 2Fe-2S/FAD and MoCo domains between monkey and rat. Chimeric monkey/rat AO was referred to one with monkey type 2Fe-2S/FAD domains and a rat type MoCo domain. Rat/monkey AO was vice versa. AO-catalyzed 2-oxidation activities of (S)-RS-8359 were measured using the expressed enzyme in Escherichia coli. Substrate inhibition was seen in rat AO and chimeric monkey/rat AO, but not in monkey AO and chimeric rat/monkey AO, suggesting that the phenomenon might be dependent on the natures of MoCo domain of rat. A biphasic Eadie-Hofstee profile was observed in monkey AO and chimeric rat/monkey AO, but not rat AO and chimeric monkey/rat AO, indicating that the biphasic profile might be related to the properties of MoCo domain of monkey. Two-fold greater V-max, values were observed in monkey AO than in chimeric rat/monkey AO, and in chimeric monkey/rat AO than in rat AO, suggesting that monkey has the more effective electron transfer system than rat. Thus, the use of chimeric enzymes revealed that 2Fe-2S/FAD and MoCo domains affect the velocity and the quantitative profiles of AO-catalyzed (S)-RS-8359 2-oxidation, respectively

Itoh, K., Asakawa, T., Hoshino, K., Adachi, M., Fukiya, K., Watanabe, N., and Tanaka, Y., Functional Analysis of Aldehyde Oxidase Using Expressed Chimeric Enzyme between Monkey and Rat, Biological & Pharmaceutical Bulletin, 2009, 32, 31-35.

Enzyme kinetics, inhibition, and regioselectivity of aldehyde oxidase

The aldehyde oxidase (AO) enzyme family plays an increasing role in drug development. However, a number of compounds that are AO substrates have failed in the clinic because the clearance or toxicity is underestimated by preclinical species. Human AO is much more active than rodent AO, and dogs do not have functional AO. While AOs normally make non-reactive metabolites such as lactams, the metabolic products often have much lower solubility that can lead to renal failure. While an endogenous substrate for the oxidation reaction is not known, electron acceptors for the reductive part of the reaction include oxygen and nitrites. Reduction of oxygen leads to the reactive oxygen species (ROS) superoxide radical anion, and hydrogen peroxide. Reduction of nitrite leads to the formation of nitric oxide with potential pharmacological implications. To date, no clinically important drug-drug interactions (DDIs) have been observed for AOs. However, the inhibition kinetics are complex, and multiple probe substrates should be used when assessing the potential for DDIs. Finally, AO appears to be amenable to computational predictions of both regioselectivity and rates of reaction, which holds promise for virtual screening

Barr, J. T., Choughule, K., and Jones, J. P., Enzyme Kinetics, Inhibition, and Regioselectivity of Aldehyde Oxidase, Enzyme Kinetics in Drug Metabolism: Fundamentals and Applications Se Methods in Molecular Biology, 2014, 1113, 167-186.

Structural data on the periplasmic aldehyde oxidoreductase PaoABC from escherichia coli: SAXS and Preliminary X-ray crystallography analysis

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

Otrelo-Cardoso, A. R., Correia, M. A. D., Schwuchow, V., Svergun, D. I., Romao, M. J., Leimkuhler, S., and Santos-Silva, T., Structural Data on the Periplasmic Aldehyde Oxidoreductase PaoABC from Escherichia coli: SAXS and Preliminary X-ray Crystallography Analysis, International Journal of Molecular Sciences, 2014, 15, 2223-2236.