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Aldehyde oxidase (AOX) is a molybdoenzyme that is primarily expressed in the liver and is involved in the metabolism of drugs and other xenobiotics. AOX-mediated metabolism can result in unexpected outcomes, such as the production of toxic metabolites and high metabolic clearance, which can lead to the clinical failure of novel therapeutic agents. Computational models can assist medicinal chemists in rapidly evaluating the AOX metabolic risk of compounds during the early phases of drug discovery and provide valuable clues for manipulating AOX-mediated metabolism liability. In this study, we developed a novel graph neural network called AOMP for predicting AOX-mediated metabolism. AOMP integrated the tasks of metabolic substrate/non-substrate classification and metabolic site prediction, while utilizing transfer learning from 13C nuclear magnetic resonance data to enhance its performance on both tasks. AOMP significantly outperformed the benchmark methods in both cross-validation and external testing. Using AOMP, we systematically assessed the AOX-mediated metabolism of common fragments in kinase inhibitors and successfully identified four new scaffolds with AOX metabolism liability, which were validated through in vitro experiments. Furthermore, for the convenience of the community, we established the first online service for AOX metabolism prediction based on AOMP, which is freely available at https://aomp.alphama.com.cn.

Abscisic acid (ABA) plays a crucial role in plant defense mechanisms under adverse environmental conditions, but its metabolism and perception in response to heavy metals are largely unknown. In Pisum sativum exposed to CdCl2, an accumulation of free ABA was detected in leaves at different developmental stages (A, youngest, unexpanded; B1, youngest, fully expanded; B2, mature; C, old), with the highest content found in A and B1 leaves. In turn, the content of ABA conjugates, which was highest in B2 and C leaves under control conditions, increased only in A leaves and decreased in leaves of later developmental stages after Cd treatment. Based on the expression of PsNCED2, PsNCED3 (9-cis-epoxycarotenoid dioxygenase), PsAO3 (aldehyde oxidase) and PsABAUGT1 (ABA-UDP-glucosyltransferase), and the activity of PsAOγ, B2 and C leaves were found to be the main sites of Cd-induced de novo synthesis of ABA from carotenoids and ABA conjugation with glucose. In turn, β-glucosidase activity and the expression of genes encoding ABA receptors (PsPYL2, PsPYL4, PsPYL8, PsPYL9) suggest that in A and B1 leaves, Cd-induced release of ABA from inactive ABA-glucosyl esters and enhanced ABA perception comes to the forefront when dealing with Cd toxicity. The distinct role of leaves at different developmental stages in defense against the harmful effects of Cd is discussed.

Nitric oxide (NO) plays numerous essential physiological functions in terrestrial animals. In mammals, NO production from l-arginine is catalysed by the enzyme NO synthase (NOS). In recent years, data have begun to emerge on NOS expression and the physiological significance of NO in ectothermic vertebrates such as fish. However, there are relatively fewer data compared to the mammalian system. Although it is already well known that animal molybdoenzymes can convert nitrate and nitrite into NO, there is almost no information on the content and properties of molybdoenzymes in fish organs in the scientific literature. In this regard, the objectives of the present work were to detect the activity of classical molybdoenzymes xanthine oxidase (XO) and aldehyde oxidase (AO) in the liver and to study their possible activity to reduce nitrate and nitrite to nitrogen monoxide. In this work, the intrinsic activity of XO and AO was examined by using their substrates. At the same time, their nitrate (NR) - and nitrite reductase (NiR) activity were determined. It was determined that XO and AO in the fish liver are mainly represented by the molybdenum-free forms. The presence of an additional source of molybdenum can activate both the intrinsic and the NR and NiR activities. The NiR activity of XO and AO was higher than their NR activity. The data shows that treatment at a concentration of sodium molybdate and glutathione of 1.0 mM increases all activities of XO and AO. The optimal conditions for maximum activation of exogenous molybdate for XO and AO were reached by heating at 70 °C for 5 minutes. The activity of XO increased almost 4.7 times, and the activity of AO 7.7 times compared with its intrinsic activity without heat treatment. NO is formed from nitrite by the enzymes XO and AO much more than from nitrate.

