Pathogenic yeasts Candida albicans and Candida parapsilosis possess a ß-type carbonic anhydrase Nce103p, which is involved in CO2 hydration and signaling. C. albicans lacking Nce103p cannot survive in low CO2 concentrations, e.g., in atmospheric growth conditions. Candida carbonic anhydrases are orthologous to the Saccharomyces cerevisiae enzyme, which had originally been detected as a substrate of a non-classical export pathway. However, experimental evidence on localization of C. albicans and C. parapsilosis carbonic anhydrases has not been reported to date. Immunogold labeling and electron microscopy used in the present study showed that carbonic anhydrases are localized in the cell wall and plasmatic membrane of both Candida species. This localization was confirmed by Western blot and mass spectrometry analyses of isolated cell wall and plasma membrane fractions. Further analysis of C. albicans and C. parapsilosis subcellular fractions revealed presence of carbonic anhydrases also in the cytosolic and mitochondrial fractions of Candida cells cultivated in shaken liquid cultures, under the atmospheric conditions.
- Klíčová slova
- Candida albicans, Candida parapsilosis, Nce103p, carbonic anhydrase, cell wall, electron microscopy, localization, mass spectrometry,
- MeSH
- buněčná membrána enzymologie MeSH
- buněčná stěna enzymologie MeSH
- Candida albicans enzymologie růst a vývoj MeSH
- Candida parapsilosis enzymologie růst a vývoj MeSH
- cytosol enzymologie MeSH
- elektronová mikroskopie MeSH
- fungální proteiny metabolismus MeSH
- hmotnostní spektrometrie MeSH
- karboanhydrasy metabolismus MeSH
- mitochondrie enzymologie MeSH
- techniky vsádkové kultivace MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- fungální proteiny MeSH
- karboanhydrasy MeSH
α-Galactosidases are assigned to the class of hydrolases and the subclass of glycoside hydrolases (GHs). They belong to six GH families and include the only characterized α-galactosidases from yeasts (GH 27, Saccharomyces cerevisiae). The present study focuses on an investigation of the lactose-inducible α-galactosidase produced by Papiliotrema flavescens. The enzyme was present on the surface of cells and in the cytosol. Its temperature optimum was about 60 °C and the pH optimum was 4.8; the pH stability ranged from 3.2 to 6.6. This α-galactosidase also exhibited transglycosylation activity. The cytosol α-galactosidase with a molecular weight about 110 kDa, was purified using a combination of liquid chromatography techniques. Three intramolecular peptides were determined by the partial structural analysis of the sequences of the protein isolated, using MALDI-TOF/TOF mass spectrometry. The data obtained recognized the first yeast α-galactosidase, which belongs to the GH 36 family. The bioinformatics analysis and homology modeling of a 210 amino acids long C-terminal sequence (derived from cDNA) confirmed the correctness of these findings. The study was also supplemented by the screening of capsular cryptococcal yeasts, which produce the surface lactose-inducible α- and β-galactosidases. The production of the lactose-inducible α-galactosidases was not found to be a general feature within the yeast strains examined and, therefore, the existing hypothesis on the general function of this enzyme in cryptococcal capsule rearrangement cannot be confirmed.
