RESUMO
A recombinant L-rhamnose isomerase (L-RhI) from probiotic Lactobacillus rhamnosus Probio-M9 (L. rhamnosus Probio-M9) was expressed. L. rhamnosus Probio-M9 was isolated from human colostrum and identified as a probiotic lactic acid bacterium, which can grow using L-rhamnose. L-RhI is one of the enzymes involved in L-rhamnose metabolism and catalyzes the reversible isomerization between L-rhamnose and L-rhamnulose. Some L-RhIs were reported to catalyze isomerization not only between L-rhamnose and L-rhamnulose but also between D-allulose and D-allose, which are known as rare sugars. Those L-RhIs are attractive enzymes for rare sugar production and have the potential to be further improved by enzyme engineering; however, the known crystal structures of L-RhIs recognizing rare sugars are limited. In addition, the optimum pH levels of most reported L-RhIs are basic rather than neutral, and such a basic condition causes non-enzymatic aldose-ketose isomerization, resulting in unexpected by-products. Herein, we report the crystal structures of L. rhamnosus Probio-M9 L-RhI (LrL-RhI) in complexes with L-rhamnose, D-allulose, and D-allose, which show enzyme activity toward L-rhamnose, D-allulose, and D-allose in acidic conditions, though the activity toward D-allose was low. In the complex with L-rhamnose, L-rhamnopyranose was found in the catalytic site, showing favorable recognition for catalysis. In the complex with D-allulose, D-allulofuranose and ring-opened D-allulose were observed in the catalytic site. However, bound D-allose in the pyranose form was found in the catalytic site of the complex with D-allose, which was unfavorable for recognition, like an inhibition mode. The structure of the complex may explain the low activity toward D-allose. KEY POINTS: ⢠Crystal structures of LrL-RhI in complexes with substrates were determined. ⢠LrL-RhI exhibits enzyme activity toward L-rhamnose, D-allulose, and D-allose. ⢠The LrL-RhI is active in acidic conditions.
Assuntos
Aldose-Cetose Isomerases , Lacticaseibacillus rhamnosus , Humanos , Raios X , Ramnose , MonossacarídeosRESUMO
Recent studies have shown that D-allose, a rare sugar, elicits antitumor effects on different types of solid cancers, such as hepatocellular carcinoma, non-small-cell lung cancer, and squamous cell carcinoma of the head and neck. In this study, we examined the effects of D-allose on the proliferation of human glioblastoma (GBM) cell lines (i.e., U251MG and U87MG) in vitro and in vivo and the underlying mechanisms. D-allose treatment inhibited the proliferation of U251MG and U87MG cells in a dose-dependent manner (3-50 mM). However, D-allose treatment did not affect cell cycles or apoptosis in these cells but significantly decreased the cell division frequency in both GBM cell lines. In a subcutaneous U87MG cell xenograft model, intraperitoneal injection of D-allose (100 mg/kg/day) significantly reduced the tumor volume in 28 days. These data indicate that D-allose-induced reduction in cell proliferation is associated with a subsequent decrease in the number of cell divisions, independent of cell-cycle arrest and apoptosis. Thus, D-allose could be an attractive additive to therapeutic strategies for GBM.
Assuntos
Carcinoma Pulmonar de Células não Pequenas , Glioblastoma , Neoplasias Hepáticas , Neoplasias Pulmonares , Humanos , Glioblastoma/tratamento farmacológico , Proliferação de Células , Glucose/metabolismo , Divisão Celular , Apoptose , Linhagem Celular TumoralRESUMO
Allitol is a hexitol produced by reducing the rare sugar D-allulose with a metal catalyst under hydrogen gas. To confirm the safe level of allitol, we conducted a series of safety assessments. From the results of Ames mutagenicity assay using Salmonella typhimurium strains TA98, TA100, TA1535, and TA1537, Escherichia coli strain WP2uvrA, and an in vitro chromosomal aberration test on cultured Chinese hamster cells, allitol did not show any significant genotoxic effect. No significant effects on general condition, urinalysis, hematology, physiology, histopathology, or at necropsy were observed at a dose of 1500 mg/kg body weight of allitol in the acute and 90-day subchronic oral-toxicity assessments for rats. A further study performed on healthy adult humans showed that the acute use level of allitol for diarrhea was 0.2 g/kg body weight for both men and women. The results of current safety assessment studies suggest that allitol is safe for human consumption.
