Health, Safety & Environment

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.

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.