Abstract Background Allopurinol is used to treat hyperuricemia and gout. It is metabolized to oxypurinol by xanthine oxidase (XO), and aldehyde oxidase (AO). Allopurinol and oxypurinol are potent XO inhibitors that reduce the plasma uric acid levels. Although oxypurinol levels show large inter-individual variations, high concentrations of oxypurinol can cause various adverse effects. Therefore, it is important to understand allopurinol metabolism by XO and AO. In this study we aimed to estimate the role of AO and XO in allopurinol metabolism by pre-administering Crl:CD and Jcl:SD rats, which have known strain differences in AO activity, with XO inhibitor febuxostat. Methods Allopurinol (30 or 100 mg/kg) was administered to Crl:CD and Jcl:SD rats with low and high AO activity, respectively, after pretreatment with or without febuxostat. The serum concentrations of allopurinol and oxypurinol were measured, and the area under the concentration-time curve (AUC) was calculated from the 48 h serum concentration-time profile. In vivo metabolic activity was measured as the ratio AUCoxypurinol /AUCallopurinol. Results Although no strain-specific differences were observed in the AUCoxypurinol/AUCallopurinol ratio in the allopurinol (30 mg/kg)-treated group, the ratio in Jcl:SD rats was higher than that in Crl:CD rats after febuxostat pretreatment. Contrastingly, the AUC ratio of allopurinol (100 mg/kg) was approximately 2-fold higher in Jcl:SD rats than that in Crl:CD rats. These findings showed that Jcl:SD rats had higher intrinsic AO activity than Crl:CD rats did. However, febuxostat pretreatment substantially decreased the activity, as measured by the AUC ratio using allopurinol (100 mg/kg), to 46 and 63% in Crl:CD rats and Jcl:SD rats, respectively, compared to the control group without febuxostat pretreatment. Conclusions We elucidated the role of XO and AO in allopurinol metabolism in Crl:CD and Jcl:SD rats. Notably, AO can exert a proportionately greater impact on allopurinol metabolism at high allopurinol concentrations. AO’s impact on allopurinol metabolism is meaningful enough that individual differences in AO may explain allopurinol toxicity events. Considering the inter-individual differences in AO activity, these findings can aid to dose adjustment of allopurinol to avoid potential adverse effects.

In the treatment of COVID-19, the antiviral medication Favipiravir has proven to be quite successful. Its metabolism is mediated by the enzymes aldehyde oxidase (AO) and xanthine oxidase (XO). This research investigated the potential drug-drug interaction between favipiravir and amlodipine in hypertensive rabbits. Twenty local adult male rabbits (aged between 10 and 12 months and weighing between 2 and 2.5 kg) were induced with hypertension by 20 mg/kg BW of desoxycorticosterone acetate subcutaneously for three weeks and then divided randomly into two groups of ten. The first group received a single oral dose of 40 mg/kg BW of favipiravir, while the second group received 5 mg/kg of amlodipine orally for 14 consecutive days to inhibit AO before receiving a single oral dose of 40 mg/kg BW of favipiravir. Blood samples were collected from the marginal ear vein at 15, 30, 45 min, and 1, 2, 4, 8, 12, 24, 48, 36, 48, and 72 h. High-performance liquid chromatography (HPLC) was used to determine the concentration of favipiravir in the plasma. The results showed that co-administration of amlodipine prolonged the time taken for favipiravir (Tmax) to reach maximum plasma concentration (Cmax) and decreased its elimination half-life, while increasing the area under the curve (AUC). Amlodipine also prolonged the elimination of favipiravir by reducing the clearance per unit time (Cl/f). Additionally, hypertension potentiated the effect of amlodipine on the absorption, distribution, metabolism, and excretion of favipiravir. In conclusion, concomitant use of favipiravir with other drugs that affect AO enzyme activity may alter the pharmacokinetic profile of the drug. Therefore, adjusting the dose of favipiravir administered to hypertensive patients receiving amlodipine is recommended.