Assuntos
Aberrações Cromossômicas , Escherichia coli , Masculino , Cricetinae , Ratos , Humanos , Feminino , Animais , Ratos Sprague-Dawley , Testes de Mutagenicidade/métodos , Cricetulus , Escherichia coli/genética , Peso Corporal , Ingestão de AlimentosRESUMO
d-Aldotetroses are rare sugars that are obtained via chemical synthesis in low yield. In this study, we demonstrated that d-aldotetroses could be produced using 3 isomerases. First, l-erythrulose was epimerized using d-tagatose 3-epimerase from Pseudomonas cichorii ST-24. The specific optical rotation of the reaction solution gradually decreased to zero, indicating that approximately 50% of the l-erythrulose was converted to d-erythrulose. d, l-Erythrulose mixture was isomerized with d-arabinose isomerase from Klebsiella pneumoniae 40bXX to produce d-threose, resulting in a conversion rate of 9.35%. d-Erythrose production using l-rhamnose isomerase from Pseudomonas stutzeri LL172 resulted in a conversion rate of 12.9%. Because of the low purity of the purchased d-erythrose, the product was reduced by the Raney nickel catalyst compared with authentic erythritol. We confirmed the products using HPLC and 13C-NMR spectra. This is the first report of d-aldotetrose production using an enzymatic reaction.
Assuntos
Aldose-Cetose Isomerases , Tetroses , Hexoses , Isomerases , Racemases e EpimerasesRESUMO
D-Allulose is a rare sugar that exists in nature. It is a food ingredient with nearly zero calories (<0.4 kcal/g) and has many physiological functionalities such as attenuation of postprandial blood glucose levels, attenuation of postprandial fat mass accumulation, and anti-aging property. This study focused on the postprandial blood glucose changes in healthy humans by a systematic review and meta-analysis. They were chosen because of its importance to a prevention from diabetes. The study objective was to examine acute blood glucose concentrations of healthy humans after the meal with and without allulose. The study collected all D-allulose related studies from various databases. A forest plot of the comparison between an allulose intake group and the control group showed both 5g and 10g intake groups have the significantly smaller area under the curve of postprandial blood glucose levels. It means that D-Allulose attenuates postprandial blood glucose concentrations in healthy humans. As the result, D-Allulose is a valuable blood glucose management tool for healthy humans and diabetes patients. Allulose Diet enables reduction of sucrose intake through Sugar Reformulation in the future diet.
Assuntos
Glicemia , Frutose , Humanos , DietaRESUMO
Transketolase is a key enzyme in the pentose phosphate pathway in all organisms, recognizing sugar phosphates as substrates. Transketolase with a cofactor of thiamine pyrophosphate catalyzes the transfer of a 2-carbon unit from D-xylulose-5-phosphate to D-ribose-5-phosphate (5-carbon aldose), giving D-sedoheptulose-7-phosphate (7-carbon ketose). Transketolases can also recognize non-phosphorylated monosaccharides as substrates, and catalyze the formation of non-phosphorylated 7-carbon ketose (heptulose), which has attracted pharmaceutical attention as an inhibitor of sugar metabolism. Here, we report the structural and biochemical characterizations of transketolase from Thermus thermophilus HB8 (TtTK), a well-characterized thermophilic Gram-negative bacterium. TtTK showed marked thermostability with maximum enzyme activity at 85 °C, and efficiently catalyzed the formation of heptuloses from lithium hydroxypyruvate and four aldopentoses: D-ribose, L-lyxose, L-arabinose, and D-xylose. The X-ray structure showed that TtTK tightly forms a homodimer with more interactions between subunits compared with transketolase from other organisms, contributing to its thermal stability. A modeling study based on X-ray structures suggested that D-ribose and L-lyxose could bind to the catalytic site of TtTK to form favorable hydrogen bonds with the enzyme, explaining the high conversion rates of 41% (D-ribose) and 43% (L-lyxose) to heptulose. These results demonstrate the potential of TtTK as an enzyme producing a rare sugar of heptulose. KEY POINTS: ⢠Transketolase catalyzes the formation of a 7-carbon sugar phosphate ⢠Structural and biochemical characterizations of thermophilic transketolase were done ⢠The enzyme could produce non-phosphorylated 7-carbon ketoses from sugars.
