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1.
Sheng Wu Gong Cheng Xue Bao ; 36(7): 1450-1458, 2020 Jul 25.
Artigo em Chinês | MEDLINE | ID: mdl-32748603

RESUMO

Heparin and heparan sulfate are a class of glycosaminoglycans for clinical anticoagulation. Heparosan N-sulfate-glucuronate 5-epimerase (C5, EC 5.1.3.17) is a critical modifying enzyme in the synthesis of heparin and heparan sulfate, and catalyzes the inversion of carboxyl group at position 5 on D-glucuronic acid (D-GlcA) of N-sulfoheparosan to form L-iduronic acid (L-IdoA). In this study, the heparin C5 epimerase gene Glce from zebrafish was expressed and molecularly modified in Escherichia coli. After comparing three expression vectors of pET-20b (+), pET-28a (+) and pCold Ⅲ, C5 activity reached the highest ((1 873.61±5.42) U/L) with the vector pCold Ⅲ. Then we fused the solution-promoting label SET2 at the N-terminal for increasing the soluble expression of C5. As a result, the soluble protein expression was increased by 50% compared with the control, and the enzyme activity reached (2 409±6.43) U/L. Based on this, site-directed mutations near the substrate binding pocket were performed through rational design, the optimal mutant (V153R) enzyme activity and specific enzyme activity were (5 804±5.63) U/L and (145.1±2.33) U/mg, respectively 2.41-fold and 2.28-fold of the original enzyme. Modification and expression optimization of heparin C5 epimerase has laid the foundation for heparin enzymatic catalytic biosynthesis.


Assuntos
Carboidratos Epimerases , Regulação Enzimológica da Expressão Gênica , Heparina , Racemases e Epimerases , Animais , Carboidratos Epimerases/genética , Heparina/metabolismo , Heparitina Sulfato/metabolismo , Ácido Idurônico/metabolismo , Racemases e Epimerases/genética
2.
Gene ; 741: 144522, 2020 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-32145329

RESUMO

Virus-induced gene silencing (VIGS) is a transient based reverse genetic tool used to elucidate the function of novel gene in N. benthamiana. In current study, 14 UDP-D-glucuronate 4-epimerase (GAE) family members were identified and their gene structure, phylogeny and expression pattern were analyzed. VIGS system was optimized for the functional characterization of NbGAE6 homologous genes in N. benthamiana. Whilst the GAE family is well-known for the interconversion of UDP-D-GlcA and UDP-D-GalA during pectin synthesis. Our results revealed that the downregulation of these genes significantly reduced the amount of GalA in the homogalacturunan which is the major component of pectin found in primary cell wall. Biphenyl assay and high performance liquid chromatography analysis (HPLC) depicted that the level of 'GalA' monosaccharide reduced to 40-51% in VIGS plants as compared to the wild type plants. Moreover, qRT-PCR also confirmed the downregulation of the NbGAE6 mRNA in VIGS plants. In all, this is the first comprehensive study of the optimization of VIGS system for the provision of rapid silencing of GAE family members in N. benthamiana, eliminating the need of stable transformants.


Assuntos
Proteínas de Arabidopsis/genética , Carboidratos Epimerases/genética , Parede Celular/metabolismo , Pectinas/genética , Tabaco/genética , Arabidopsis/genética , Parede Celular/genética , Parede Celular/virologia , Regulação da Expressão Gênica de Plantas , Inativação Gênica , Vetores Genéticos/genética , Monossacarídeos/metabolismo , Pectinas/biossíntese , Peptídeos , Vírus de Plantas/genética , RNA Mensageiro/genética , Tabaco/virologia
3.
J Environ Sci Health B ; 55(3): 265-272, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-31762384

RESUMO

Food contaminated with Shiga toxin-producing Escherichia coli (STEC) represents a hazardous public health problem worldwide. Therefore, the present study was performed to elucidate the virulent and antimicrobial resistance characteristics of STEC isolated from milk and dairy products marketed in Egypt. A total of 125 samples (raw market milk, bulk tank milk, Kareish cheese, white soft cheese, and small scale-produced ice cream, 25 each) were collected for determination the prevalence and antimicrobial resistance profiling of STEC. Thirty-six STEC isolates were recovered from milk and dairy products. Serological analysis illustrated that three isolates were E. coli O157:H7 and 33 isolates belonged to different serotypes. Molecular examination indicated that all isolates harboured stx1 and/or stx2 genes, 14 isolates expressed eaeA gene and 3 isolates possessed rfbE gene. Antimicrobial resistance profiling of the isolates was both phenotypically and genetically examined. Interestingly, 31 out of 36 (86.11%) isolates were multidrug-resistant and harboured the extended-spectrum ß-lactamase encoding genes, namely, blaCTX-M-15, blaSHV-12 and blaCTX-M-14. Moreover, 12 isolates (33.33%) harboured plasmid-mediated quinolone resistant gene, qnrS. The overall conclusion of the current investigation indicated insufficient hygienic measures adopted during milking, handling, and processing leading to development of pathogenic and multidrug-resistant STEC.