Lactiplantibacillus plantarum‎, one of lactic acid bacteria (LAB), is found in various foods, ‎‎including dairy products, meat, and vegetables, and most of these bacteria offer beneficial ‎‎effects to humans and animals as potential probiotics with broad-spectrum antimicrobial ‎‎activities. The aim of this study was evaluating the antibacterial efficacy of L. ‎plantarum‎ ‎against ‎some foodborne bacteria isolated from dairy products. This research involved 34 dairy ‎products, ‎including local and imported milk, cheese, and yogurt sold locally in Baghdad ‎province, Iraq, during May ‎‎2022. For the isolation of L. ‎plantarum‎, a special medium called ‎MRS (de Man Rogosa and ‎Sharpe) was applied. Colonies were purified and identified by routine ‎bacteriological methods, ‎Vitek2 system, and confirmed by the polymerase chain reaction (PCR) ‎targeting the ‎16S rRNA‎ ‎gene followed by the amplicon sequencing. Other aerobic bacteria ‎contaminating dairy products ‎were also isolated onto sterile selective media specific for each ‎microorganism, and the isolates ‎were identified by routine diagnostics tests followed by ‎verification with Vitek2 system. Then, ‎the culture supernatant of L. ‎plantarum‎ was tested for its ‎antagonistic activity toward foodborne ‎bacteria by the use of agar well diffusion assay. The ‎findings showed the isolation of 2 L. ‎plantarum‎‎, 3 Pseudomonas aeruginosa‎, 4 Escherichia coli, ‎one isolate of Bacillus subtilis, and ‎another Staphylococcus hominis. The filtered supernatant of ‎L. ‎plantarum‎ was significantly ‎efficient in inhibiting the growth of the above bacteria. Each of E. ‎coli and B. subtilis‎ revealed ‎zones of inhibition of 36 and 38 mm in diameter, respectively, ‎while P. aeruginosa‎ and S. ‎hominis had inhibition zones diameters of 27 and 29 mm, ‎respectively. This suggests that the L. ‎plantarum‎‎ supernatant possesses a broad-spectrum ‎activity against foodborne bacteria. To ‎conclude, locally made dairy products can hold different ‎contaminating bacteria, which can be ‎eliminated by using probiotics, such as L. ‎plantarum‎, to ‎avoid foodborne diseases onset‎.

Molybdenum-containing enzymes of the xanthine oxidase (XO) family are well known to catalyse oxygen atom transfer reactions, with the great majority of the characterised enzymes catalysing the insertion of an oxygen atom into the substrate. Although some family members are known to catalyse the “reverse” reaction, the capability to abstract an oxygen atom from the substrate molecule is not generally recognised for these enzymes. Hence, it was with surprise and scepticism that the “molybdenum community” noticed the reports on the mammalian XO capability to catalyse the oxygen atom abstraction of nitrite to form nitric oxide (NO). The lack of precedent for a molybdenum- (or tungsten) containing nitrite reductase on the nitrogen biogeochemical cycle contributed also to the scepticism. It took several kinetic, spectroscopic and mechanistic studies on enzymes of the XO family and also of sulfite oxidase and DMSO reductase families to finally have wide recognition of the molybdoenzymes’ ability to form NO from nitrite. Herein, integrated in a collection of “personal views” edited by Professor Ralf Mendel, is an overview of my personal journey on the XO and aldehyde oxidase-catalysed nitrite reduction to NO. The main research findings and the path followed to establish XO and AO as competent nitrite reductases are reviewed. The evidence suggesting that these enzymes are probable players of the mammalian NO metabolism is also discussed.

Aldehyde oxidases (AOXs) are common detoxifying enzymes in several organisms. In insects, AOXs act in xenobiotic metabolism and as odorant-degrading enzymes (ODEs). These last appear as crucial enzymes in the life cycle of insects, helping to reset their olfactory system, particularly in lepidopterans, which fulfill important ecological roles (e.g., pollination or destructive life cycles). A comprehensive understanding of their olfactory system has provided opportunities to study key chemosensory proteins. However, no significant advance has been made around lepidopteran AOXs research, and even less around butterflies, a recently evolved lineage. In this study we have identified novel AOX gene families in moths and butterflies in order to understand their role as ODEs. Eighteen genomes from both moths and butterflies were used for phylogenetics, molecular evolution and sequence analyses. We identified 164 AOXs, from which 91 are new. Their phylogeny showed two main clades that are potentially related to odorant-degrading function, where both moths and butterflies have AOXs. A first ODE-related clade seems to have a non-ditrysian origin, likely related to plant volatiles. A second ODE-related clade could be more pheromone-biased. Molecular evolution analysis suggests a slight purifying selection process, though a number of sites appeared under positive selection. ODE-related AOXs have changed a phenylalanine residue by proline in the active site. Finally, this study could serve as a reference for further evolutionary and functional studies around Lepidopteran AOXs.