- Klíčová slova
- Cryptococcus flavescens, GH 36 family, Lactose-inducible, Long-chain, Papiliotrema flavescens, α-Galactosidase,
- MeSH
- alfa-galaktosidasa chemie genetika izolace a purifikace metabolismus MeSH
- Basidiomycota klasifikace enzymologie genetika růst a vývoj MeSH
- Cryptococcus MeSH
- cytosol enzymologie MeSH
- DNA fungální genetika MeSH
- fungální proteiny chemie genetika izolace a purifikace metabolismus MeSH
- geny hub genetika MeSH
- glykosidhydrolasy metabolismus MeSH
- komplementární DNA MeSH
- koncentrace vodíkových iontů MeSH
- konformace proteinů MeSH
- laktosa metabolismus MeSH
- molekulární modely MeSH
- molekulová hmotnost MeSH
- sekvence aminokyselin MeSH
- sekvenční analýza proteinů MeSH
- sekvenční seřazení MeSH
- stabilita enzymů MeSH
- substrátová specifita MeSH
- teplota MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alfa-galaktosidasa MeSH
- DNA fungální MeSH
- fungální proteiny MeSH
- glykosidhydrolasy MeSH
- komplementární DNA MeSH
- laktosa MeSH
Tiaprofenic acid is a widely used anti-inflammatory drug; however, the reductive metabolism of tiaprofenic acid is not yet well understood. Here, we compared the reduction of tiaprofenic acid in microsomes and cytosol from the human liver. The microsomes exhibited lower Km value toward tiaprofenic acid than the cytosol (Km = 164 ± 18 μM vs. 569 ± 74 μM, respectively), whereas the cytosol showed higher specific activity during reduction than the microsomes (Vmax = 728 ± 52 pmol mg of protein-1 min-1 vs. 285 ± 11 pmol mg of protein-1 min-1, respectively). Next, a panel of recombinant carbonyl reducing enzymes from AKR and SDR superfamilies has been studied to find the enzymes responsible for the cytosolic reduction of tiaprofenic acid. CBR1 was identified as the reductase of tiaprofenic acid with high specific activity (56,965 ± 6741 pmol mg of protein-1 min-1). Three other enzymes, AKR1A1, AKR1B10, and AKR1C4, were also able to reduce tiaprofenic acid, but with very low activity. Thus, CBR1 was shown to be a tiaprofenic acid reductase in vitro and was also suggested to be the principal tiaprofenic acid reductase in vivo.
- Klíčová slova
- AKR, Carbonyl reducing enzymes (CREs), Carbonyl reductase, Reductive drug metabolism, Short-chain dehydrogenase/reductase, Tiaprofenic acid,
- MeSH
- alkoholoxidoreduktasy chemie genetika metabolismus MeSH
- biokatalýza MeSH
- cytosol enzymologie MeSH
- dehydrogenasy/reduktasy s krátkým řetězcem chemie genetika metabolismus MeSH
- játra enzymologie metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- mikrozomy enzymologie MeSH
- propionáty chemie metabolismus MeSH
- rekombinantní proteiny biosyntéza chemie izolace a purifikace MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alkoholoxidoreduktasy MeSH
- CBR1 protein, human MeSH Prohlížeč
- dehydrogenasy/reduktasy s krátkým řetězcem MeSH
- propionáty MeSH
- rekombinantní proteiny MeSH
- tiaprofenic acid MeSH Prohlížeč
Fenofibric acid is a hypolipidemic drug that is used as an active ingredient per se or is administered in the form of fenofibrate that releases fenofibric acid after absorption. The metabolism of fenofibric acid is mediated primarily by glucuronidation. However, the other part of fenofibric acid is excreted as reduced fenofibric acid. Enzymes responsible for the formation of reduced fenofibric acid as well as their subcellular localization have remained unknown until now. We have found that the predominant site of fenofibric acid reduction is the human liver cytosol, whereas liver microsomes reduced fenofibric acid to a lower extent and exhibited a lower affinity for this drug (Km > 1000 μM). Of nine carbonyl-reducing enzymes (CREs) tested, CBR1 exhibited the greatest activity for fenofibric acid reduction (CLint = 85.975 μl/mg protein/min). CBR1 predominantly formed (-)-enantiomers of reduced fenofibric acid similar to liver cytosol and in accordance with the in vivo data. AKR1C1, AKR1C2, AKR1C3 and AKR1B1 were also identified as reductases of fenofibric acid but are expected to play only a minor role in fenofibric acid metabolism.