Assuntos
Laticínios/microbiologia , Farmacorresistência Bacteriana/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/efeitos dos fármacos , Escherichia coli Shiga Toxigênica/patogenicidade , Adesinas Bacterianas/genética , Animais , Carboidratos Epimerases/genética , Queijo/microbiologia , Farmacorresistência Bacteriana/genética , Egito , Escherichia coli O157/efeitos dos fármacos , Escherichia coli O157/isolamento & purificação , Escherichia coli O157/patogenicidade , Proteínas de Escherichia coli/genética , Microbiologia de Alimentos , Sorvetes/microbiologia , Testes de Sensibilidade Microbiana , Leite/microbiologia , Plasmídeos/efeitos dos fármacos , Plasmídeos/genética , Prevalência , Escherichia coli Shiga Toxigênica/isolamento & purificação , Transaminases/genética , Virulência/genética , beta-Lactamases/genética
4.
Enzyme Microb Technol ; 132: 109441, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31731964

RESUMO

D-allulose has received increasing attention due to its excellent physiological properties and commercial potential. The D-allulose 3-epimerase from Rhodopirellula baltica (RbDAEase) catalyzes the conversion of D-fructose to D-allulose. However, its poor thermostability has hampered its industrial application. Site-directed mutagenesis based on homologous structures in which the residuals on high flexible regions were substituted according to B-factors analysis, is an effective way to improve the thermostability and robustness of an enzyme. RbDAEase showed substrate specificity toward D-allulose with a Km of 58.57 mM and kcat of 1849.43 min-1. It showed a melting temperature (Tm) of 45.7 °C and half-life (t1/2) of 52.3 min at pH 8.0, 60 °C with 1 mM Mn2+. The Site-directed mutation L144 F strengthened the thermostability to a Δt1/2 of 50.4 min, ΔTm of 12.6 °C, and ΔT5060 of 22 °C. It also improved the conversion rate to 28.6%. Structural analysis reveals that a new hydrophobic interaction was formed by the mutation. Thus, site-directed mutagenesis based on B-factors analysis would be an efficient strategy to enhance the thermostability of designed ketose 3-epimerases.


Assuntos
Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Mutagênese Sítio-Dirigida , Planctomycetales/enzimologia , Planctomycetales/genética , Engenharia de Proteínas , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Temperatura
5.
PLoS Genet ; 15(12): e1008526, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31834878

RESUMO

Pathological phosphorylated TDP-43 protein (pTDP) deposition drives neurodegeneration in amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). However, the cellular and genetic mechanisms at work in pathological TDP-43 toxicity are not fully elucidated. To identify genetic modifiers of TDP-43 neurotoxicity, we utilized a Caenorhabditis elegans model of TDP-43 proteinopathy expressing human mutant TDP-43 pan-neuronally (TDP-43 tg). In TDP-43 tg C. elegans, we conducted a genome-wide RNAi screen covering 16,767 C. elegans genes for loss of function genetic suppressors of TDP-43-driven motor dysfunction. We identified 46 candidate genes that when knocked down partially ameliorate TDP-43 related phenotypes; 24 of these candidate genes have conserved homologs in the human genome. To rigorously validate the RNAi findings, we crossed the TDP-43 transgene into the background of homozygous strong genetic loss of function mutations. We have confirmed 9 of the 24 candidate genes significantly modulate TDP-43 transgenic phenotypes. Among the validated genes we focused on, one of the most consistent genetic modifier genes protecting against pTDP accumulation and motor deficits was the heparan sulfate-modifying enzyme hse-5, the C. elegans homolog of glucuronic acid epimerase (GLCE). We found that knockdown of human GLCE in cultured human cells protects against oxidative stress induced pTDP accumulation. Furthermore, expression of glucuronic acid epimerase is significantly decreased in the brains of FTLD-TDP cases relative to normal controls, demonstrating the potential disease relevance of the candidate genes identified. Taken together these findings nominate glucuronic acid epimerase as a novel candidate therapeutic target for TDP-43 proteinopathies including ALS and FTLD-TDP.


Assuntos
Proteínas de Caenorhabditis elegans/genética , Carboidratos Epimerases/genética , Proteínas de Ligação a DNA/genética , Proteinopatias TDP-43/genética , Animais , Animais Geneticamente Modificados , Autopsia , Encéfalo/metabolismo , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/metabolismo , Carboidratos Epimerases/metabolismo , Linhagem Celular , Proteínas de Ligação a DNA/metabolismo , Modelos Animais de Doenças , Regulação para Baixo , Técnicas de Silenciamento de Genes , Células HEK293 , Humanos , Interferência de RNA , Genética Reversa , Proteinopatias TDP-43/metabolismo
6.
Appl Microbiol Biotechnol ; 103(21-22): 8753-8761, 2019 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-31637494

