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1.
Arch Microbiol ; 202(1): 197-203, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31372664

RESUMO

Galactofuranose (Galf)-containing glycostructures are important to secure the integrity of the fungal cell wall. Golgi-localized Galf-transferases (Gfs) have been identified in Aspergillus nidulans and Aspergillus fumigatus. BLASTp searches identified three putative Galf-transferases in Aspergillus niger. Phylogenetic analysis showed that they group in three distinct groups. Characterization of the three Galf-transferases in A. niger by constructing single, double, and triple mutants revealed that gfsA is most important for Galf biosynthesis. The growth phenotypes of the ΔgfsA mutant are less severe than that of the ΔgfsAC mutant, indicating that GfsA and GfsC have redundant functions. Deletion of gfsB did not result in any growth defect and combining ΔgfsB with other deletion mutants did not exacerbate the growth phenotype. RT-qPCR experiments showed that induction of the agsA gene was higher in the ΔgfsAC and ΔgfsABC compared to the single mutants, indicating a severe cell wall stress response after multiple gfs gene deletions.


Assuntos
Aspergillus niger/enzimologia , Aspergillus niger/genética , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Transferases/genética , Transferases/metabolismo , Aspergillus fumigatus/classificação , Aspergillus fumigatus/enzimologia , Aspergillus fumigatus/genética , Aspergillus nidulans/classificação , Aspergillus nidulans/enzimologia , Aspergillus nidulans/genética , Aspergillus niger/classificação , Parede Celular , Deleção de Genes , Mutação , Filogenia
2.
Nat Commun ; 10(1): 2917, 2019 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-31266949

RESUMO

Novel antibacterial agents are needed to address the emergence of global antibiotic resistance. MraY is a promising candidate for antibiotic development because it is the target of five classes of naturally occurring nucleoside inhibitors with potent antibacterial activity. Although these natural products share a common uridine moiety, their core structures vary substantially and they exhibit different activity profiles. An incomplete understanding of the structural and mechanistic basis of MraY inhibition has hindered the translation of these compounds to the clinic. Here we present crystal structures of MraY in complex with representative members of the liposidomycin/caprazamycin, capuramycin, and mureidomycin classes of nucleoside inhibitors. Our structures reveal cryptic druggable hot spots in the shallow inhibitor binding site of MraY that were not previously appreciated. Structural analyses of nucleoside inhibitor binding provide insights into the chemical logic of MraY inhibition, which can guide novel approaches to MraY-targeted antibiotic design.


Assuntos
Antibacterianos/química , Bactérias/enzimologia , Proteínas de Bactérias/química , Produtos Biológicos/química , Inibidores Enzimáticos/química , Nucleosídeos/antagonistas & inibidores , Transferases/química , Aminoglicosídeos/química , Arginina/análogos & derivados , Arginina/química , Bactérias/química , Bactérias/genética , Proteínas de Bactérias/antagonistas & inibidores , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Sítios de Ligação , Cristalografia por Raios X , Transferases/antagonistas & inibidores , Transferases/genética , Transferases/metabolismo
3.
Chemistry ; 25(51): 11945-11954, 2019 Sep 12.
Artigo em Inglês | MEDLINE | ID: mdl-31294500

RESUMO

Acyl transferase from Mycobacterium smegmatis (MsAcT) is a promising biocatalyst because it catalyzes an acyl transfer reaction in aqueous solution, thereby accepting many primary and secondary alcohols as substrates. MsAcT also exhibits high enantioselectivity for a selected number of secondary alcohols. To increase the applicability of this enzyme for the production of optically active compounds, a detailed understanding of the reaction mechanism and the factors that affect enantioselectivity is essential. Herein, quantum chemical calculations are employed to study the reactions of two secondary alcohols, 1-isopropyl propargyl alcohol and 2-hydroxy propanenitrile, for which the enzyme displays opposite enantiopreference, favoring the S enantiomer in the former case and R enantiomer in the latter. A model of the active site has been designed and for both substrates various binding modes are evaluated and the intermediates and transition states along the reaction path are then located. The calculated energy profiles agree with the experimental observations, and reproduce the selectivity outcome. Through a detailed analysis of the geometries of key transition states, insights into the origins of the enantiopreference are obtained.


