The catalytic reaction of cytokinin oxidase/dehydrogenase (EC 1.5.99.12) was studied in detail using the recombinant flavoenzyme from maize. Determination of the redox potential of the covalently linked flavin cofactor revealed a relatively high potential dictating the type of electron acceptor that can be used by the enzyme. Using 2,6-dichlorophenol indophenol, 2,3-dimethoxy-5-methyl-1,4-benzoquinone or 1,4-naphthoquinone as electron acceptor, turnover rates with N6-(2-isopentenyl)adenine of approx. 150 s(-1) could be obtained. This suggests that the natural electron acceptor of the enzyme is quite probably a p-quinone or similar compound. By using the stopped-flow technique, it was found that the enzyme is rapidly reduced by N6-(2-isopentenyl)adenine (k(red)=950 s(-1)). Re-oxidation of the reduced enzyme by molecular oxygen is too slow to be of physiological relevance, confirming its classification as a dehydrogenase. Furthermore, it was established for the first time that the enzyme is capable of degrading aromatic cytokinins, although at low reaction rates. As a result, the enzyme displays a dual catalytic mode for oxidative degradation of cytokinins: a low-rate and low-substrate specificity reaction with oxygen as the electron acceptor, and high activity and strict specificity for isopentenyladenine and analogous cytokinins with some specific electron acceptors.

• MeSH
• aldehydy metabolismus   MeSH
• chinony metabolismus   MeSH
• cytokininy metabolismus   MeSH
• elektrochemie MeSH
• flavinadenindinukleotid metabolismus   MeSH
• katalýza MeSH
• kinetika MeSH
• kukuřice setá enzymologie   MeSH
• oxidace-redukce MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• rekombinantní proteiny metabolismus   MeSH
• rostlinné proteiny chemie   metabolismus   MeSH
• substrátová specifita MeSH
• terciární struktura proteinů MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• srovnávací studie MeSH
• Názvy látek
• aldehydy MeSH
• chinony MeSH
• cytokinin oxidase MeSH Prohlížeč
• cytokininy MeSH
• flavinadenindinukleotid MeSH
• oxidoreduktasy MeSH
• rekombinantní proteiny MeSH
• rostlinné proteiny MeSH

Isocitrate dehydrogenase is an enzyme converting isocitrate to α-ketoglutarate in the canonical tricarboxylic acid (TCA) cycle. There are three different types of isocitrate dehydrogenase documented in eukaryotes. Our study points out the complex evolutionary history of isocitrate dehydrogenases across kinetoplastids, where the common ancestor of Trypanosomatidae and Bodonidae was equipped with two isoforms of the isocitrate dehydrogenase enzyme: the NADP+-dependent isocitrate dehydrogenase 1 with possibly dual localization in the cytosol and mitochondrion and NADP+-dependent mitochondrial isocitrate dehydrogenase 2. In the extant trypanosomatids, isocitrate dehydrogenase 1 is present only in a few species suggesting that it was lost upon separation of Trypanosoma spp. and replaced by the mainly NADP+-dependent cytosolic isocitrate dehydrogenase 3 of bacterial origin in all the derived lineages. In this study, we experimentally demonstrate that the omnipresent isocitrate dehydrogenase 2 has a dual localization in both mitochondrion and cytosol in at least four species that possess only this isoform. The apparent lack of the NAD+-dependent isocitrate dehydrogenase activity in trypanosomatid mitochondrion provides further support to the existence of the noncanonical TCA cycle across trypanosomatids and the bidirectional activity of isocitrate dehydrogenase 3 when operating with NADP+ cofactor instead of NAD+. This observation can be extended to all 17 species analyzed in this study, except for Leishmania mexicana, which showed only low isocitrate dehydrogenase activity in the cytosol. The variability in isocitrate oxidation capacity among species may reflect the distinct metabolic strategies and needs for reduced cofactors in particular environments.