Abstract Metopimazine (MPZ) is a peripherally restricted, dopamine D2 receptor antagonist used for four decades to treat acute nausea and vomiting. MPZ is currently under clinical investigation for the treatment of gastroparesis (GP). MPZ undergoes high first‐pass metabolism that produces metopimazine acid (MPZA), the major circulating metabolite in humans. Despite a long history of use, the enzymes involved in the metabolism of MPZ have not been identified. Here we report a series of studies designed to identify potential MPZ metabolites in vitro, determine their clinical relevance in humans, and elucidate the enzymes responsible for their formation. The findings demonstrated that the formation of MPZA was primarily catalyzed by human liver microsomal amidase. Additionally, human liver cytosolic aldehyde oxidase (AO) catalyzes the formation of MPZA, in vitro, although to a much lesser extent. Neither cytochrome P450 enzymes nor flavin‐monooxygenases (FMO) were involved in the formation MPZA, although two minor oxidative pathways were catalyzed by CYP3A4 and CYP2D6 in vitro. Analysis of plasma samples from subjects dosed 60 mg of MPZ verified that these oxidative pathways are very minor and that CYP enzyme involvement was negligible compared to microsomal amidase/hydrolase in overall MPZ metabolism in humans. The metabolism by liver amidase, an enzyme family not well defined in small molecule drug metabolism, with minimal metabolism by CYPs, differentiates this drug from current D2 antagonists used or in development for the treatment of GP.

Immune response to SARS-CoV-2 and ensuing inflammation pose a huge challenge to the host’s nicotinamide adenine dinucleotide (NAD+) metabolism. Humans depend on vitamin B3 for biosynthesis of NAD+, indispensable for many metabolic and NAD+-consuming signaling reactions. The balance between its utilization and resynthesis is vitally important. Many extra-pulmonary symptoms of COVID-19 strikingly resemble those of pellagra, vitamin B3 deficiency (e.g., diarrhoea, dermatitis, oral cavity and tongue manifestations, loss of smell and taste, mental confusion). In most developed countries, pellagra is successfully eradicated by vitamin B3 fortification programs. Thus, conceivably, it has not been suspected as a cause of COVID-19 symptoms. Here, the deregulation of the NAD+ metabolism in response to the SARS-CoV-2 infection is reviewed, with special emphasis on the differences in the NAD+ biosynthetic pathway’s efficiency in conditions predisposing for the development of serious COVID-19. SARS-CoV-2 infection-induced NAD+ depletion and the elevated levels of its metabolites contribute to the development of a systemic disease. Acute liberation of nicotinamide (NAM) in antiviral NAD+-consuming reactions potentiates “NAM drain”, cooperatively mediated by nicotinamide N-methyltransferase and aldehyde oxidase. “NAM drain” compromises the NAD+ salvage pathway’s fail-safe function. The robustness of the host’s NAD+ salvage pathway, prior to the SARS-CoV-2 infection, is an important determinant of COVID-19 severity and persistence of certain symptoms upon resolution of infection.

Abstract Aldehyde Oxidase (hAOX1) is a cytosolic enzyme involved in the metabolism of drugs and xenobiotic compounds. The enzyme belongs to the xanthine oxidase (XO) family of Mo containing enzyme and is a homo‐dimer of two 150 kDa monomers. Nonsynonymous Single Nucleotide Polymorphisms (nsSNPs) of hAOX1 have been reported as affecting the ability of the enzyme to metabolize different substrates. Some of these nsSNPs have been biochemically and structurally characterized but the lack of a systematic and comprehensive study regarding all described and validated nsSNPs is urgent, due to the increasing importance of the enzyme in drug development, personalized medicine and therapy, as well as in pharmacogenetic studies. The objective of the present work was to collect all described nsSNPs of hAOX1 and utilize a series of bioinformatics tools to predict their effect on protein structure stability with putative implications on phenotypic functional consequences. Of 526 nsSNPs reported in NCBI‐dbSNP, 119 are identified as deleterious whereas 92 are identified as nondeleterious variants. The stability analysis was performed for 119 deleterious variants and the results suggest that 104 nsSNPs may be responsible for destabilizing the protein structure, whereas five variants may increase the protein stability. Four nsSNPs do not have any impact on protein structure (neutral nsSNPs) of hAOX1. The prediction results of the remaining six nsSNPs are nonconclusive. The in silico results were compared with available experimental data. This methodology can also be used to identify and prioritize the stabilizing and destabilizing variants in other enzymes involved in drug metabolism.