Assuntos
Thermus thermophilus , Transcetolase , Transcetolase/química , Transcetolase/metabolismo , Ribose , Monossacarídeos , Fosfatos , Cetoses , CarbonoRESUMO
D-allose is a rare sugar that has been reported to up-regulate thioredoxin-interacting protein (TXNIP) expression and affect the production of intracellular reactive oxygen species (ROS). However, the antitumor effect of D-allose is unknown. This study aimed to determine whether orally administered D-allose could be a candidate drug against bladder cancer (BC). To this end, BC cell lines were treated with varying concentrations of D-allose (10, 25, and 50 mM). Cell viability and intracellular ROS levels were assessed using cell viability assay and flow cytometry. TXNIP expression was evaluated using Western blotting. The antitumor effect of orally administered D-allose was assessed using a xenograft mouse model. D-allose reduced cell viability and induced intracellular ROS production in BC cells. Moreover, D-allose stimulated TXNIP expression in a dose-dependent manner. Co-treatment of D-allose and the antioxidant L-glutathione canceled the D-allose-induced reduction in cell viability and intracellular ROS elevation. Furthermore, oral administration of D-allose inhibited tumor growth without adverse effects (p < 0.05). Histopathological findings in tumor tissues showed that D-allose decreased the nuclear fission rate from 4.1 to 1.1% (p = 0.004). Oral administration of D-allose suppressed BC growth in a preclinical mouse model, possibly through up-regulation of TXNIP expression followed by an increase in intracellular ROS. Therefore, D-allose is a potential therapeutic compound for the treatment of BC.
Assuntos
Açúcares , Neoplasias da Bexiga Urinária , Animais , Linhagem Celular Tumoral , Glucose/metabolismo , Humanos , Camundongos , Espécies Reativas de Oxigênio , Neoplasias da Bexiga Urinária/tratamento farmacológicoRESUMO
Some rare sugars can be potently medicinal, such as l-gulose. In this study, we present a novel alditol oxidase (fAldOx) from the soil fungus Penicillium sp. KU-1, and its application for the effective production of l-gulose. To the best of our knowledge, this is the first report of a successful direct conversion of d-sorbitol to l-gulose. We further purified it to homogeneity with a â¼108-fold purification and an overall yield of 3.26%, and also determined the effectors of fAldOx. The enzyme possessed broad substrate specificity for alditols such as erythritol (kcat/KM, 355 m-1 s-1), thus implying that the effective production of multiple rare sugars for medicinal applications may be possible.
Assuntos
Oxirredutases do Álcool/metabolismo , Proteínas Fúngicas/metabolismo , Hexoses/química , Penicillium/enzimologia , Sorbitol/metabolismo , Álcoois Açúcares/metabolismo , Açúcares/química , Oxirredutases do Álcool/química , Bioengenharia , Proteínas Fúngicas/química , Hexoses/metabolismo , Especificidade por Substrato , Açúcares/metabolismoRESUMO
We found that l-gulose, a rare sugar, was produced from d-sorbitol efficiently, using a wheat-bran culture extract of the fungus Penicillium sp. KU-1 isolated from soil. The culture extract showed enzyme activity for the oxidation of d-sorbitol to produce l-gulose; a high production yield of approximately 94% was achieved.