Assuntos
Álcoois/química , Mycobacterium smegmatis/química , Transferases/metabolismo , Catálise , Domínio Catalítico , Estereoisomerismo
4.
Plant Physiol Biochem ; 142: 94-105, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31279136

RESUMO

1-Deoxy-D-xylulose-5-phosphate synthasse (DXS) and 1-deoxy-D-xylulose-5-phosphate reductoisomerase (DXR) are key enzymes in terpenoid biosynthesis. DXS catalyzes the formation of 1-deoxy-D-xylulose 5-phosphate (DXP) from pyruvate and D-glyceraldehyde-3-phosphate. DXR catalyzes the formation of 2-C-methyl-D-erythritol 4-phosphate (MEP) from DXP. Previous studies of the DXS and DXR genes have focused on herbs, such as Arabidopsis thaliana, Salvia miltiorrhiza, and Amomum villosum, but few studies have been conducted on woody plants. For that reason, we chose Populus trichocarpa as a model woody plant for investigating the DXS and DXR genes. PtDXS exhibited the highest expression level in leaves and the lowest expression in roots. PtDXR showed maximum expression in young leaves, and the lowest expression in mature leaves. The expression profiles revealed by RT-PCR following different elicitor treatments such as abscisic acid, NaCl, PEG6000, H2O2, and cold stress showed that PtDXS and PtDXR were elicitor-responsive genes. Our results showed that the PtDXS gene exhibited diurnal changes, but PtDXR did not. Moreover, overexpression of PtDXR in transgenic poplars improved tolerance to abiotic and biotic stresses. Those results showed that the PtDXR encoded a functional protein, and widely participates in plant growth and development, stress physiological process.


Assuntos
Aldose-Cetose Isomerases/genética , Proteínas de Plantas/genética , Populus/genética , Transferases/genética , Ácido Abscísico/farmacologia , Aldose-Cetose Isomerases/metabolismo , Ritmo Circadiano , Secas , Perfilação da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Peróxido de Hidrogênio/farmacologia , Doenças das Plantas/genética , Doenças das Plantas/microbiologia , Proteínas de Plantas/metabolismo , Plantas Geneticamente Modificadas , Populus/efeitos dos fármacos , Populus/fisiologia , Saccharomycetales/patogenicidade , Tolerância ao Sal/genética , Cloreto de Sódio/farmacologia , Estresse Fisiológico , Transferases/metabolismo
5.
Molecules ; 24(15)2019 Jul 26.
Artigo em Inglês | MEDLINE | ID: mdl-31357567

RESUMO

A group of prenyltransferases catalyze chain elongation of farnesyl diphosphate (FPP) to designated lengths via consecutive condensation reactions with specific numbers of isopentenyl diphosphate (IPP). cis-Prenyltransferases, which catalyze cis-double bond formation during IPP condensation, usually synthesize long-chain products as lipid carriers to mediate peptidoglycan biosynthesis in prokaryotes and protein glycosylation in eukaryotes. Unlike only one or two cis-prenyltransferases in bacteria, yeast, and animals, plants have several cis-prenyltransferases and their functions are less understood. As reported here, a cis-prenyltransferase from Lilium longiflorum anther, named LLA66, was expressed in Saccharomyces cerevisiae and characterized to produce C40/C45 products without the capability to restore the growth defect from Rer2-deletion, although it was phylogenetically categorized as a long-chain enzyme. Our studies suggest that evolutional mutations may occur in the plant cis-prenyltransferase to convert it into a shorter-chain enzyme.


Assuntos
Lilium/química , Lilium/enzimologia , Transferases/química , Transferases/metabolismo , Lilium/classificação , Lilium/genética , Modelos Moleculares , Filogenia , Conformação Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Relação Estrutura-Atividade , Transferases/genética
6.
Mol Biol Rep ; 46(5): 5175-5184, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-31313133