• Klíčová slova
• Krebs cycle, NAD+, NADP+, TCA cycle, cofactor preference, isocitrate dehydrogenase,
• MeSH
• isocitrátdehydrogenasa * genetika   metabolismus   MeSH
• isocitráty metabolismus   MeSH
• NAD * metabolismus   MeSH
• NADP metabolismus   MeSH
• protein - isoformy MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• isocitrátdehydrogenasa * MeSH
• isocitráty MeSH
• isocitric acid MeSH Prohlížeč
• NAD * MeSH
• NADP MeSH
• protein - isoformy MeSH

Most of the phosphatases of human fungal pathogens Candida albicans and C. parapsilosis have never been experimentally characterised, although dephosphorylation reactions are central to many biological processes. PHO15 genes of these yeasts have been annotated as the sequences encoding 4-nitrophenyl phosphatase, on the basis of homology to PHO13 gene from the bakers' yeast Saccharomyces cerevisiae. To examine the real function of these potential phosphatases from Candida spp., CaPho15p and CpPho15p were prepared using expression in Escherichia coli and characterised. They share the hallmark motifs of the haloacid dehalogenase superfamily, readily hydrolyse 4-nitrophenyl phosphate at pH 8-8.3 and require divalent cations (Mg2+, Mn2+ or Co2+) as cofactors. CaPho15p and CpPho15p did not dephosphorylate phosphopeptides, but rather hydrolysed molecules related to carbohydrate metabolism. The preferred substrate for the both phosphatases was 2-phosphoglycolate. Among the other molecules tested, CaPho15 showed preference for glyceraldehyde phosphate and ß-glycerol phosphate, while CpPho15 dephosphorylated mainly 1,3-dihydroxyacetone phosphate. This type of substrate specificity indicates that CaPho15 and CpPho15 may be a part of metabolic repair system of C. albicans and C. parapsilosis.

• MeSH
• aminokyselinové motivy MeSH
• biotransformace MeSH
• Candida albicans enzymologie   MeSH
• Candida parapsilosis enzymologie   MeSH
• Escherichia coli genetika   metabolismus   MeSH
• exprese genu MeSH
• fosfatasy genetika   metabolismus   MeSH
• fungální proteiny genetika   metabolismus   MeSH
• glykoláty metabolismus   MeSH
• klonování DNA MeSH
• koenzymy analýza   MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• substrátová specifita MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• fosfatasy MeSH
• fungální proteiny MeSH
• glykoláty MeSH
• koenzymy MeSH
• phosphoglycolate MeSH Prohlížeč
• rekombinantní proteiny MeSH

Glucocorticoids promote the development of many organs including intestine. At the cellular level, the activity of glucocorticoids is regulated by 11 beta-hydroxysteroid dehydrogenase (11 beta HSD) which converts active glucocorticoids to inactive metabolites. As 11 beta HSD is also expressed in the intestine, this enzyme may be an important regulator of intestinal maturation. To investigate this, we have performed the systematic study of the development of intestinal 11 beta HSD activity and its cofactor preference as well as of the effect of 11 beta HSD inhibition by carbenoxolone on postnatal development of sucrase, alkaline phosphatase and Na,K-ATPase in the intestine. The activity of 11 beta HSD was low in ileum of suckling rats and significantly increased during the weaning period. In colon, the activity was already high in suckling rats and gradually rose during the postnatal development. 11 beta HSD activity was undetectable in jejunum both in young and adult rats. At 14.5 nM corticosterone, colonic 11 beta HSD utilized predominantly NAD as a cofactor, but displayed significant sensitivity also to NADP. Ileal 11 beta HSD had similar sensitivity to both cofactors. With NAD as a cofactor, ileal 11 beta HSD had a Km (59 +/- 10 nM) compatible with the colonic enzyme (81 +/- 14 nM). Carbenoxolone administration to suckling and weanling rats in vivo did not result in any changes of sucrase activity in jejunum and ileum, alkaline phosphatase activity in ileum and distal colon or Na,K-ATPase activity in ileum. However, carbenoxolone significantly increased Na,K-ATPase activity in distal colon. Our results indicate that the high-affinity type of 11 beta HSD is expressed not only in colon but also in ileum and that 11 beta HSD is an important factor in the regulation of tissue levels of active glucocorticoids in developing colon but not in the small intestine.