Assuntos
Fibras na Dieta/metabolismo , Hexoses/biossíntese , Penicillium/metabolismo , Meios de Cultura , Fermentação , Sorbitol/metabolismoRESUMO
d-Allulose has potential as a low-calorie sweetener which can suppress fat accumulation. Several enzymes capable of d-allulose production have been isolated, including d-tagatose 3-epimerases. Here, we report the isolation of a novel protein from Methylomonas sp. expected to be a putative enzyme based on sequence similarity to ketose 3-epimerase. The synthesized gene encoding the deduced ketose 3-epimerase was expressed as a recombinant enzyme in Escherichia coli, and it exhibited the highest enzymatic activity toward l-ribulose, followed by d-ribulose and d-allulose. The X-ray structure analysis of l-ribulose 3-epimerase from Methylomonas sp. (MetLRE) revealed a homodimeric enzyme, the first reported structure of dimeric l-ribulose 3-epimerase. The monomeric structure of MetLRE is similar to that of homotetrameric l-ribulose 3-epimerases, but the short C-terminal α-helix of MetLRE is unique and different from those of known l-ribulose 3 epimerases. The length of the C-terminal α-helix was thought to be involved in tetramerization and increasing stability; however, the addition of residues to MetLRE at the C terminus did not lead to tetramer formation. MetLRE is the first dimeric l-ribulose 3-epimerase identified to exhibit high relative activity toward d-allulose.
Assuntos
Methylomonas/enzimologia , Pentoses/química , Racemases e Epimerases/química , Cristalografia por Raios X , Modelos Moleculares , Pentoses/metabolismo , Racemases e Epimerases/metabolismoRESUMO
The rare sugar D-tagatose is a safe natural product used as a commercial food ingredient. Here, we show that D-tagatose controls a wide range of plant diseases and focus on downy mildews to analyze its mode of action. It likely acts directly on the pathogen, rather than as a plant defense activator. Synthesis of mannan and related products of D-mannose metabolism are essential for development of fungi and oomycetes; D-tagatose inhibits the first step of mannose metabolism, the phosphorylation of D-fructose to D-fructose 6-phosphate by fructokinase, and also produces D-tagatose 6-phosphate. D-Tagatose 6-phosphate sequentially inhibits phosphomannose isomerase, causing a reduction in D-glucose 6-phosphate and D-fructose 6-phosphate, common substrates for glycolysis, and in D-mannose 6-phosphate, needed to synthesize mannan and related products. These chain-inhibitory effects on metabolic steps are significant enough to block initial infection and structural development needed for reproduction such as conidiophore and conidiospore formation of downy mildew.
Assuntos
Fungos/efeitos dos fármacos , Hexoses/farmacologia , Doenças das Plantas/prevenção & controle , Substâncias Protetoras/farmacologia , Agroquímicos/química , Agroquímicos/farmacologia , Fungos/patogenicidade , Fungicidas Industriais/química , Fungicidas Industriais/farmacologia , Hexosefosfatos/genética , Hexoses/química , Fosforilação/efeitos dos fármacos , Doenças das Plantas/microbiologiaRESUMO
Host cell surface glycans play critical roles in influenza virus A (IVA) infection ranging from modulation of IVA attachment to membrane fusion and host tropism. Approaches for quick and sensitive profile of viral avidity toward a specific type of host cell glycan can contribute to the understanding of tropism switching among different IVA strains. Here, we developed a method based on chemoenzymatic glycan engineering to investigate the possible involvement of α1-2-fucosides in IVA infections. Using a truncated human fucosyltransferase 1 (hFUT1), we created α1-2-fucosides in situ on host cells to assess their influence on the host cell binding to IVA hemagglutinin and the susceptibility of host cells toward IVA-induced killing. We discovered that the newly created α1-2-fucosides on host cells enhanced the infection of several human pandemic IVA subtypes either directly or indirectly. These findings suggest that glycan epitopes other than sialic acid should also be considered for assessing the human pandemic risk of this viral pathogen.