- Klíčová slova
- AKR, Carbonyl reducing enzymes, Carbonyl reductase, Fenofibric acid, Reductive drug metabolism, Short-chain dehydrogenase/reductase,
- MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- biokatalýza účinky léků MeSH
- cytosol účinky léků enzymologie MeSH
- fenofibrát analogy a deriváty chemie metabolismus MeSH
- játra účinky léků metabolismus MeSH
- kinetika MeSH
- lidé MeSH
- methanol farmakologie MeSH
- oxidace-redukce MeSH
- stereoizomerie MeSH
- subcelulární frakce metabolismus MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- Názvy látek
- alkoholoxidoreduktasy MeSH
- CBR1 protein, human MeSH Prohlížeč
- fenofibrát MeSH
- fenofibric acid MeSH Prohlížeč
- methanol MeSH
This work describes novel in vitro inhibitors of human mitochondrial (mdN) and cytosolic (cdN) 5'(3')-deoxynucleotidases. We designed a series of derivatives of the lead compound (S)-1-[2-deoxy-3,5-O-(phosphonobenzylidene)-β-d-threo-pentofuranosyl]thymine bearing various substituents in the para position of the benzylidene moiety. Detailed kinetic study revealed that certain para substituents increase the inhibitory potency (iodo derivative; K = 2.71 μM) and some induce a shift in selectivity toward cdN (carboxy derivative, K = 11.60 μM; iodoxy derivative, K = 6.60 μM). Crystal structures of mdN in complex with three of these compounds revealed that various para substituents lead to two alternative inhibitor binding modes within the enzyme active site. Two binding modes were also identified for cdN complexes by heteronuclear NMR spectroscopy.
- MeSH
- 5'-nukleotidasa antagonisté a inhibitory metabolismus MeSH
- cytosol enzymologie MeSH
- inhibitory enzymů chemická syntéza chemie farmakologie MeSH
- lidé MeSH
- mitochondrie enzymologie MeSH
- molekulární konformace MeSH
- organofosfonáty chemická syntéza chemie farmakologie MeSH
- vztah mezi dávkou a účinkem léčiva MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- 5'-nukleotidasa MeSH
- 5'(3')-nucleotidase MeSH Prohlížeč
- inhibitory enzymů MeSH
- organofosfonáty MeSH
Bupropion is widely used as an antidepressant drug and also as a smoking cessation aid. In humans, this drug is extensively metabolized to form several metabolites. Oxidised hydroxybupropion and two reduced metabolites, threohydrobupropion and erythrohydrobupropion, are major metabolites. All of these metabolites are considered to be active. Although the oxidative metabolic pathway and the central role of CYP2B6 are known, the enzymes that participate in the reduction have not been identified to date. The aim of this study was to confirm the role of human liver subcellular fractions in the metabolism of bupropion and elucidate the contribution of particular carbonyl-reducing enzymes. An HPLC method for the determination of bupropion metabolites was utilised. Bupropion is reduced to threohydrobupropion and less to erythrohydrobupropion in human liver cytosol, microsomes and also mitochondria. Surprisingly, intrinsic clearance for formation of both metabolites is the highest in mitochondrial fraction. Moreover this study provides the first direct evidence that 11β-hydroxysteroid dehydrogenase 1, AKR1C1, AKR1C2, AKR1C3 and CBR1 participate in the reducing biotransformation of bupropion in vitro. The enzyme kinetics of all of these reductases was investigated and kinetic parameters were calculated.
- MeSH
- alkoholoxidoreduktasy genetika metabolismus MeSH
- antidepresiva druhé generace metabolismus MeSH
- biotransformace MeSH
- bupropion analogy a deriváty metabolismus MeSH
- cytosol enzymologie MeSH
- hydroxysteroidní dehydrogenasy genetika metabolismus MeSH
- jaterní mikrozomy enzymologie MeSH
- jaterní mitochondrie enzymologie MeSH
- játra enzymologie MeSH
- kinetika MeSH
- lidé MeSH
- oxidace-redukce MeSH
- rekombinantní proteiny metabolismus MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkoholoxidoreduktasy MeSH
- antidepresiva druhé generace MeSH
- bupropion MeSH
- CBR1 protein, human MeSH Prohlížeč
- hydrobupropion MeSH Prohlížeč
- hydroxysteroidní dehydrogenasy MeSH
- rekombinantní proteiny MeSH
Oxcarbazepine, a second generation antiepileptic drug belonging to the family of dibenz[b,f]azepines, is subjected to a rapid and extensive biotransformation. Oxcarbazepine demonstrates a low potential for drug interactions because its biotransformation is mainly mediated by the reduction pathway instead of oxidative pathways, which are very susceptible to drug interactions. The reductive metabolism of oxcarbazepine yields a 10-monohydroxy derivative (10,11-dihydro-10-hydroxy-carbazepine), which is responsible for the pharmacological activity. The identity and localization of enzymes participating in the reduction of oxcarbazepine in response to this active metabolite have remained unknown until now. Thus, we investigated the reductive metabolism of oxcarbazepine in human liver subcellular fractions and using recombinant carbonyl reducing enzymes. The reduction of oxcarbazepine was shown to occur largely in the liver cytosol rather than liver microsomes. Furthermore, the activity and stereospecificity of cytosolic carbonyl reducing enzymes toward oxcarbazepine were assessed. Of the eight tested enzymes, six reductases were identified to contribute to the reduction of oxcarbazepine. The highest activities were demonstrated by AKR1C1, AKR1C2, AKR1C3, and AKR1C4. The contribution of CBR1 and CBR3 to the reduction of oxcarbazepine was also significant, although their role in oxcarbazepine metabolism in vivo is unclear.