RESUMO

D-Mannose is an epimer of glucose at the C-2 position and exists in nature as a component of mannan. It has 60 and 86% sweetness than that of sucrose and D-glucose, respectively. Because of its low-calorie and nontoxic features, D-mannose is used widely in food, medicine, cosmetic, and food-additive industries. Besides, it exhibits many physiologic benefits on health: immune system, diabetes mellitus, intestinal diseases, and urinary tract infections. It is used as a starting material to synthesize immunostimulatory agents, anti-tumor agents, vitamins, and D-mannitol. However, D-mannose production using chemical synthesis and plant extraction cannot meet the requirements of the industry. This article presents recent research on the biological production of D-mannose. The physiologic benefits and applications of D-mannose are summarized. Besides, different D-mannose-producing enzymes from various sources are discussed in detail with regard to their biochemical characteristics, catalytic efficiency, and reaction kinetics for D-mannose production. Furthermore, attempts to use enzymatic conversion to produce D-mannose are reviewed.


Assuntos
Bactérias/enzimologia , Bactérias/metabolismo , Reatores Biológicos/microbiologia , Manose/metabolismo , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/química , Glucose/química , Plantas/química
7.
Molecules ; 24(17)2019 Aug 22.
Artigo em Inglês | MEDLINE | ID: mdl-31443364

RESUMO

Isorhamnetin-3-O-rhamnoside was synthesized by a highly efficient three-enzyme (rhamnosyltransferase, glycine max sucrose synthase and uridine diphosphate (UDP)-rhamnose synthase) cascade using a UDP-rhamnose regeneration system. The rhamnosyltransferase gene (78D1) from Arabidopsis thaliana was cloned, expressed, and characterized in Escherichia coli. The optimal activity was at pH 7.0 and 45 °C. The enzyme was stable over the pH range of 6.5 to 8.5 and had a 1.5-h half-life at 45 °C. The Vmax and Km for isorhamnetin were 0.646 U/mg and 181 µM, respectively. The optimal pH and temperature for synergistic catalysis were 7.5 and 25 °C, and the optimal concentration of substrates were assayed, respectively. The highest titer of isorhamnetin-3-O-rhamnoside production reached 231 mg/L with a corresponding molar conversion of 100%. Isorhamnetin-3-O-rhamnoside was purified and the cytotoxicity against HepG2, MCF-7, and A549 cells were evaluated. Therefore, an efficient method for isorhamnetin-3-O-rhamnoside production described herein could be widely used for the rhamnosylation of flavonoids.


Assuntos
Carboidratos Epimerases/química , Técnicas de Química Sintética , Flavonóis/síntese química , Glucosiltransferases/química , Hexosiltransferases/química , Açúcares de Uridina Difosfato/química , Antineoplásicos/síntese química , Antineoplásicos/farmacologia , Catálise , Linhagem Celular Tumoral , Flavonóis/farmacologia , Humanos
8.
Int J Biol Macromol ; 138: 536-545, 2019 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-31330210

RESUMO

Recently, rare sugars have caused extensively attention due to their beneficial physiological functions and potential applications in food systems and medical fields. Ketose 3-epimerase (KEase) can catalyze reversibly the epimerization between ketoses which is the pivotal enzyme in Izumoring strategy and an effective tool for biological production of rare sugars. In this work, a KEase from Caballeronia fortuita was recombined and characterized as a d-tagatose 3-epimerase (DTEase, EC 5.1.3.31). The recombinant DTEase displayed the highest activity at pH7.5 and 65°C in the presence of Co2+. The recombinant DTEase displayed the relatively high thermostability and the half-life (t1/2) was determined to be 7.13, 5.13, and 1.05h at 50, 55, and 60°C, respectively. The recombinant DTEase had a wide substrate specificity and the specific activities towards d-tagatose, d-allulose, d-fructose and l-sorbose were measured to be 801±2.3, 450±2.7, 270±1.5 and 55±1.8Umg-1, respectively. So far, the recombinant DTEase exhibited the highest specific activity towards d-tagatose compared with other reported KEases. Furthermore, the recombinant DTEase could produce 314.2g/L d-sorbose from 500g/L d-tagatose and 147.0g/L d-allulose from 500g/L d-fructose, with a transformation ratio of 68.2% and 29.4%, respectively. The recombinant DTEase could realize effectively the transformations between various ketoses and was a prominent candidate for production of rare sugars.