• MeSH
• 11-beta-hydroxysteroiddehydrogenasy MeSH
• alkalická fosfatasa metabolismus   MeSH
• hydroxysteroiddehydrogenasy antagonisté a inhibitory   fyziologie   MeSH
• inhibitory enzymů farmakologie   MeSH
• karbenoxolon farmakologie   MeSH
• krysa rodu Rattus MeSH
• odstavení MeSH
• potkani Wistar MeSH
• sacharasa metabolismus   MeSH
• sodíko-draslíková ATPasa metabolismus   MeSH
• střeva enzymologie   růst a vývoj   MeSH
• střevní sliznice enzymologie   růst a vývoj   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 11-beta-hydroxysteroiddehydrogenasy MeSH
• alkalická fosfatasa MeSH
• hydroxysteroiddehydrogenasy MeSH
• inhibitory enzymů MeSH
• karbenoxolon MeSH
• sacharasa MeSH
• sodíko-draslíková ATPasa MeSH

The Nuclear Exosome Targeting (NEXT) complex is a key cofactor of the mammalian nuclear exosome in the removal of Promoter Upstream Transcripts (PROMPTs) and potentially aberrant forms of other noncoding RNAs, such as snRNAs. NEXT is composed of three subunits SKIV2L2, ZCCHC8 and RBM7. We have recently identified the NEXT complex in our screen for oligo(U) RNA-binding factors. Here, we demonstrate that NEXT displays preference for U-rich pyrimidine sequences and this RNA binding is mediated by the RNA recognition motif (RRM) of the RBM7 subunit. We solved the structure of RBM7 RRM and identified two phenylalanine residues that are critical for interaction with RNA. Furthermore, we showed that these residues are required for the NEXT interaction with snRNAs in vivo. Finally, we show that depletion of components of the NEXT complex alone or together with exosome nucleases resulted in the accumulation of mature as well as extended forms of snRNAs. Thus, our data suggest a new scenario in which the NEXT complex is involved in the surveillance of snRNAs and/or biogenesis of snRNPs.

• MeSH
• aminokyselinové motivy MeSH
• HEK293 buňky MeSH
• HeLa buňky MeSH
• lidé MeSH
• oligoribonukleotidy metabolismus   MeSH
• podjednotky proteinů chemie   metabolismus   MeSH
• proteiny vázající RNA analýza   chemie   metabolismus   MeSH
• RNA malá jaderná chemie   metabolismus   MeSH
• sekvence nukleotidů MeSH
• uracilnukleotidy metabolismus   MeSH
• vazba proteinů MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• oligo(U) MeSH Prohlížeč
• oligoribonukleotidy MeSH
• podjednotky proteinů MeSH
• proteiny vázající RNA MeSH
• RBM7 protein, human MeSH Prohlížeč
• RNA malá jaderná MeSH
• uracilnukleotidy MeSH

Using the 32P-postlabeling assay, we investigated the ability of quaternary benzo[c]phenanthridine alkaloids, sanguinarine, chelerythrine and fagaronine, to form DNA adducts in vitro. Two enhanced versions of the assay (enrichment by nuclease P1 and 1-butanol extraction) were utilized in the study. Hepatic microsomes of rats pre-treated with beta-naphthoflavone or those of uninduced rats, used as metabolic activators, were incubated in the presence of calf thymus DNA and the alkaloids, with NADPH used as a cofactor. Under these conditions sanguinarine and chelerythrine, but not fagaronine, formed DNA adducts detectable by 32P-postlabeling. DNA adduct formation by both alkaloids was found to be concentration dependent. When analyzing different atomic and bond indices of the C11-C12 bond (ring B) in alkaloid molecules we found that fagaronine behaved differently from sanguinarine and chelerythrine. While sanguinarine and chelerythrine showed a preference for electrophilic attack indicating higher potential to be activated by cytochrome P450, fagaronine exhibited a tendency for nucleophilic attack. Our results demonstrate that sanguinarine and chelerythrine are metabolized by hepatic microsomes to species, which generate DNA adducts.