Following oral administration, the bioavailability of progesterone is low and highly variable. As a result, no clinically relevant, natural progesterone oral formulation is available. After oral delivery, first-pass metabolism initially occurs in the intestines; however, very little information on progesterone metabolism in this organ currently exists. The aim of this study is to investigate the contributions of liver and intestine to progesterone clearance. In the presence of NADPH, a rapid clearance of progesterone was observed in human and rat liver samples (t1/2 2.7 and 2.72 min, respectively). The rate of progesterone depletion in intestine was statistically similar between rat and human (t1/2 197.6 min in rat and 157.2 min in human). However, in the absence of NADPH, progesterone was depleted at a significantly lower rate in rat intestine compared to human. The roles of aldo keto reductases (AKR), xanthine oxidase (XAO) and aldehyde oxidase (AOX) in progesterone metabolism were also investigated. The rate of progesterone depletion was found to be significantly reduced by AKR1C, 1D1 and 1B1 in human liver and by AKR1B1 in human intestine. The inhibition of AOX also caused a significant reduction in progesterone degradation in human liver, whereas no change was observed in the presence of an XAO inhibitor. Understanding the kinetics of intestinal as well as liver metabolism is important for the future development of progesterone oral formulations. This novel information can inform decisions on the development of targeted formulations and help predict dosage regimens.

The activity of nicotinamide N-methyltransferase (NNMT) is tightly linked to the maintenance of the nicotinamide adenine dinucleotide (NAD+) level. This enzyme catalyzes methylation of nicotinamide (NAM) into methyl nicotinamide (MNAM), which is either excreted or further metabolized to N1-methyl-2-pyridone-5-carboxamide (2-PY) and H2O2. Enzymatic activity of NNMT is important for the prevention of NAM-mediated inhibition of NAD+-consuming enzymes poly–adenosine -diphosphate (ADP), ribose polymerases (PARPs), and sirtuins (SIRTs). Inappropriately high expression and activity of NNMT, commonly present in various types of cancer, has the potential to disrupt NAD+ homeostasis and cellular methylation potential. Largely overlooked, in the context of cancer, is the inhibitory effect of 2-PY on PARP-1 activity, which abrogates NNMT’s positive effect on cellular NAD+ flux by stalling liberation of NAM and reducing NAD+ synthesis in the salvage pathway. This review describes, and discusses, the mechanisms by which NNMT promotes NAD+ depletion and epigenetic reprogramming, leading to the development of metabolic plasticity, evasion of a major tumor suppressive process of cellular senescence, and acquisition of stem cell properties. All these phenomena are related to therapy resistance and worse clinical outcomes.

Currently no approved treatment exists for fibrodysplasia ossificans progressiva (FOP) patients, and disease progression results in severe restriction of joint function and premature mortality. LDN-193189 has been demonstrated to be efficacious in a mouse FOP disease model after oral administration. To support species selection for drug safety evaluation and to guide structure optimization for back-up compounds, in vitro metabolism of LDN-193189 was investigated in liver microsome and cytosol fractions of mouse, rat, dog, rabbit, monkey and human. Metabolism studies included analysis of reactive intermediate formation using glutathione and potassium cyanide (KCN) and analysis of non-P450 mediated metabolites in cytosol fractions of various species. Metabolite profiles and metabolic soft spots of LDN-193189 were elucidated using LC/UV and mass spectral techniques. The in vitro metabolism of LDN-193189 was significantly dependent on aldehyde oxidase, with formation of the major NIH-Q55 metabolite. The piperazinyl moiety of LDN-193189 was liable to NADPH-dependent metabolism which generated reactive iminium intermediates, as confirmed through KCN trapping experiments, and aniline metabolites (M337 and M380), which brought up potential drug safety concerns. Subsequently, strategies were employed to avoid metabolic liabilities leading to the synthesis of Compounds 1, 2, and 3. This study demonstrated the importance of metabolite identification for the discovery of novel and safe drug candidates for the treatment of FOP and helped medicinal chemists steer away from potential metabolic liabilities.