Assuntos
Burkholderiaceae/enzimologia , Carboidratos Epimerases/química , Açúcares/metabolismo , Biotransformação , Burkholderiaceae/classificação , Burkholderiaceae/genética , Carboidratos Epimerases/genética , Carboidratos Epimerases/isolamento & purificação , Clonagem Molecular , Ativação Enzimática , Fermentação , Expressão Gênica , Hexoses/química , Concentração de Íons de Hidrogênio , Cinética , Filogenia , Análise de Sequência de DNA , Especificidade por Substrato , Temperatura
9.
Int J Mol Sci ; 20(14)2019 Jul 19.
Artigo em Inglês | MEDLINE | ID: mdl-31330931

RESUMO

GDP-mannose 3,5-epimerase (GM35E) catalyzes the conversion of GDP-mannose towards GDP-l-galactose and GDP-l-gulose. Although this reaction represents one of the few enzymatic routes towards the production of l-sugars and derivatives, it has not yet been exploited for that purpose. One of the reasons is that so far only GM35Es from plants have been characterized, yielding biocatalysts that are relatively unstable and difficult to express heterologously. Through the mining of sequence databases, we succeeded in identifying a promising bacterial homologue. The gene from the thermophilic organism Methylacidiphilum fumariolicum was codon optimized for expression in Escherichia coli, resulting in the production of 40 mg/L of recombinant protein. The enzyme was found to act as a self-sufficient GM35E, performing three chemical reactions in the same active site. Furthermore, the biocatalyst was highly stable at temperatures up to 55 °C, making it well suited for the synthesis of new carbohydrate products with application in the pharma industry.


Assuntos
Proteínas de Bactérias , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Sequência de Aminoácidos , Catálise , Ativação Enzimática , Estabilidade Enzimática , Guanosina Difosfato Manose/química , Guanosina Difosfato Manose/metabolismo , Concentração de Íons de Hidrogênio , Modelos Moleculares , Estrutura Molecular , Conformação Proteica , Proteínas Recombinantes , Relação Estrutura-Atividade , Termodinâmica
10.
World J Gastroenterol ; 25(23): 2947-2960, 2019 Jun 21.
Artigo em Inglês | MEDLINE | ID: mdl-31249452

RESUMO

BACKGROUND: Changes in N-linked glycosylation have been observed in the circulation of individuals with hepatocellular carcinoma. In particular, an elevation in the level of core fucosylation has been observed. However, the mechanisms through which core fucose is increased are not well understood. We hypothesized that a review of the literature and related bioinformatic review regarding six genes known to be involved in the attachment of core fucosylation, the synthesis of the fucosylation substrate guanosine diphosphate (GDP)-fucose, or the transport of the substrate into the Golgi might offer mechanistic insight into the regulation of core fucose levels. AIM: To survey the literature to capture the involvement of genes regulating core N-linked fucosylation in hepatocellular carcinoma. METHODS: The PubMed biomedical literature database was searched for the association of hepatocellular carcinoma and each of the core fucose-related genes and their protein products. We also queried The Cancer Genome Atlas Liver hepatocellular carcinoma (LIHC) dataset for genetic, epigenetic and gene expression changes for the set of six genes using the tools at cBioportal. RESULTS: A total of 27 citations involving one or more of the core fucosylation-related genes (FPGT, FUK, FUT8, GMDS, SLC35C1, TSTA3) and hepatocellular carcinoma were identified. The same set of gene symbols was used to query the 371 patients with liver cancer in the LIHC dataset to identify the frequency of mRNA over or under expression, as well as non-synonymous mutations, copy number variation and methylation level. Although all six genes trended to more samples displaying over expression relative to under-expression, it was noted that a number of tumor samples had undergone amplification of the genes of the de novo synthesis pathway, GMDS (27 samples) and TSTA3 (78 samples). In contrast, the other four genes had undergone amplification in 2 or fewer samples. CONCLUSION: Amplification of genes involved in the de novo pathway for generation of GDP-fucose, GMDS and TSTA3, likely contributes to the elevated core fucose observed in hepatocellular carcinoma.


Assuntos
Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Redes e Vias Metabólicas/genética , Carboidratos Epimerases/metabolismo , Carcinoma Hepatocelular/patologia , Variações do Número de Cópias de DNA , Metilação de DNA , Glicoproteínas/metabolismo , Glicosilação , Guanosina Difosfato Fucose/metabolismo , Humanos , Hidroliases/metabolismo , Cetona Oxirredutases/metabolismo , Neoplasias Hepáticas/patologia , Mutação
11.
Appl Microbiol Biotechnol ; 103(16): 6559-6570, 2019 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-31201453

RESUMO

Carbohydrate epimerases and isomerases are essential for the metabolism and synthesis of carbohydrates. In this study, Runella slithyformis Runsl_4512 and Dyadobacter fermentans Dfer_5652 were characterized from a cluster of uncharacterized proteins of the acylglucosamine 2-epimerase (AGE) superfamily. These proteins catalyzed the intramolecular conversion of D-mannose to D-glucose, whereas they did not act on ß-(1 → 4)-mannobiose, N-acetyl-D-glucosamine, and D-fructose, which are substrates of known AGE superfamily members. The kcat/Km values of Runsl_4512 and Dfer_5652 for D-mannose epimerization were 3.89 and 3.51 min-1 mM-1, respectively. Monitoring the Runsl_4512 reaction through 1H-NMR showed the formation of ß-D-glucose and ß-D-mannose from D-mannose and D-glucose, respectively. In the reaction with ß-D-glucose, ß-D-mannose was produced at the initial stage of the reaction, but not in the reaction with α-D-glucose. These results indicate that Runsl_4512 catalyzed the 2-epimerization of the ß-anomer substrate with a net retention of the anomeric configuration. Since 2H was obviously detected at the 2-C position of D-mannose and D-glucose in the equilibrated reaction mixture produced by Runsl_4512 in 2H2O, this enzyme abstracts 2-H from the substrate and adds another proton to the intermediate. This mechanism is in accordance with the mechanism proposed for the reactions of other epimerases of the AGE superfamily, that is, AGE and cellobiose 2-epimerase. Upon reaction with 500 g/L D-glucose at 50 °C and pH 8.0, Runsl_4512 and Dfer_5652 produced D-mannose with a 24.4 and 22.8% yield, respectively. These D-mannose yields are higher than those of other enzyme systems, and ME acts as an efficient biocatalyst for producing D-mannose.