• MeSH
• adukty DNA biosyntéza   MeSH
• alkaloidy metabolismus   farmakologie   MeSH
• benzofenantridiny MeSH
• fenantridiny metabolismus   farmakologie   MeSH
• fytogenní protinádorové látky metabolismus   farmakologie   MeSH
• interkalátory metabolismus   farmakologie   MeSH
• isochinoliny MeSH
• jaterní mikrozomy účinky léků   metabolismus   MeSH
• krysa rodu Rattus MeSH
• kvantová teorie MeSH
• potkani Sprague-Dawley MeSH
• radioizotopy fosforu analýza   MeSH
• systém (enzymů) cytochromů P-450 metabolismus   MeSH
• zvířata MeSH
• Check Tag
• krysa rodu Rattus MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• adukty DNA MeSH
• alkaloidy MeSH
• benzofenantridiny MeSH
• chelerythrine MeSH Prohlížeč
• fagaronine MeSH Prohlížeč
• fenantridiny MeSH
• fytogenní protinádorové látky MeSH
• interkalátory MeSH
• isochinoliny MeSH
• radioizotopy fosforu MeSH
• sanguinarine MeSH Prohlížeč
• systém (enzymů) cytochromů P-450 MeSH

Dehydrogenase/reductase (SDR family) member 7 (DHRS7, retSDR4, SDR34C1) is a previously uncharacterized member of the short-chain dehydrogenase/reductase (SDR) superfamily. While human SDR members are known to play an important role in various (patho)biochemical pathways including intermediary metabolism and biotransformation of xenobiotics, only 20% of them are considered to be well characterized. Based on phylogenetic tree and SDR sequence clusters analysis DHRS7 is a close relative to well-known SDR member 11β-hydroxysteroid dehydrogenase 1 (11β-HSD1) that participates in metabolism of endogenous and xenobiotic substances with carbonyl group. The aim of present study is to determine the basic biochemical properties of DHRS7 and its possible involvement in metabolism of substrates with carbonyl group. For the first time the computational predictions of this membrane protein and membrane topology were experimentally confirmed. DHRS7 has been demonstrated to be an integral protein facing the lumen of the endoplasmic reticulum with lack of posttranscriptional glycosylation modification. Subsequently, NADP(H) cofactor preference and enzymatic reducing activity of DHRS7 was determined towards endogenous substrates with a steroid structure (cortisone, 4-androstene-3,17-dion) and also toward relevant exogenous substances bearing a carbonyl group harmful to human health (1,2-naphtoquinone, 9,10-phenantrenequinone). In addition to 11β-HSD1, DHRS7 is another enzyme from SDR superfamily that have been proved, at least in vitro, to contribute to the metabolism of xenobiotics with carbonyl group.