Background: Molybdenum hydroxylases have been implicated as key oxidative enzymes in some diseases. Methods: Twenty 2-(substituted-thio)-quinazolin-4-one derivatives recently synthesized in our laboratory were examined for their inhibitory activity toward molybdenum hydroxylases. Results and conclusion: The tested quinazolines inhibited both xanthine oxidase and aldehyde oxidase enzymes in a competitive pattern with Ki values range of 66–753 μM. Pharmacophore prediction methodology was used to study the structure requirements of those inhibitors.

Iron sulfur (Fe-S) clusters and the molybdenum cofactor (Moco) are present at enzyme sites, where the active metal facilitates electron transfer. Such enzyme systems are soluble in the mitochondrial matrix, cytosol and nucleus, or embedded in the inner mitochondrial membrane, but virtually absent from the cell secretory pathway. They are of ancient evolutionary origin supporting respiration, DNA replication, transcription, translation, the biosynthesis of steroids, heme, catabolism of purines, hydroxylation of xenobiotics, and cellular sulfur metabolism. Here, Fe-S cluster and Moco biosynthesis in Drosophila melanogaster is reviewed and the multiple biochemical and physiological functions of known Fe-S and Moco enzymes are described. We show that RNA interference of Mocs3 disrupts Moco biosynthesis and the circadian clock. Fe-S-dependent mitochondrial respiration is discussed in the context of germ line and somatic development, stem cell differentiation and aging. The subcellular compartmentalization of the Fe-S and Moco assembly machinery components and their connections to iron sensing mechanisms and intermediary metabolism are emphasized. A biochemically active Fe-S core complex of heterologously expressed fly Nfs1, Isd11, IscU, and human frataxin is presented. Based on the recent demonstration that copper displaces the Fe-S cluster of yeast and human ferredoxin, an explanation for why high dietary copper leads to cytoplasmic iron deficiency in flies is proposed. Another proposal that exosomes contribute to the transport of xanthine dehydrogenase from peripheral tissues to the eye pigment cells is put forward, where the Vps16a subunit of the HOPS complex may have a specialized role in concentrating this enzyme within pigment granules. Finally, we formulate a hypothesis that (i) mitochondrial superoxide mobilizes iron from the Fe-S clusters in aconitase and succinate dehydrogenase; (ii) increased iron transiently displaces manganese on superoxide dismutase, which may function as a mitochondrial iron sensor since it is inactivated by iron; (iii) with the Krebs cycle thus disrupted, citrate is exported to the cytosol for fatty acid synthesis, while succinyl-CoA and the iron are used for heme biosynthesis; (iv) as iron is used for heme biosynthesis its concentration in the matrix drops allowing for manganese to reactivate superoxide dismutase and Fe-S cluster biosynthesis to reestablish the Krebs cycle.

The antimicrobial, antioxidant, brine shrimp toxicity and enzyme inhibitory activities of the essential oil and extracts of Salvia spinosa were investigated. GC-FID and GC-MS analysis of the essential oil resulted in identification of 19 compounds representing 98.5% of the oil. The major compounds of the essential oil were identified as caryophyllene oxide (63.0%), spathulenol (23.0%), linalool (3.9%) and trans-caryophyllene (3.0%). Essential oil showed strong aldehyde oxidase inhibitory activity with IC50 value of 40 µg/ml. Methanol extract displayed the strongest DPPH radical scavenging, brine shrimp toxicity and α-glucosidase inhibitory activities. Total phenolic and flavonoid contents of the plant extracts were 36–377 mg GAE/g and 13–134 mg QE/g, respectively. Methanol extract exhibited high amounts of phenolic and flavonoid compounds. The results showed that S. spinosa has great potential for several uses in food and pharmaceutical industries.