RESUMO

Isoprenoids, the largest and most diverse class of secondary metabolites in plants, play an important role in plant growth and development. Isoprenoids can be synthesized by two distinct pathways: the methylerythritol-4-phosphate (MEP) pathway and the mevalonate (MVA) pathway. 1-Deoxy-D-xylulose-5-phosphate synthase (DXS) is the first step and a key regulatory enzyme of the MEP pathway in plants. The DXS gene has been reported to play a key role in seedling development, flowering, and fruit quality in plants of the Solanaceae, such as tomato, potato and tobacco. However, to improve our understanding and utilization of DXS genes, a thorough bioinformatics study is needed. In this study, 48 DXS genes were aligned and analyzed by computational tools to predict their protein properties, including molecular mass, theoretical isoelectric point (pI), signal peptides, transmembrane and conserved domains, and expression patterns. Sequence comparison analysis revealed strong conservation among the 48 DXS genes. Phylogenetic analysis indicated that all DXS genes were derived from one ancestor and could be classified into three groups with different expression patterns. Moreover, the functional divergence of DXS was restricted after gene duplication. The results suggested that the function and evolution of the DXS gene family were highly conserved and that the DXS genes of Group I may play a more important role than those of other groups.


Assuntos
Biologia Computacional/métodos , Solanaceae/enzimologia , Transferases/genética , Transferases/metabolismo , Evolução Molecular , Redes e Vias Metabólicas , Família Multigênica , Filogenia , Proteínas de Plantas/química , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Metabolismo Secundário , Solanaceae/genética , Solanaceae/crescimento & desenvolvimento , Terpenos/metabolismo , Transferases/química
7.
Sheng Wu Gong Cheng Xue Bao ; 35(5): 857-870, 2019 May 25.
Artigo em Chinês | MEDLINE | ID: mdl-31223004

RESUMO

To investigate the effects of genistein (Gen) on the biosynthesis of N-glycolylneuraminic acid (Neu5Gc) in rats, 80 4-week-old male SD rats were randomly equally into the control and genistein groups. The rats of control and genistein groups were fed 5% ethanol and 300 mg/(kg·d) genistein respectively by gavage. The contents of Neu5Gc in hind leg muscle, kidney and liver tissues of rats were measured by using high performance liquid chromatography coupled with fluorescence detector (HPLC/FLD), and the mechanism of inhibition of Neu5Gc synthesis was investigated by using the molecular docking of Gen and sialyltransferase. On the 15th day, the content of Neu5Gc in hind leg muscle and liver tissues decreased 13.77% and 15.45%, respectively, and there was no significant change in the content of Neu5Gc in kidney tissues. On the 30th day, the content of Neu5Gc in liver tissues decreased 13.35%, however, there was no significant change in the content of Neu5Gc in kidney tissues and Neu5Gc was not detected in hind leg muscle. The content of Neu5Gc in hind leg muscle, kidney and liver tissues decreased respectively 32.65%, 32.78%, 16.80% and 12.72%, 11.42%, 12.30% while rats fed on the 45th and the 60th days. Genistein has formed the hydrogen bond with sialyltransferase activity site residues His319, Ser151, Gly293, Thr328 and formed a hydrophobic interactions with the residues His302, His301, Trp300, Ser271, Phe292, Thr328, Ser325 and Ile274. The results of molecular docking indicated that the weak intermolecular interaction was the main cause of genistein inhibiting sialyltransferase activity. The research results provided an experimental basis for the subsequent reduction of Neu5Gc in red meat before slaughter.


Assuntos
Regulação Enzimológica da Expressão Gênica , Genisteína , Ácidos Neuramínicos , Transferases , Animais , Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Genisteína/farmacologia , Masculino , Simulação de Acoplamento Molecular , Ácidos Neuramínicos/metabolismo , Distribuição Aleatória , Ratos , Ratos Sprague-Dawley , Transferases/metabolismo
8.
JAMA Netw Open ; 2(6): e195844, 2019 06 05.
Artigo em Inglês | MEDLINE | ID: mdl-31199452