Assuntos
Carboidratos Epimerases/metabolismo , Cytophagaceae/enzimologia , Manose/metabolismo , Glucose/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Especificidade por Substrato , Temperatura
12.
J Agric Food Chem ; 67(22): 6285-6291, 2019 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-31117501

RESUMO

N-Acetyl-d-neuraminic acid (Neu5Ac) is a potential baby nutrient and the key precursor of antiflu medicine Zanamivir. The Neu5Ac chemoenzymatic synthesis consists of N-acetyl-d-glucosamine epimerase (AGE)-catalyzed epimerization of N-acetyl-d-glucosamine (GlcNAc) to N-acetyl-d-mannosamine (ManNAc) and aldolase-catalyzed condensation between ManNAc and pyruvate. Herein, we cloned and characterized BT0453, a novel AGE, from a human gut symbiont Bacteroides thetaiotaomicron. BT0453 shows the highest soluble fraction among the AGEs tested. With GlcNAc and sodium pyruvate as substrates, Neu5Ac production by coupling whole cells expressing BT0453 and Escherichia coli N-acetyl-d-neuraminic acid aldolase was explored. After 36 h, a 53.6% molar yield, 3.6 g L-1 h-1 productivity and 42.9 mM titer of Neu5Ac were obtained. Furthermore, for the first time, the T7- BT0453-T7- nanA polycistronic unit was integrated into the E. coli genome, generating a chromosome-based biotransformation system. BT0453 protein engineering and metabolic engineering studies hold potential for the industrial production of Neu5Ac.


Assuntos
Aldeído Liases/genética , Proteínas de Bactérias/genética , Bacteroides thetaiotaomicron/enzimologia , Carboidratos Epimerases/genética , Proteínas de Transporte/genética , Proteínas de Escherichia coli/genética , Escherichia coli/enzimologia , Ácido N-Acetilneuramínico/biossíntese , Aldeído Liases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/química , Carboidratos Epimerases/metabolismo , Proteínas de Transporte/química , Proteínas de Transporte/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Expressão Gênica , Cinética , Engenharia Metabólica
13.
J Biol Chem ; 294(23): 9172-9185, 2019 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-31010825

RESUMO

The 6-deoxy sugar l-rhamnose (l-Rha) is found widely in plant and microbial polysaccharides and natural products. The importance of this and related compounds in host-pathogen interactions often means that l-Rha plays an essential role in many organisms. l-Rha is most commonly biosynthesized as the activated sugar nucleotide uridine 5'-diphospho-ß-l-rhamnose (UDP-ß-l-Rha) or thymidine 5'-diphospho-ß-l-rhamnose (TDP-ß-l-Rha). Enzymes involved in the biosynthesis of these sugar nucleotides have been studied in some detail in bacteria and plants, but the activated form of l-Rha and the corresponding biosynthetic enzymes have yet to be explored in algae. Here, using sugar-nucleotide profiling in two representative algae, Euglena gracilis and the toxin-producing microalga Prymnesium parvum, we show that levels of UDP- and TDP-activated l-Rha differ significantly between these two algal species. Using bioinformatics and biochemical methods, we identified and characterized a fusion of the RmlC and RmlD proteins, two bacteria-like enzymes involved in TDP-ß-l-Rha biosynthesis, from P. parvum Using this new sequence and also others, we explored l-Rha biosynthesis among algae, finding that although most algae contain sequences orthologous to plant-like l-Rha biosynthesis machineries, instances of the RmlC-RmlD fusion protein identified here exist across the Haptophyta and Gymnodiniaceae families of microalgae. On the basis of these findings, we propose potential routes for the evolution of nucleoside diphosphate ß-l-Rha (NDP-ß-l-Rha) pathways among algae.