• Klíčová slova
• 11β-HSD1, 11β-hydroxysteroid dehydrogenase, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanol, 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone, DHRS7, ER, Membrane topology, NNAL, NNK, Oxidoreductase activity, SDR, SDR34C1, Short-chain dehydrogenases/reductases, dehydrogenase/reductase (SDR family) member 7, endoplasmic reticulum, short-chain dehydrogenases/reductases,
• MeSH
• 11-beta-hydroxysteroiddehydrogenasa typ 1 chemie   metabolismus   MeSH
• benzaldehydy metabolismus   MeSH
• fluorescenční protilátková technika MeSH
• intracelulární membrány metabolismus   MeSH
• izoenzymy chemie   metabolismus   MeSH
• jaterní mikrozomy enzymologie   MeSH
• kinetika MeSH
• lidé MeSH
• molekulární sekvence - údaje MeSH
• NAD metabolismus   MeSH
• NADP metabolismus   MeSH
• nitrosaminy chemie   metabolismus   MeSH
• oxidoreduktasy chemie   metabolismus   MeSH
• sekvence aminokyselin MeSH
• Sf9 buňky MeSH
• spektrofotometrie MeSH
• substrátová specifita MeSH
• ultracentrifugace MeSH
• western blotting MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• 11-beta-hydroxysteroiddehydrogenasa typ 1 MeSH
• 4-(N-methyl-N-nitrosamino)-1-(3-pyridyl)-1-butanone MeSH Prohlížeč
• 4-nitrobenzaldehyde MeSH Prohlížeč
• benzaldehydy MeSH
• DHRS7 protein, human MeSH Prohlížeč
• DHRS7c protein, human MeSH Prohlížeč
• izoenzymy MeSH
• NAD MeSH
• NADP MeSH
• nitrosaminy MeSH
• oxidoreduktasy MeSH

Dehydrogenase/reductase (SDR family) member 3 (DHRS3), also known as retinal short-chain dehydrogenase/reductase (retSDR1) is a member of SDR16C family. This family is thought to be NADP(H) dependent and to have multiple substrates; however, to date, only all-trans-retinal has been identified as a DHRS3 substrate. The reductive reaction catalysed by DHRS3 seems to be physiological, and recent studies proved the importance of DHRS3 for maintaining suitable retinoic acid levels during embryonic development in vivo. Although it seems that DHRS3 is an important protein, knowledge of the protein and its properties is quite limited, with the majority of information being more than 15 years old. This study aimed to generate a more comprehensive characterisation of the DHRS3 protein. Recombinant enzyme was prepared and demonstrated to be a microsomal, integral-membrane protein with the C-terminus oriented towards the cytosol, consistent with its preference of NADPH as a cofactor. It was determined that DHRS3 also participates in the metabolism of other endogenous compounds, such as androstenedione, estrone, and DL-glyceraldehyde, and in the biotransformation of xenobiotics (e.g., NNK and acetohexamide) in addition to all-trans-retinal. Purified and reconstituted enzyme was prepared for the first time and will be used for further studies. Expression of DHRS3 was shown at the level of both mRNA and protein in the human liver, testis and small intestine. This new information could open other areas of DHRS3 protein research.

• Klíčová slova
• DHRS3, Expression, Membrane topology, Reductase activity, retSDR1,
• MeSH
• alkoholoxidoreduktasy metabolismus   MeSH
• cytosol metabolismus   MeSH
• jaterní mikrozomy enzymologie   metabolismus   MeSH
• játra enzymologie   metabolismus   MeSH
• lidé MeSH
• membránové proteiny metabolismus   MeSH
• NADH, NADPH oxidoreduktasy metabolismus   MeSH
• NADP metabolismus   MeSH
• Sf9 buňky MeSH
• Spodoptera metabolismus   MeSH
• syntázy mastných kyselin metabolismus   MeSH
• tenké střevo enzymologie   metabolismus   MeSH
• testis enzymologie   metabolismus   MeSH
• tretinoin metabolismus   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• mužské pohlaví MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• alkoholoxidoreduktasy MeSH
• membránové proteiny MeSH
• NADH, NADPH oxidoreduktasy MeSH
• NADP MeSH
• short chain trans-2-enoyl-CoA reductase MeSH Prohlížeč
• syntázy mastných kyselin MeSH
• tretinoin MeSH