RESUMO

Importance: The prevalence of high-intensity binge drinking (HIBD), defined as consuming 2 or more times the binge threshold defined by the National Institute on Alcohol Abuse and Alcoholism (NIAAA), is rapidly increasing in the United States. While the relationship between alcohol consumption and lipid and liver function enzyme (LFT) biomarkers has been previously examined, the associations of HIBD with those biomarkers remain unknown. Objective: To examine associations of HIBD with lipid and LFT levels in a cross-sectional sample enriched with participants who engage in HIBD. Design, Setting, and Participants: Cross-sectional study using data from the NIAAA clinical sample collected from March 3, 2005, to August 21, 2017, with participants recruited for either the NIAAA screening protocols or inpatient alcohol treatment program. For this study, participants were stratified by self-reported alcohol consumption into 4 sex-specific binge levels: nonbinge and 1, 2, and 3 or more times the binge threshold (levels I, II, and III). Multivariable analyses examined the odds of clinically high levels of lipids and LFTs across binge levels. Analyses were performed from December 3, 2018, to January 30, 2019. Main Outcomes and Measures: Serum levels of high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol, total cholesterol, triglycerides, alanine aminotransferase, aspartate aminotransferase, and γ-glutamyltransferase. Results: A total of 2065 participants underwent protocol screening; 1519 with data available on alcohol consumption, body mass index, lipid levels, and LFT levels were included in the final analyses. Mean (SD) age was 39.7 (12.1) years; mean (SD) body mass index was 26.6 (5.1); 978 (64.4%) were male; 718 (47.3%) were white; and 578 (31.1%) consumed alcohol at the nonbinge level, 321 (21.2%) at level I, 239 (15.7%) at level II, and 318 (25.1%) at level III. High-intensity binge drinking was associated with 2- to 8-fold increased odds for clinically high levels of HDL-C, total cholesterol, triglycerides, and all LFTs (eg, for HDL-C: level III odds ratio [OR], 8.65; 95% CI, 4.75-15.77 and for γ-glutamyltransferase: level III OR, 8.21; 95% CI, 5.90-11.43). Increased HIBD frequency (days consuming at levels II and III) was associated with increased odds for clinically high levels of HDL-C, total cholesterol, and all LFTs (per unit increase in days consuming at the respective binge level) (eg, for HDL-C: level II OR, 1.025; 95% CI, 1.014-1.036 and level III OR, 1.033; 95% CI, 1.019-1.047 and for γ-glutamyltransferase: level II OR, 1.028; 95% CI, 1.019-1.037 and level III OR, 1.033; 95% CI, 1.019-1.047). Conclusions and Relevance: High-impact binge drinking was significantly associated with increased odds for clinically high levels of lipids and LFTs. Given that HIBD is increasingly common among US adults, targeted interventions aimed at reducing HIBD may have important health benefits.


Assuntos
Bebedeira/fisiopatologia , Metabolismo dos Lipídeos/fisiologia , Transferases/metabolismo , Adulto , Alanina Transaminase/metabolismo , Aspartato Aminotransferases/metabolismo , Bebedeira/sangue , Bebedeira/enzimologia , Biomarcadores/metabolismo , HDL-Colesterol/metabolismo , LDL-Colesterol/metabolismo , Estudos Transversais , Feminino , Humanos , Fígado/enzimologia , Testes de Função Hepática , Masculino , Triglicerídeos/metabolismo , gama-Glutamiltransferase/metabolismo
9.
Reprod Biol ; 19(2): 173-178, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31151753

RESUMO

The tumor suppressor gene KCTD11 plays a critical role in cell proliferation, differentiation and invasion. The current study investigated the regulation and the spatiotemporal expression pattern of Kctd11 in the rat ovary during the periovulatory period. Ovaries, granulosa cells, or theca-interstitial cells were collected at various times after hCG administration using an established gonadotropin-primed immature rat model that induces follicular development and ovulation. Real-time quantitative PCR analysis revealed that mRNA for Kctd11 was significantly induced both in theca-intersititial and granulosa cells after hCG treatment although their temporal expression patterns differed. In situ hybridization analysis demonstrated that Kctd11 mRNA expression was induced in theca-intersititial cells at 6 h after hCG, and the expression remained elevated until 12 h after hCG. Kctd11 mRNA was stimulated in granulosa cells at 6 h and reached the highest expression at 12 h. There was negligible Kctd11 mRNA signal observed in newly forming corpora lutea. In addition, the data indicate that both the protein kinase A and the protein kinase C pathway regulate the expression of Kctd11 mRNA in granulosa cells. Either forskolin or phorbol 12 myristate 13-acetate can mimic hCG induction of Kctd11 expression. Furthermore, the stimulation of Kctd11 by hCG requires new protein synthesis. Inhibition of progesterone action and the EGF pathway blocked Kctd11 mRNA expression, whereas inhibition of prostaglandin synthesis had no effect. Our finding suggest that the induction of the Kctd11 may be important for theca and granulosa cell differentiation into luteal cells.