Aldehyde Oxidase (AO) enzyme (EC 1.2.3.1) catalyzes the final steps of carotenoid catabolism and it is a key enzyme in the abscisic acid (ABA) biosynthesis. AO isoforms are located in the cytosolic compartment of tissues in many plants, where induce the oxidation of aldehydes into carboxylic acid, and in addition, catalyze the hydroxylation of some heterocycles. The goal of the present study was to characterize the AO genes involved in the accumulation of carotenoid pigments in wheat grain, an important quantitative trait controlled by multiple genes. The cDNAs corresponding to the four AO isoforms from Arabidopsis thaliana and five AO isoforms from Brachypodium distachyon were used as query in 454 sequence assemblies data for Triticum aestivum cv. Chinese Spring (https://urgi.versailles.inra.fr/blast/blast.php) to obtain the partial or whole orthologous wheat AO sequences. Three wheat isoforms, designated AO1, AO2, and AO3 were located on the chromosome groups 2, 5, and 7, respectively, and mapped on two consensus wheat maps by SNP markers located within the AO gene sequences. To validate the possible relationships between AO3 genes and carotenoid accumulation in wheat, the expression levels of AO-A3 and AO-B3 gene were determined during the kernel maturation stage of two durum wheat cultivars, Ciccio and Svevo, characterized by a low and high carotenoid content, respectively. Different AO-A3 gene expression values were observed between the two cultivars indicating that the AO-A3 allele present in Ciccio was more active in carotenoid degradation. A gene marker was developed and can be used for marker-assisted selection in wheat breeding programs.

The endogenous vitamins such as pyridoxal (vitamin B6) and all-trans retinaldehyde (vitamin A) are metabolized to more or less toxic metabolites by drug-metabolizing enzymes including aldehyde oxidase (AO; EC 1.2.3.1). To better understand this function, the specificity of the rabbit liver aldehyde oxidase enzyme toward endogenous vitamins was quantitatively studied. Therefore, the present study showed the kinetic parameters of AO for the oxidation of vitamin B6 and vitamin A were measured in partially purified rabbit liver fraction. Km values of AO endogenous vitamin were observed with pyridoxal (21 ± 6.4 μM) and all-trans-retinal (46 ± 9.1 μM) respectively for partially purified rabbit liver fraction. AO from rabbit liver fraction showed high Vmax with vitamin B6 and vitamin A (1.84 ± 0.2 and 1.28 ± 0.1 nmol/min/mg protein, respectively). Therefore, the present study showed the kinetic parameters of AO for the oxidation of vitamin B6 and vitamin A were measured in partially purified rabbit liver fraction. A high affinity and low Km values of AO endogenous vitamin were observed with pyridoxal (21 ± 6.4 μM) and all-trans-retinal (46 ± 9.1 μM), respectively for partially purified rabbit liver fraction. Pyridoxal and all-trans-retinal oxidized to their metabolites (25.2 ± 12.7 and 13.3 ± 4.1 nmol/min/mg protein, respectively) by partially purified rabbit liver aldehyde oxidase. These results confirmed that the hydrophobicity enhances affinity of pyridoxal and all-trans-retinal (aromatic aldehyde) toward AO as excellent substrates. It is concluded these results presented serve as a guide for predicting the susceptibility of endogenous to oxidation by rabbit liver AO.

In the form of molybdate the transition metal molybdenum is essential for plants as it is required by a number of enzymes that catalyze key reactions in nitrogen assimilation, purine degradation, phytohormone synthesis, and sulfite detoxification. However, molybdate itself is biologically inactive and needs to be complexed by a specific organic pterin in order to serve as a permanently bound prosthetic group, the molybdenum cofactor, for the socalled molybdo-enyzmes. While the synthesis of molybdenum cofactor has been intensively studied, only little is known about the uptake of molybdate by the roots, its transport to the shoot and its allocation and storage within the cell. Yet, recent evidence indicates that intracellular molybdate levels are tightly controlled by molybdate transporters, in particular during plant development. Moreover, a tight connection between molybdenum and iron metabolisms is presumed because (i) uptake mechanisms for molybdate and iron affect each other, (ii) most molybdo-enzymes do also require iron-containing redox groups such as iron-sulfur clusters or heme, (iii) molybdenum metabolism has recruited mechanisms typical for iron-sulfur cluster synthesis, and (iv) both molybdenum cofactor synthesis and extramitochondrial iron-sulfur proteins involve the function of a specific mitochondrial ABC-type transporter.