Assuntos
Proteínas de Algas/metabolismo , Carboidratos Epimerases/metabolismo , Haptófitas/metabolismo , Ramnose/biossíntese , Proteínas de Algas/genética , Carboidratos Epimerases/classificação , Carboidratos Epimerases/genética , Filogenia , Plastídeos/metabolismo , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Ramnose/química , Simbiose
14.
Mol Biotechnol ; 61(6): 432-441, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-30963480

RESUMO

D-Allulose is a rare monosaccharide that exists in extremely small quantities in nature, and it is also hard to prepare at a large scale via chemical or enzyme synthetic route due to low conversion and downstream separation complexity. Using D-psicose epimerase and L-rhamnulose kinase, a method enabling high conversion of D-allulose from D-fructose without the need for a tedious isomer separation step was established recently. However, this method requires expensive ATP to facilitate the reaction. In the present study, an ATP regenerate system was developed coupling with polyphosphate kinase. In our optimized reaction with purified enzymes, the conversion rate of 99% D-fructose was achieved at the concentrations of 2 mM ATP, 5 mM polyphosphate, 20 mM D-fructose, and 20 mM Mg2+ when incubated at 50 °C and at pH 7.5. ATP usage can be reduced to 10% of the theoretical amount compared to that without the ATP regeneration system. A fed-batch mode was also studied to minimize the inhibitory effect of polyphosphate. The biosynthetic system reported here offers a potential and promising platform for the conversion of D-fructose into D-allulose at reduced ATP cost.


Assuntos
Trifosfato de Adenosina/metabolismo , Carboidratos Epimerases/metabolismo , Frutose/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Biotransformação , Carboidratos Epimerases/genética , Cátions Bivalentes , Clonagem Molecular , Ensaios Enzimáticos , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/biossíntese , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Magnésio/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Polifosfatos/metabolismo , Proteínas Recombinantes de Fusão/genética , Frações Subcelulares/química , Frações Subcelulares/metabolismo , Thermotoga maritima/genética , Thermotoga maritima/metabolismo
15.
J Bacteriol ; 201(10)2019 05 15.
Artigo em Inglês | MEDLINE | ID: mdl-30833352

RESUMO

Sinorhizobium meliloti produces multiple extracellular glycans, including among others, lipopolysaccharides (LPS), and the exopolysaccharides (EPS) succinoglycan (SG) and galactoglucan (GG). These polysaccharides serve cell protective roles. Furthermore, SG and GG promote the interaction of S. meliloti with its host Medicago sativa in root nodule symbiosis. ExoB has been suggested to be the sole enzyme catalyzing synthesis of UDP-galactose in S. meliloti (A. M. Buendia, B. Enenkel, R. Köplin, K. Niehaus, et al. Mol Microbiol 5:1519-1530, 1991, https://doi.org/10.1111/j.1365-2958.1991.tb00799.x). Accordingly, exoB mutants were previously found to be affected in the synthesis of the galactose-containing glycans LPS, SG, and GG and consequently, in symbiosis. Here, we report that the S. meliloti Rm2011 uxs1-uxe-apsS-apsH1-apsE-apsH2 (SMb20458-63) gene cluster directs biosynthesis of an arabinose-containing polysaccharide (APS), which contributes to biofilm formation, and is solely or mainly composed of arabinose. Uxe has previously been identified as UDP-xylose 4-epimerase. Collectively, our data from mutational and overexpression analyses of the APS biosynthesis genes and in vitro enzymatic assays indicate that Uxe functions as UDP-xylose 4- and UDP-glucose 4-epimerase catalyzing UDP-xylose/UDP-arabinose and UDP-glucose/UDP-galactose interconversions, respectively. Overexpression of uxe suppressed the phenotypes of an exoB mutant, evidencing that Uxe can functionally replace ExoB. We suggest that under conditions stimulating expression of the APS biosynthesis operon, Uxe contributes to the synthesis of multiple glycans and thereby to cell protection, biofilm formation, and symbiosis. Furthermore, we show that the C2H2 zinc finger transcriptional regulator MucR counteracts the previously reported CuxR-c-di-GMP-mediated activation of the APS biosynthesis operon. This integrates the c-di-GMP-dependent control of APS production into the opposing regulation of EPS biosynthesis and swimming motility in S. meliloti IMPORTANCE Bacterial extracellular polysaccharides serve important cell protective, structural, and signaling roles. They have particularly attracted attention as adhesives and matrix components promoting biofilm formation, which significantly contributes to resistance against antibiotics. In the root nodule symbiosis between rhizobia and leguminous plants, extracellular polysaccharides have a signaling function. UDP-sugar 4-epimerases are important enzymes in the synthesis of the activated sugar substrates, which are frequently shared between multiple polysaccharide biosynthesis pathways. Thus, these enzymes are potential targets to interfere with these pathways. Our finding of a bifunctional UDP-sugar 4-epimerase in Sinorhizobium meliloti generally advances the knowledge of substrate promiscuity of such enzymes and specifically of the biosynthesis of extracellular polysaccharides involved in biofilm formation and symbiosis in this alphaproteobacterium.