Assuntos
Proteínas de Ciclo Celular/metabolismo , Ovário/metabolismo , Ovulação/fisiologia , Transferases/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Proteínas de Ciclo Celular/genética , Gonadotropina Coriônica/farmacologia , Feminino , Regulação da Expressão Gênica/efeitos dos fármacos , Células da Granulosa/fisiologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Células Tecais/fisiologia , Transferases/genética , Proteínas Supressoras de Tumor/genética
10.
Nat Chem Biol ; 15(6): 632-640, 2019 06.
Artigo em Inglês | MEDLINE | ID: mdl-31036922

RESUMO

Several important Gram-negative bacterial pathogens possess surface capsular layers composed of hypervariable long-chain polysaccharides linked via a conserved 3-deoxy-ß-D-manno-oct-2-ulosonic acid (ß-Kdo) oligosaccharide to a phosphatidylglycerol residue. The pathway for synthesis of the terminal glycolipid was elucidated by determining the structures of reaction intermediates. In Escherichia coli, KpsS transfers a single Kdo residue to phosphatidylglycerol; this primer is extended using a single enzyme (KpsC), possessing two cytidine 5'-monophosphate (CMP)-Kdo-dependent glycosyltransferase catalytic centers with different linkage specificities. The structure of the N-terminal ß-(2→4) Kdo transferase from KpsC reveals two α/ß domains, supplemented by several helices. The N-terminal Rossmann-like domain, typically responsible for acceptor binding, is severely reduced in size compared with canonical GT-B folds in glycosyltransferases. The similar structure of the C-terminal ß-(2→7) Kdo transferase indicates a past gene duplication event. Both Kdo transferases have a narrow active site tunnel, lined with key residues shared with GT99 ß-Kdo transferases. This enzyme provides the prototype for the GT107 family.


Assuntos
Cápsulas Bacterianas/metabolismo , Glicolipídeos/biossíntese , Bactérias Gram-Negativas/metabolismo , Transferases/metabolismo , Modelos Moleculares , Estrutura Molecular , Transferases/química
12.
Molecules ; 24(9)2019 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-31064128

RESUMO

A novel recombinant strain has been constructed for converting glycerol into a specific conjugated linoleic acid isomer (trans-10, cis-12 CLA) using Yarrowia lipolytica as host. The lipid accumulation pathway was modified for increasing lipid content. Overexpression of the diacylglycerol transferase (DGA1) gene improved the intracellular lipid yield by approximately 45% as compared to the original strain. The corresponding intracellular lipid yield of recombinant strain WXYL037 reached 52.2% of the cell dry weight. In combination with integration of Δ12 desaturase from Mortierella alpina (MA12D) and DGA1, the linoleic acid (LA) production content reached 0.88 g/L, which was 2-fold that of the original strain. Furthermore, with overexpressed DGA1, MA12D and Propionibacterium acnes isomerase (PAI), the titer of trans-10, cis-12 CLA in WXYL037 reached 110.6 mg/L after 72 h of shake flask culture, representing a 201.8% improvement when compared with that attained in the WXYL030 strain, which manifested overexpressed PAI. With optimal medium, the maximum CLA content and lipid yield of Y. lipolytica Po1g were 132.6 mg/L and 2.58 g/L, respectively. This is the first report of the production of trans-10, cis-12 CLA by the oleaginous yeast Y. lipolytica using glycerol as the sole carbon source through expression of DGA1 combined with MA12D and PAI.


Assuntos
Ácidos Linoleicos Conjugados/biossíntese , Transferases/genética , Transferases/metabolismo , Yarrowia/metabolismo , Extratos Celulares/química , Escherichia coli/metabolismo , Fermentação , Glicerol/química , Isomerismo , Lipídeos/química , Propionibacterium acnes/genética , Saccharomyces cerevisiae/metabolismo , Transdução de Sinais
13.
J Biosci Bioeng ; 128(5): 585-592, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31122725