Assuntos
Fucose/química , Fucosiltransferases/química , Glicosídeos/metabolismo , Vírus da Influenza A/patogenicidade , Influenza Humana/etiologia , Técnicas Microbiológicas/métodos , Animais , Células CHO , Engenharia Celular , Sobrevivência Celular , Cricetulus , Cães , Epitopos/química , Epitopos/metabolismo , Glicosídeos/química , Glicoproteínas de Hemaglutininação de Vírus da Influenza/metabolismo , Interações Hospedeiro-Patógeno , Humanos , Vírus da Influenza A/metabolismo , Vírus da Influenza A/fisiologia , Células Madin Darby de Rim Canino , Tropismo Viral , Galactosídeo 2-alfa-L-FucosiltransferaseRESUMO
A practical synthesis of the very rare sugar d-idose and the stable building blocks for d-idose, d-iduronic, and d-idonic acids from ido-heptonic acid requires only isopropylidene protection, Shing silica gel-supported periodate cleavage of the C6-C7 bond of the heptonic acid, and selective reduction of C1 and/or C6. d-Idose is the most unstable of all the aldohexoses and a stable precursor which be stored and then converted under very mild conditions into d-idose is easily prepared.
Assuntos
Hexoses/síntese química , Ácido Idurônico/síntese química , Açúcares Ácidos/síntese química , Configuração de Carboidratos , Glucose/química , Heptoses/química , Hexoses/química , Ácido Idurônico/química , Estrutura Molecular , Açúcares Ácidos/químicaRESUMO
The biological activities of deoxy sugars (deoxy monosaccharides) have remained largely unstudied until recently. We compared the growth inhibition by all 1-deoxyketohexoses using the animal model Caenorhabditis elegans. Among the eight stereoisomers, 1-deoxy-d-allulose (1d-d-Alu) showed particularly strong growth inhibition. The 50% inhibition of growth (GI50) concentration by 1d-d-Alu was estimated to be 5.4â¯mM, which is approximately 10 times lower than that of d-allulose (52.7â¯mM), and even lower than that of the potent glycolytic inhibitor, 2-deoxy-d-glucose (19.5â¯mM), implying that 1d-d-Alu has a strong growth inhibition. In contrast, 5-deoxy- and 6-deoxy-d-allulose showed no growth inhibition of C. elegans. The inhibition by 1d-d-Alu was alleviated by the addition of d-ribose or d-fructose. Our findings suggest that 1d-d-Alu-mediated growth inhibition could be induced by the imbalance in d-ribose metabolism. To our knowledge, this is the first report of biological activity of 1d-d-Alu which may be considered as an antimetabolite drug candidate.
Assuntos
Caenorhabditis elegans/crescimento & desenvolvimento , Frutose/química , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/metabolismo , Animais , Tamanho Corporal/efeitos dos fármacos , Caenorhabditis elegans/efeitos dos fármacos , Desoxiaçúcares/química , Desoxiaçúcares/farmacologia , Desoxiglucose/farmacologia , Sinergismo Farmacológico , Frutose/farmacologia , Ribose/farmacologia , EstereoisomerismoRESUMO
D-Allose (D-All), C-3 epimer of D-glucose, is a rare sugar known to suppress reactive oxygen species generation and prevent hypertension. We previously reported that D-allulose, a structural isomer of D-All, prolongs the lifespan of the nematode Caenorhabditis elegans. Thus, D-All was predicted to affect longevity. In this study, we provide the first empirical evidence that D-All extends the lifespan of C. elegans. Lifespan assays revealed that a lifespan extension was induced by 28 mM D-All. In particular, a lifespan extension of 23.8 % was achieved (p < 0.0001). We further revealed that the effects of D-All on lifespan were dependent on the insulin gene daf-16 and the longevity gene sir-2.1, indicating a distinct mechanism from those of other hexoses, such as D-allulose, with previously reported antiaging effects.