- Klíčová slova
- AKR, Carbonyl reducing enzymes, Carbonyl reductase, Oxcarbazepine, Reductive drug metabolism, Short-chain dehydrogenase/reductase,
- MeSH
- alkoholoxidoreduktasy metabolismus MeSH
- antikonvulziva metabolismus MeSH
- cytosol enzymologie MeSH
- jaterní mikrozomy metabolismus MeSH
- játra enzymologie MeSH
- karbamazepin analogy a deriváty metabolismus MeSH
- lidé MeSH
- oxidace-redukce MeSH
- oxkarbazepin MeSH
- vysokoúčinná kapalinová chromatografie MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- alkoholoxidoreduktasy MeSH
- antikonvulziva MeSH
- CBR1 protein, human MeSH Prohlížeč
- karbamazepin MeSH
- oxkarbazepin MeSH
UNLABELLED: Aristolochic acid is the cause of aristolochic acid nephropathy (AAN) and Balkan endemic nephropathy (BEN) and their associated urothelial malignancies. Using Western blotting, we investigated the expression of NAD(P)H: quinone oxidoreductase (NQO1), the most efficient cytosolic enzyme that reductively activates aristolochic acid I (AAI) in mice and rats. In addition, the effect of AAI on the expression of the NQO1 protein and its enzymatic activity in these experimental animal models was examined. We found that NQO1 protein levels in cytosolic fractions isolated from liver, kidney and lung of mice differed from those expressed in these organs of rats. In mice, the highest levels of NQO1 protein and NQO1 activity were found in the kidney, followed by lung and liver. In contrast, the NQO1 protein levels and enzyme activity were lowest in rat-kidney cytosol, whereas the highest amounts of NQO1 protein and activity were found in lung cytosols, followed by those of liver. NQO1 protein and enzyme activity were induced in liver and kidney of AAI-pretreated mice compared with those of untreated mice. NQO1 protein and enzyme activity were also induced in rat kidney by AAI. Furthermore, the increase in hepatic and renal NQO1 enzyme activity was associated with AAI bio-activation and elevated AAI-DNA adduct levels were found in ex vivo incubations of cytosolic fractions with DNA and AAI. In conclusion, our results indicate that AAI can increase its own metabolic activation by inducing NQO1, thereby enhancing its own genotoxic potential.