Sufficient structural information on mammalian cytochromes P450 has now been published (including seventeen X-ray structures of these enzymes by June 2006) to allow characteristic features of these enzymes to be identified, including: (i) the presence of a common fold, typical of all P450s, (ii) similarities in the positioning of the heme cofactor, (iii) the spatial arrangement of certain structural elements, and (iv) the access/egress paths for substrates and products, (v) probably common orientation in the membrane, (vi) characteristic properties of the active sites with networks of water molecules, (vii) mode of interaction with redox partners and (viii) a certain degree of flexibility of the structure and active site determining the ease with which the enzyme may bind the substrates. As well as facilitating the identification of common features, comparison of the available structures allows differences among the structures to be identified, including variations in: (i) preferred access/egress paths to/from the active site, (ii) the active site volume and (iii) flexible regions. The availability of crystal structures provides opportunities for molecular dynamic simulations, providing data that are apparently complementary to experimental findings but also allow the dynamic behavior of access/egress paths and other dynamic features of the enzymes to be explored.

• MeSH
• konformace proteinů MeSH
• lidé MeSH
• membránové proteiny chemie   MeSH
• molekulární modely * MeSH
• molekulární sekvence - údaje MeSH
• savci MeSH
• sbalování proteinů * MeSH
• sekvence aminokyselin MeSH
• sekvenční homologie aminokyselin MeSH
• systém (enzymů) cytochromů P-450 chemie   MeSH
• terciární struktura proteinů MeSH
• vazebná místa MeSH
• voda chemie   MeSH
• zvířata MeSH
• Check Tag
• lidé MeSH
• zvířata MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• přehledy MeSH
• Názvy látek
• membránové proteiny MeSH
• systém (enzymů) cytochromů P-450 MeSH
• voda MeSH

The World Health Organization (WHO) identifies several bunyaviruses as significant threats to global public health security. Developing effective therapies against these viruses is crucial to combat future outbreaks and mitigate their impact on patient outcomes. Here, we report the synthesis of some isoindol-1-one derivatives and explore their inhibitory properties over an indispensable metal-dependent cap-snatching endonuclease (Cap-ENDO) shared among evolutionary divergent bunyaviruses. The compounds suppressed RNA hydrolysis by Cap-ENDOs, with IC50 values predominantly in the lower μM range. Molecular docking studies revealed the interactions with metal ions to be essential for the 2,3-dihydro-6,7-dihydroxy-1H-isoindol-1-one scaffold activity. Calorimetric analysis uncovered Mn2+ ions to have the highest affinity for sites within the targets, irrespective of aminoacidic variations influencing metal cofactor preferences. Interestingly, spectrophotometric findings unveiled sole dinuclear species formation between the scaffold and Mn2+. Moreover, the complexation of two Mn2+ ions within the viral enzymes appears to be favourable, as indicated by the binding of compound 11 to TOSV Cap-ENDO (Kd = 28 ± 3 μM). Additionally, the tendency of compound 11 to stabilize His+ more than His- Cap-ENDOs suggests exploitable differences in their catalytic pockets relevant to improving specificity. Collectively, our results underscore the isoindolinone scaffold's potential as a strategic starting point for the design of pan-antibunyavirus drugs.

• Klíčová slova
• Bunyavirales, Cap-snatching endonuclease inhibitor, Isoindolinone scaffold, Metal chelation, Pan-antivirals,
• MeSH
• antivirové látky farmakologie   chemie   chemická syntéza   MeSH
• Bunyaviridae účinky léků   metabolismus   MeSH
• endonukleasy * metabolismus   antagonisté a inhibitory   MeSH
• inhibitory enzymů farmakologie   chemie   chemická syntéza   MeSH
• isoindoly chemická syntéza   farmakologie   chemie   MeSH
• lidé MeSH
• molekulární struktura MeSH
• racionální návrh léčiv * MeSH
• simulace molekulového dockingu * MeSH
• vztah mezi dávkou a účinkem léčiva MeSH
• vztahy mezi strukturou a aktivitou MeSH
• Check Tag
• lidé MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• antivirové látky MeSH
• endonukleasy * MeSH
• inhibitory enzymů MeSH
• isoindoly MeSH