RESUMO

The common proteins rubber elongation factor (REF) and small rubber particle protein (SRPP) are associated with Hevea brasiliensis (Hb) rubber particles. They are involved in the stability of rubber particles and natural rubber biosynthesis. Recently, we cloned cDNAs encoding REF and SRPP from laticifers of Ficus carica (Fc). In the present study, we overexpressed REF/SRPPs (HbREF, FcREF, and FcSRPP) in Saccharomyces cerevisiae in anticipation of future rubber biosynthesis in recombinant yeast. The proteins were localized in the endoplasmic reticulum and lipid droplets (LDs), and affected LD morphology. Furthermore, their overexpression resulted in an accumulation of neutral lipids and a decrease in yeast cell size. This suggests that REF/SRPPs affect lipid metabolism and lead to a decline in the phospholipid content of yeast. We also found that expression of these proteins induced accumulation of steryl esters and triacylglycerols in yeast. This suggests that the coexpression of REF/SRPPs with key enzymes for the biosynthesis of target lipids in yeast is a promising way of increasing production of important lipids like triacylglycerols and terpenes, and that a protein complex consisting of cis-prenyltransferase (CPT), CPT-like protein, and REF/SRPPs for rubber biosynthesis could be reconstituted on yeast lipid droplets.


Assuntos
Ficus/metabolismo , Hevea/metabolismo , Metabolismo dos Lipídeos , Fatores de Alongamento de Peptídeos/metabolismo , Saccharomyces cerevisiae/metabolismo , DNA Complementar/metabolismo , Ficus/genética , Hevea/genética , Fatores de Alongamento de Peptídeos/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Saccharomyces cerevisiae/genética , Transferases/genética , Transferases/metabolismo
14.
Eur J Med Chem ; 171: 462-474, 2019 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-30933853

RESUMO

The present status of antibiotic resistant requires an urgent invention of novel agents that act on clinically unexplored antibacterial targets. The enzyme MraY (phospho-MurNAc-pentapeptide translocase), essential for bacterial cell wall synthesis, fulfils this criterion as it has not been explored as a target in a clinical context. Specifically, the enzyme is involved in the lipid-linked cycle of peptidoglycan biosynthesis and is reportedly targeted by naturally-occurring nucleoside antibiotics. The antimicrobial 'caprazamycin' class of nucleoside antibiotics targets Mycobacterium tuberculosis and clinically relevant Gram-negative bacteria such as Pseudomonas aeruginosa besides various drug resistant strains and is therefore an eligible starting point for the development of novel agents. In this review, we aim to summarise the structure-activity relationships of the natural, semi-synthetic as well as synthetic analogues of nucleoside antibiotic caprazamycins. This review highlights caprazamycins as promising lead structures for development of potent and selective antimicrobial agents that target MraY, the bacterial enzyme involved in the first membrane-dependent step in bacterial peptidoglycan assembly.


Assuntos
Antibacterianos/farmacologia , Azepinas/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Produtos Biológicos/farmacologia , Mycobacterium tuberculosis/efeitos dos fármacos , Transferases/antagonistas & inibidores , Uridina/análogos & derivados , Antibacterianos/química , Azepinas/química , Proteínas de Bactérias/metabolismo , Produtos Biológicos/química , Relação Dose-Resposta a Droga , Estrutura Molecular , Mycobacterium tuberculosis/enzimologia , Relação Estrutura-Atividade , Transferases/metabolismo , Uridina/química , Uridina/farmacologia
15.
Neurochem Res ; 44(10): 2307-2313, 2019 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30847858

RESUMO

Dihydrolipoamide dehydrogenase (LADH, E3) deficiency is a rare (autosomal, recessive) genetic disorder generally presenting with an onset in the neonatal age and early death; the highest carrier rate has been found among Ashkenazi Jews. Acute clinical episodes usually involve severe metabolic decompensation and lactate acidosis that result in neurological, cardiological, and/or hepatological manifestations. Clinical severity is due to the fact that LADH is a common E3 subunit to the alpha-ketoglutarate, pyruvate, alpha-ketoadipate, and branched-chain alpha-keto acid dehydrogenase complexes, and is also a constituent in the glycine cleavage system, thus a loss in LADH function adversely affects multiple key metabolic routes. However, the severe clinical pictures frequently still do not parallel the LADH activity loss, which implies the involvement of auxiliary biochemical mechanisms; enhanced reactive oxygen species generation as well as affinity loss for multienzyme complexes proved to be key auxiliary exacerbating pathomechanisms. This review provides an overview and an up-to-date molecular insight into the pathomechanisms of this disease in light of the structural conclusions drawn from the first crystal structure of a disease-causing hE3 variant determined recently in our laboratory.