Assuntos
Carboidratos Epimerases/metabolismo , Polissacarídeos Bacterianos/biossíntese , Sinorhizobium meliloti/enzimologia , Sinorhizobium meliloti/metabolismo , Carboidratos Epimerases/genética , Sinorhizobium meliloti/genética , Uridina Difosfato Galactose/metabolismo , Uridina Difosfato Glucose/metabolismo , Açúcares de Uridina Difosfato/metabolismo , Uridina Difosfato Xilose/metabolismo
16.
Glycobiology ; 29(6): 479-489, 2019 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-30869121

RESUMO

A class of carbohydrate-modified proteins, heparan sulfate proteoglycans (HSPGs), play critical roles both in normal development and during disease. Genetic studies using a model organism, Drosophila, have been contributing to understanding the in vivo functions of HSPGs. Despite the many strengths of the Drosophila model for in vivo studies, biochemical analysis of Drosophila HS is somewhat limited, mainly due to the insufficient amount of the material obtained from the animal. To overcome this obstacle, we generated mutant cell lines for four HS modifying enzymes that are critical for the formation of ligand binding sites on HS, Hsepi, Hs2st, Hs6st and Sulf1, using a recently established method. Morphological and immunological analyses of the established lines suggest that they are spindle-shaped cells of mesodermal origin. The disaccharide profiles of HS from these cell lines showed characteristics of lack of each enzyme as well as compensatory modifications by other enzymes. Metabolic radiolabeling of HS allowed us to assess chain length and net charge of the total population of HS in wild-type and Hsepi mutant cell lines. We found that Drosophila HS chains are significantly shorter than those from mammalian cells. BMP signaling assay using Hs6st cells indicates that molecular phenotypes of these cell lines are consistent with previously known in vivo phenomena. The established cell lines will provide us with a direct link between detailed structural information of Drosophila HS and a wealth of knowledge on biological phenotypic data obtained over the last two decades using this animal model.


Assuntos
Carboidratos Epimerases/genética , Linhagem Celular , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteoglicanas de Heparan Sulfato/metabolismo , Mutação , Sulfatases/genética , Sulfotransferases/genética , Animais , Carboidratos Epimerases/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Fenótipo , Sulfatases/metabolismo , Sulfotransferases/metabolismo
17.
Proc Natl Acad Sci U S A ; 116(14): 6760-6765, 2019 04 02.
Artigo em Inglês | MEDLINE | ID: mdl-30872481

RESUMO

Heparan sulfate (HS) is a linear, complex polysaccharide that modulates the biological activities of proteins through binding sites made by a series of Golgi-localized enzymes. Of these, glucuronyl C5-epimerase (Glce) catalyzes C5-epimerization of the HS component, d-glucuronic acid (GlcA), into l-iduronic acid (IdoA), which provides internal flexibility to the polymer and forges protein-binding sites to ensure polymer function. Here we report crystal structures of human Glce in the unbound state and of an inactive mutant, as assessed by real-time NMR spectroscopy, bound with a (GlcA-GlcNS)n substrate or a (IdoA-GlcNS)n product. Deep infiltration of the oligosaccharides into the active site cleft imposes a sharp kink within the central GlcNS-GlcA/IdoA-GlcNS trisaccharide motif. An extensive network of specific interactions illustrates the absolute requirement of N-sulfate groups vicinal to the epimerization site for substrate binding. At the epimerization site, the GlcA/IdoA rings are highly constrained in two closely related boat conformations, highlighting ring-puckering signatures during catalysis. The structure-based mechanism involves the two invariant acid/base residues, Glu499 and Tyr578, poised on each side of the target uronic acid residue, thus allowing reversible abstraction and readdition of a proton at the C5 position through a neutral enol intermediate, reminiscent of mandelate racemase. These structures also shed light on a convergent mechanism of action between HS epimerases and lyases and provide molecular frameworks for the chemoenzymatic synthesis of heparin or HS analogs.


Assuntos
Carboidratos Epimerases/química , Ácido Glucurônico/química , Heparina/química , Oligossacarídeos/química , Sítios de Ligação , Carboidratos Epimerases/genética , Catálise , Cristalografia por Raios X , Células HEK293 , Humanos , Relação Estrutura-Atividade , Especificidade por Substrato
18.
Microb Cell Fact ; 18(1): 59, 2019 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-30909913

RESUMO

BACKGROUND: A novel D-allulose 3-epimerase from Staphylococcus aureus (SaDAE) has been screened as a D-allulose 3-epimerase family enzyme based on its high specificity for D-allulose. It usually converts both D-fructose and D-tagatose to respectively D-allulose and D-sorbose. We targeted potential biocatalysts for the large-scale industrial production of rare sugars. RESULTS: SaDAE showed a high activity on D-allulose with an affinity of 41.5 mM and catalytic efficiency of 1.1 s-1 mM-1. Four residues, Glu146, Asp179, Gln205, and Glu240, constitute the catalytic tetrad of SaDAE. Glu146 and Glu240 formed unique interactions with substrates based on the structural model analysis. The redesigned SaDAE_V105A showed an improvement of relative activity toward D-fructose of 68%. The conversion rate of SaDAE_V105A reached 38.9% after 6 h. The triple mutant S191D/M193E/S213C showed higher thermostability than the wild-type enzyme, exhibiting a 50% loss of activity after incubation for 60 min at 74.2 °C compared with 67 °C for the wild type. CONCLUSIONS: We redesigned SaDAE for thermostability and biocatalytic production of D-allulose. The research will aid the development of industrial biocatalysts for D-allulose.