RESUMO
The X-ray structure of ketose 3-epimerase from Arthrobacter globiformis M30, which was previously reported to be a D-allulose 3-epimerase (AgD-AE), was determined at 1.96â Å resolution. The crystal belonged to the hexagonal space group P6522, with unit-cell parameters a = b = 103.98, c = 256.53â Å. The structure was solved by molecular replacement using the structure of Mesorhizobium loti L-ribulose 3-epimerase (MlL-RE), which has 41% sequence identity, as a search model. A hexagonal crystal contained two molecules in the asymmetric unit, and AgD-AE formed a homotetramer with twofold symmetry. The overall structure of AgD-AE was more similar to that of MlL-RE than to the known structures of D-psicose (alternative name D-allulose) 3-epimerases (D-PEs or D-AEs), although AgD-AE and MlL-RE have different substrate specificities. Both AgD-AE and MlL-RE have long helices in the C-terminal region that would contribute to the stability of the homotetramer. AgD-AE showed higher enzymatic activity for L-ribulose than D-allulose; however, AgD-AE is stable and is a unique useful enzyme for the production of D-allulose from D-fructose.
Assuntos
Arthrobacter/química , Proteínas de Bactérias/química , Carboidratos Epimerases/química , Frutose/química , Cetoses/química , Sequência de Aminoácidos , Arthrobacter/enzimologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Carboidratos Epimerases/genética , Carboidratos Epimerases/metabolismo , Domínio Catalítico , Clonagem Molecular , Cristalografia por Raios X , Escherichia coli/genética , Escherichia coli/metabolismo , Frutose/metabolismo , Expressão Gênica , Vetores Genéticos/química , Vetores Genéticos/metabolismo , Cetoses/metabolismo , Mesorhizobium/química , Mesorhizobium/enzimologia , Modelos Moleculares , Pentoses/química , Pentoses/metabolismo , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Multimerização Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alinhamento de Sequência , Homologia Estrutural de Proteína , Especificidade por SubstratoRESUMO
Dietary restriction (DR) is an effective intervention known to increase lifespan in a wide variety of organisms. DR also delays the onset of aging-associated diseases. DR mimetics, compounds that can mimic the effects of DR, have been intensively explored. d-Allulose (d-Alu), the C3-epimer of d-fructose, is a rare sugar that has various health benefits, including anti-hyperglycemia and anti-obesity effects. Here, we report that d-Alu increased the lifespan of Caenorhabditis elegans both under monoxenic and axenic culture conditions. d-Alu did not further extend the lifespan of the long-lived DR model eat-2 mutant, strongly indicating that the effect is related to DR. However, d-Alu did not reduce the food intake of wild-type C. elegans. To explore the mechanisms of the d-Alu longevity effect, we examined the lifespan of d-Alu-treated mutants deficient for nutrient sensing pathway-related genes daf-16, sir-2.1, aak-2, and skn-1. As a result, d-Alu increased the lifespan of the daf-16, sir-2.1, and skn-1 mutants, but not the aak-2 mutant, indicating that the lifespan extension was dependent on the energy sensor, AMP-activated protein kinase (AMPK). d-Alu also enhanced the mRNA expression and enzyme activities of superoxide dismutase (SOD) and catalase. From these findings, we conclude that d-Alu extends lifespan by increasing oxidative stress resistance through a DR mechanism, making it a candidate DR mimetic.