Assuntos
Acidose Láctica/metabolismo , Citocromo-B(5) Redutase/deficiência , Di-Hidrolipoamida Desidrogenase/metabolismo , Doença da Urina de Xarope de Bordo/metabolismo , Metemoglobinemia/metabolismo , Espécies Reativas de Oxigênio/metabolismo , Aminoácido Oxirredutases/metabolismo , Citocromo-B(5) Redutase/metabolismo , Humanos , Complexos Multienzimáticos/metabolismo , Ácido Pirúvico/metabolismo , Transferases/metabolismo
16.
J Enzyme Inhib Med Chem ; 34(1): 490-499, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30724623

RESUMO

The use of natural systems, such as outer membrane protein A (OmpA), phosphoporin E (PhoE), ice nucleation protein (INP), etc., has been proved very useful for the surface exposure of proteins on the outer membrane of Gram-negative bacteria. These strategies have the clear advantage of unifying in a one-step the production, the purification and the in vivo immobilisation of proteins/biocatalysts onto a specific biological support. Here, we introduce the novel Anchoring-and-Self-Labelling-protein-tag (ASLtag), which allows the in vivo immobilisation of enzymes on E. coli surface and the labelling of the neosynthesised proteins with the engineered alkylguanine-DNA-alkyl-transferase (H5) from Sulfolobus solfataricus. Our results demonstrated that this tag enhanced the overexpression of thermostable enzymes, such as the carbonic anhydrase (SspCA) from Sulfurihydrogenibium yellowstonense and the ß-glycoside hydrolase (SsßGly) from S. solfataricus, without affecting their folding and catalytic activity, proposing a new tool for the improvement in the utilisation of biocatalysts of biotechnological interest.


Assuntos
Proteínas da Membrana Bacteriana Externa/metabolismo , Enzimas Imobilizadas/metabolismo , Escherichia coli/enzimologia , Transferases/metabolismo , Enzimas Imobilizadas/química , Escherichia coli/metabolismo , Humanos , Coloração e Rotulagem , Propriedades de Superfície , Transferases/química
17.
Artif Cells Nanomed Biotechnol ; 47(1): 256-259, 2019 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-30688096

RESUMO

We herein describe a simple but efficient method for the determination of terminal transferase (TdT) activity, which relies on our finding that Fe(III)-quenched boron-dipyrromethene (BODIPY)-ATP is utilized as a switch-on monomer for polymerization and enables the facile synthesis of fluorescence oligonucleotides without additional, post-processing steps. As TdT carries out the synthesis of DNA by adding the monomers into growing chains, Fe(III) is displaced from BODIPY with the release of pyrophosphate group, which consequently leads to the generation of highly fluorescent long oligonucleotides. With this strategy, we selectively detected the TdT activity with high sensitivity. In addition, its practical applicability was successfully demonstrated by determining TdT activities in human serum.


Assuntos
Ensaios Enzimáticos/métodos , Polimerização , Transferases/metabolismo , Trifosfato de Adenosina/química , Compostos de Boro/química , Estudos de Viabilidade , Corantes Fluorescentes/química , Corantes Fluorescentes/metabolismo , Ferro/química , Limite de Detecção , Oligonucleotídeos/metabolismo
18.
ACS Infect Dis ; 5(3): 406-417, 2019 03 08.
Artigo em Inglês | MEDLINE | ID: mdl-30614674

RESUMO

To fight the growing threat of antibiotic resistance, new antibiotics are required that target essential bacterial processes other than protein, DNA/RNA, and cell wall synthesis, which constitute the majority of currently used antibiotics. 1-Deoxy-d-xylulose-5-phosphate (DXP) synthase is a vital enzyme in bacterial central metabolism, feeding into the de novo synthesis of thiamine diphosphate, pyridoxal phosphate, and essential isoprenoid precursors isopentenyl diphosphate and dimethylallyl diphosphate. While potent and selective inhibitors of DXP synthase in vitro activity have been discovered, their antibacterial activity is modest. To improve the antibacterial activity of selective alkyl acetylphosphonate (alkylAP) inhibitors of DXP synthase, we synthesized peptidic enamide prodrugs of alkylAPs inspired by the natural product dehydrophos, a prodrug of methyl acetylphosphonate. This prodrug strategy achieves dramatic increases in activity against Gram-negative pathogens for two alkylAPs, butyl acetylphosphonate and homopropargyl acetylphosphonate, decreasing minimum inhibitory concentrations against Escherichia coli by 33- and nearly 2000-fold, respectively. Antimicrobial studies and LC-MS/MS analysis of alkylAP-treated E. coli establish that the increased potency of prodrugs is due to increased accumulation of alkylAP inhibitors of DXP synthase via transport of the prodrug through the OppA peptide permease and subsequent amide hydrolysis. This work demonstrates the promise of targeting DXP synthase for the development of novel antibacterial agents.