Assuntos
Carboidratos Epimerases , Frutose/biossíntese , Engenharia Metabólica , Staphylococcus aureus , Proteínas de Bactérias/biossíntese , Proteínas de Bactérias/genética , Carboidratos Epimerases/biossíntese , Carboidratos Epimerases/química , Carboidratos Epimerases/genética , Concentração de Íons de Hidrogênio , Cinética , Staphylococcus aureus/enzimologia , Staphylococcus aureus/genética , Especificidade por Substrato
19.
Biomacromolecules ; 20(4): 1613-1622, 2019 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-30844259

RESUMO

With the present accessibility of algal raw material, microbial alginates as a source for strong gelling material are evaluated as an alternative for advanced applications. Recently, we have shown that alginate from algal sources all contain a fraction of very long G-blocks (VLG), that is, consecutive sequences of guluronic acid (G) residues of more than 100 residues. By comparing the gelling properties of these materials with in vitro epimerized polymannuronic acid (poly-M) with shorter G-blocks, but comparable with the G-content, we could demonstrate that VLG have a large influence on gelling properties. Hypothesized to function as reinforcement bars, VLG prevents the contraction of the gels during formation (syneresis) and increases the Young's modulus (strength of the gel). Here we report that these VLG structures are also present in alginates from Azotobacter vinelandii and that these polymers consequently form stable, low syneretic gels with calcium, comparable in mechanical strength to algal alginates with the similar monomeric composition. The bacterium expresses seven different extracellular mannuronan epimerases (AlgE1-AlgE7), of which only the bifunctional epimerase AlgE1 seems to be able to generate the long G-blocks when acting on poly-M. The data implies evidence for a processive mode of action and the necessity of two catalytic sites to obtain the observed epimerization pattern. Furthermore, poly-M epimerized with AlgE1 in vitro form gels with comparable or higher rigidity and gel strength than gels made from brown seaweed alginate with matching G-content. These findings strengthen the viability of commercial alginate production from microbial sources.


Assuntos
Alginatos/metabolismo , Azotobacter vinelandii/metabolismo , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/metabolismo , Ácidos Hexurônicos/metabolismo , Azotobacter vinelandii/genética , Proteínas de Bactérias/genética , Carboidratos Epimerases/genética
20.
Infect Immun ; 87(4)2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30718288

RESUMO

In serogroup C Neisseria meningitidis, the cssA (siaA) gene codes for an UDP-N-acetylglucosamine 2-epimerase that catalyzes the conversion of UDP-N-acetyl-α-d-glucosamine into N-acetyl-d-mannosamine and UDP in the first step in sialic acid biosynthesis. This enzyme is required for the biosynthesis of the (α2→9)-linked polysialic acid capsule and for lipooligosaccharide (LOS) sialylation. In this study, we have used a reference serogroup C meningococcal strain and an isogenic cssA knockout mutant to investigate the pathogenetic role of surface-exposed sialic acids in a model of meningitis based on intracisternal inoculation of BALB/c mice. Results confirmed the key role of surface-exposed sialic acids in meningococcal pathogenesis. The 50% lethal dose (LD50) of the wild-type strain 93/4286 was about four orders of magnitude lower than that of the cssA mutant. Compared to the wild-type strain, the ability of this mutant to replicate in brain and spread systemically was severely impaired. Evaluation of brain damage evidenced a significant reduction in cerebral hemorrhages in mice infected with the mutant in comparison with the levels in those challenged with the wild-type strain. Histological analysis showed the typical features of bacterial meningitis, including inflammatory cells in the subarachnoid, perivascular, and ventricular spaces especially in animals infected with the wild type. Noticeably, 80% of mice infected with the wild-type strain presented with massive bacterial localization and accompanying inflammatory infiltrate in the corpus callosum, indicating high tropism of meningococci exposing sialic acids toward this brain structure and a specific involvement of the corpus callosum in the mouse model of meningococcal meningitis.


Assuntos
Proteínas de Bactérias/genética , Meningite Meningocócica/microbiologia , Ácido N-Acetilneuramínico/metabolismo , Neisseria meningitidis Sorogrupo C/patogenicidade , Animais , Proteínas de Bactérias/metabolismo , Encéfalo/microbiologia , Encéfalo/patologia , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Modelos Animais de Doenças , Feminino , Técnicas de Inativação de Genes , Humanos , Meningite Meningocócica/mortalidade , Meningite Meningocócica/patologia , Camundongos , Camundongos Endogâmicos BALB C , Neisseria meningitidis Sorogrupo C/genética , Neisseria meningitidis Sorogrupo C/metabolismo , Virulência
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