Assuntos
Caenorhabditis elegans/efeitos dos fármacos , Restrição Calórica/métodos , Frutose/farmacologia , Proteínas Quinases Ativadas por AMP/metabolismo , Animais , Materiais Biomiméticos/química , Materiais Biomiméticos/farmacologia , Caenorhabditis elegans/genética , Caenorhabditis elegans/fisiologia , Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Catalase/genética , Catalase/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Ingestão de Alimentos/genética , Ingestão de Alimentos/fisiologia , Frutose/química , Genes de Helmintos , Longevidade/efeitos dos fármacos , Longevidade/genética , Longevidade/fisiologia , Mutação , Estresse Oxidativo/efeitos dos fármacos , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Receptores Nicotínicos/genética , Receptores Nicotínicos/metabolismo , Estereoisomerismo , Superóxido Dismutase/genética , Superóxido Dismutase/metabolismoRESUMO
An enzyme that catalyzes C-3 epimerization between d-fructose and d-allulose was found in Arthrobacter globiformis strain M30. Arthrobacter species have long been used in the food industry and are well-known for their high degree of safety. The enzyme was purified by ion exchange and hydrophobic interaction chromatographies and characterized as a d-allulose 3-epimerase (d-AE). The molecular weight of the purified enzyme was estimated to be 128 kDa with four identical subunits. The enzyme showed maximal activity and thermostability in the presence of Mg2+. The optimal pH and temperature for enzymatic activity were 7.0-8.0 and 70°C, respectively. The enzyme was immobilized to ion exchange resin whereupon it was stable for longer periods than the free enzyme when stored at below 10°C. In the column reaction, the enzyme activity also maintained stability for more than 4 months. Under these conditions, 215 kg of d-allulose produced per liter immobilized enzyme, and this was the highest production yield of d-allulose reported so far. These highly stable properties suggest that this enzyme represents an ideal candidate for the industrial production of d-allulose.
Assuntos
Arthrobacter/enzimologia , Frutose/metabolismo , Racemases e Epimerases/análise , Racemases e Epimerases/isolamento & purificação , Racemases e Epimerases/metabolismo , Arthrobacter/química , Estabilidade Enzimática , Frutose/biossíntese , Concentração de Íons de Hidrogênio , Cinética , Engenharia Metabólica , Peso Molecular , TemperaturaRESUMO
Ease of separation of petrol-soluble acetonides derived from the triacetonide of methyl glucoheptonate allows scalable syntheses of rare sugars containing the l-gluco or d-gulo structural motif with any oxidation level at the C6 or C1 position of the hexose, usually without chromatography: meso-d-glycero-d-guloheptitol available in two steps is an ideal entry point for the study of the biotechnological production of heptoses.
Assuntos
Hexoses/síntese química , Açúcares Ácidos/química , Açúcares/síntese química , Hexoses/química , Conformação Molecular , Estereoisomerismo , Açúcares/químicaRESUMO
Pseudomonas cichorii D-tagatose 3-epimerase (PcDTE), which has a broad substrate specificity, efficiently catalyzes the epimerization of not only D-tagatose to D-sorbose but also D-fructose to D-psicose (D-allulose) and also recognizes the deoxy sugars as substrates. In an attempt to elucidate the substrate recognition and catalytic reaction mechanisms of PcDTE for deoxy sugars, the X-ray structures of the PcDTE mutant form with the replacement of Cys66 by Ser (PcDTE_C66S) in complexes with deoxy sugars were determined. These X-ray structures showed that substrate recognition by the enzyme at the 1-, 2-, and 3-positions is responsible for enzymatic activity and that substrate-enzyme interactions at the 4-, 5-, and 6-positions are not essential for the catalytic reaction of the enzyme leading to the broad substrate specificity of PcDTE. They also showed that the epimerization site of 1-deoxy 3-keto D-galactitol is shifted from C3 to C4 and that 1-deoxy sugars may bind to the catalytic site in the inhibitor-binding mode. The hydrophobic groove that acts as an accessible surface for substrate binding is formed through the dimerization of PcDTE. In PcDTE_C66S/deoxy sugar complex structures, bound ligand molecules in both the linear and ring forms were detected in the hydrophobic groove, while bound ligand molecules in the catalytic site were in the linear form. This result suggests that the sugar-ring opening of a substrate may occur in the hydrophobic groove and also that the narrow channel of the passageway to the catalytic site allows a substrate in the linear form to pass through.