Assuntos
Antibacterianos/química , Inibidores Enzimáticos/química , Proteínas de Escherichia coli/antagonistas & inibidores , Escherichia coli/efeitos dos fármacos , Pró-Fármacos/química , Transferases/antagonistas & inibidores , Antibacterianos/farmacologia , Inibidores Enzimáticos/farmacologia , Escherichia coli/enzimologia , Escherichia coli/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Testes de Sensibilidade Microbiana , Pentosefosfatos/metabolismo , Pró-Fármacos/farmacologia , Transferases/química , Transferases/metabolismo
19.
BMC Plant Biol ; 19(1): 1, 2019 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-30606102

RESUMO

BACKGROUND: Stevia rebaudiana produces sweet-tasting steviol glycosides (SGs) in its leaves which can be used as natural sweeteners. Metabolic engineering of Stevia would offer an alternative approach to conventional breeding for enhanced production of SGs. However, an effective protocol for Stevia transformation is lacking. RESULTS: Here, we present an efficient and reproducible method for Agrobacterium-mediated transformation of Stevia. In our attempts to produce transgenic Stevia plants, we found that prolonged dark incubation is critical for increasing shoot regeneration. Etiolated shoots regenerated in the dark also facilitated subsequent visual selection of transformants by green fluorescent protein during Stevia transformation. Using this newly established transformation method, we overexpressed the Stevia 1-deoxy-d-xylulose-5-phosphate synthase 1 (SrDXS1) and kaurenoic acid hydroxylase (SrKAH), both of which are required for SGs biosynthesis. Compared to control plants, the total SGs content in SrDXS1- and SrKAH-overexpressing transgenic lines were enhanced by up to 42-54% and 67-88%, respectively, showing a positive correlation with the expression levels of SrDXS1 and SrKAH. Furthermore, their overexpression did not stunt the growth and development of the transgenic Stevia plants. CONCLUSION: This study represents a successful case of genetic manipulation of SGs biosynthetic pathway in Stevia and also demonstrates the potential of metabolic engineering towards producing Stevia with improved SGs yield.


Assuntos
Diterpenos de Caurano/metabolismo , Glucosídeos/metabolismo , Oxigenases de Função Mista/metabolismo , Proteínas de Plantas/metabolismo , Stevia/metabolismo , Transferases/metabolismo , Engenharia Genética/métodos , Oxigenases de Função Mista/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Brotos de Planta/metabolismo , Plantas Geneticamente Modificadas , Stevia/enzimologia , Stevia/genética , Transferases/genética
20.
Curr Med Chem ; 26(21): 3923-3957, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-29446729

RESUMO

Enzymes are a class of macromolecules that function as highly efficient and specific biological catalysts requiring only mild reaction conditions. Enzymes are essential to maintaining life activities, including promoting metabolism and homeostasis, and participating in a variety of physiological functions. Accordingly, enzymatic levels and activity are closely related to the health of the organism, where enzymatic dysfunctions often lead to corresponding diseases in the host. Due to this, diagnosis of certain diseases is based on the levels and activity of certain enzymes. Therefore, rapid real-time and accurate detection of enzymes in situ are important for diagnosis, monitoring, clinical treatment and pathological studies of disease. Fluorescent probes have unique advantages in terms of detecting enzymes, including being simple to use in highly sensitive and selective real-time rapid in-situ noninvasive and highly spatial resolution visual imaging. However, fluorescent probes are most commonly used to detect oxidoreductases, transferases and hydrolases due to the processes and types of enzyme reactions. This paper summarizes the application of fluorescent probes to detect these three types of enzymes over the past five years. In addition, we introduce the mechanisms underlying detection of these enzymes by their corresponding probes.


Assuntos
Corantes Fluorescentes/química , Fluorometria , Hidrolases/análise , Oxirredutases/análise , Transferases/análise , Humanos , Hidrolases/metabolismo , Oxirredutases/metabolismo , Transferases/metabolismo
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