- Klíčová slova
- Aristolochic acid nephropathy, DNA adducts, Metabolic activation, NAD(P):quinone oxidoreductase, Protein expression,
- MeSH
- aktivace enzymů účinky léků MeSH
- cytosol enzymologie MeSH
- játra enzymologie patologie MeSH
- karcinogeny farmakologie MeSH
- krysa rodu Rattus MeSH
- kyseliny aristolochové farmakologie MeSH
- ledviny enzymologie patologie MeSH
- myši knockoutované MeSH
- myši MeSH
- NAD(P)H dehydrogenasa (chinon) metabolismus MeSH
- plíce enzymologie patologie MeSH
- potkani Wistar MeSH
- zvířata MeSH
- Check Tag
- krysa rodu Rattus MeSH
- mužské pohlaví MeSH
- myši MeSH
- ženské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- srovnávací studie MeSH
- Názvy látek
- aristolochic acid I MeSH Prohlížeč
- karcinogeny MeSH
- kyseliny aristolochové MeSH
- NAD(P)H dehydrogenasa (chinon) MeSH
- Nqo1 protein, mouse MeSH Prohlížeč
- NQO1 protein, rat MeSH Prohlížeč
Naegleria gruberi is a free-living heterotrophic aerobic amoeba well known for its ability to transform from an amoeba to a flagellate form. The genome of N. gruberi has been recently published, and in silico predictions demonstrated that Naegleria has the capacity for both aerobic respiration and anaerobic biochemistry to produce molecular hydrogen in its mitochondria. This finding was considered to have fundamental implications on the evolution of mitochondrial metabolism and of the last eukaryotic common ancestor. However, no actual experimental data have been shown to support this hypothesis. For this reason, we have decided to investigate the anaerobic metabolism of the mitochondrion of N. gruberi. Using in vivo biochemical assays, we have demonstrated that N. gruberi has indeed a functional [FeFe]-hydrogenase, an enzyme that is attributed to anaerobic organisms. Surprisingly, in contrast to the published predictions, we have demonstrated that hydrogenase is localized exclusively in the cytosol, while no hydrogenase activity was associated with mitochondria of the organism. In addition, cytosolic localization displayed for HydE, a marker component of hydrogenase maturases. Naegleria gruberi, an obligate aerobic organism and one of the earliest eukaryotes, is producing hydrogen, a function that raises questions on the purpose of this pathway for the lifestyle of the organism and potentially on the evolution of eukaryotes.
- Klíčová slova
- Naegleria, hydrogen hypothesis, hydrogenase, maturases, mitochondrial evolution,
- MeSH
- cytosol enzymologie MeSH
- hydrogenasa genetika metabolismus MeSH
- mitochondrie genetika metabolismus MeSH
- Naegleria enzymologie genetika MeSH
- protozoální proteiny genetika metabolismus MeSH
- vodík metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- hydrogenasa MeSH
- protozoální proteiny MeSH
- vodík MeSH
The human 5'(3')-deoxyribonucleotidases catalyze the dephosphorylation of deoxyribonucleoside monophosphates to the corresponding deoxyribonucleosides and thus help to maintain the balance between pools of nucleosides and nucleotides. Here, the structures of human cytosolic deoxyribonucleotidase (cdN) at atomic resolution (1.08 Å) and mitochondrial deoxyribonucleotidase (mdN) at near-atomic resolution (1.4 Å) are reported. The attainment of an atomic resolution structure allowed interatomic distances to be used to assess the probable protonation state of the phosphate anion and the side chains in the enzyme active site. A detailed comparison of the cdN and mdN active sites allowed the design of a cdN-specific inhibitor.
- Klíčová slova
- 5′(3′)-deoxyribonucleotidases, enzyme inhibition, hydrolases, structure-based drug design,
- MeSH
- cytosol chemie enzymologie MeSH
- deoxyribonukleotidy chemie MeSH
- Escherichia coli genetika metabolismus MeSH
- eukaryotické buňky chemie enzymologie MeSH
- fosfáty chemie MeSH
- inhibitory enzymů chemie MeSH
- izoenzymy antagonisté a inhibitory chemie genetika MeSH
- katalytická doména MeSH
- konformace proteinů MeSH
- krystalografie rentgenová MeSH
- lidé MeSH
- mitochondrie chemie enzymologie MeSH
- molekulární modely MeSH
- nukleotidasy antagonisté a inhibitory chemie genetika MeSH
- organofosfonáty chemie MeSH
- orgánová specificita MeSH
- racionální návrh léčiv MeSH
- rekombinantní proteiny chemie genetika MeSH
- simulace molekulového dockingu MeSH
- vztahy mezi strukturou a aktivitou MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- Názvy látek
- deoxyribonukleotidy MeSH
- fosfáty MeSH
- inhibitory enzymů MeSH
- izoenzymy MeSH
- nukleotidasy MeSH
- organofosfonáty MeSH
- rekombinantní proteiny MeSH
