RESUMEN
Cellulases from GH9 family show endo-, exo- or processive endocellulase activity, but the reason behind the variation is unclear. A GH9 recombinant endoglucanase, AtGH9C-CBM3A-CBM3B from Acetivibrio thermocellus was structurally characterized for conformation, binding and dynamics assessment. Modeled AtGH9C-CBM3A-CBM3B depicted (α/α)6-barrel structure with Asp98, Asp101 and Glu489 acting as catalytic triad. CD results revealed 25.2 % α-helix, 18.4 % ß-sheet and rest 56.4 % of random coils, corroborating with predictions from PSIPRED and SOPMA. MD simulation of AtGH9C-CBM3A-CBM3B bound cellotetraose showed structural stability and global compactness with lowered RMSD values (1.5 nm) as compared with only AtGH9C-CBM3A-CBM3B (1.8 nm) for 200 ns. Higher fluctuation in RMSF values in far-positioned CBM3B pointed to its redundancy in substrate binding. Docking studies showed maximum binding with cellotetraose (ΔG = -5.05 kcal/mol), with reduced affinity towards ligands with degree of polymerization (DP) lower (DP < 4) or higher than 4 (DP > 4). Processivity index displayed the enzyme to be processive with loop 3 (342-379 aa) possibly blocking the non-reducing end of cellulose chain, resulting in cellotetraose release. SAXS analysis of AtGH9C-CBM3A-CBM3B at 5 mg/mL displayed monodispersed state with fist-and-elbow shape in solution. Negative zeta potential of -24 mV at 5 mg/mL indicated stability and free from aggregation.
Asunto(s)
Celulasa , Simulación de Dinámica Molecular , Unión Proteica , Proteínas Recombinantes , Celulasa/química , Celulasa/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/genética , Especificidad por Sustrato , Tetrosas/metabolismo , Tetrosas/química , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Celulosa/análogos & derivadosRESUMEN
Glycoside hydrolase family 5 (GH5) encompasses enzymes with several different activities, including endo-1,4-ß-mannosidases. These enzymes are involved in mannan degradation, and have a number of biotechnological applications, such as mannooligosaccharide prebiotics production, stain removal and dyes decolorization, to name a few. Despite the importance of GH5 enzymes, only a few members of subfamily 7 were structurally characterized. In the present work, biochemical and structural characterization of Bacillus licheniformis GH5 mannanase, BlMan5_7 were performed and the enzyme cleavage pattern was analyzed, showing that BlMan5_7 requires at least 5 occupied subsites to perform efficient hydrolysis. Additionally, crystallographic structure at 1.3 Å resolution was determined and mannoheptaose (M7) was docked into the active site to investigate the interactions between substrate and enzyme through molecular dynamic (MD) simulations, revealing the existence of a - 4 subsite, which might explain the generation of mannotetraose (M4) as an enzyme product. Biotechnological application of the enzyme in stain removal was investigated, demonstrating that BlMan5_7 addition to washing solution greatly improves mannan-based stain elimination.
Asunto(s)
Bacillus licheniformis , Dominio Catalítico , Mutagénesis Sitio-Dirigida , Bacillus licheniformis/enzimología , Bacillus licheniformis/genética , Cristalografía por Rayos X , Simulación de Dinámica Molecular , Manosidasas/química , Manosidasas/genética , Manosidasas/metabolismo , Especificidad por Sustrato , Hidrólisis , Tetrosas/química , Tetrosas/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Conformación Proteica , Mananos/química , Mananos/metabolismo , beta-Manosidasa/química , beta-Manosidasa/genética , beta-Manosidasa/metabolismo , Modelos Moleculares , Simulación del Acoplamiento Molecular , OligosacáridosRESUMEN
Glioblastoma (GBM) is the most frequent brain cancer and more lethal than other cancers. Characteristics of this cancer are its high drug resistance, high recurrence rate and invasiveness. Invasiveness in GBM is related to overexpression of matrix metalloproteinases (MMPs) which are mediated by wnt/ß-catenin and induced by the activation of signaling pathways extracellularly activated by the cytokine neuroleukin (NLK) in cancer stem cells (CSC). Therefore, in this work we evaluated the effect of the tetrose saccharide, erythrose (Ery), a NLK inhibitor of invasiveness and drug sensitization in glioblastoma stem cells (GSC). GSC were obtained from parental U373 cell line by a CSC phenotype enrichment protocol based on microenvironmental stress conditions such as hypoxia, hipoglycemia, drug exposition and serum starvation. Enriched fraction of GSC overexpressed the typical markers of brain CSC: low CD133+ and high CD44; in addition, epithelial to mesenchyme transition (EMT) markers and MMPs were increased several times in GSC vs. U373 correlating with higher invasiveness, elongated and tubular mitochondrion and temozolomide (TMZ) resistance. IC50 of Ery was found at nM concentration and at 24 h induced a severe diminution of EMT markers, MMPs and invasiveness in GSC. Furthermore, the phosphorylation pattern of NLK after Ery exposition also was affected. In addition, when Ery was administered to GSC at subIC50, it was capable of reverting TMZ resistance at concentrations innocuous to non-tumor cancer cells. Moreover, Ery added daily induced the death of all GSC. Those findings indicated that the phytodrug Ery could be used as adjuvant therapy in GBM.
Asunto(s)
Neoplasias Encefálicas , Glioblastoma , Humanos , Glioblastoma/genética , Tetrosas/metabolismo , Tetrosas/farmacología , Tetrosas/uso terapéutico , Línea Celular Tumoral , Temozolomida/uso terapéutico , Resistencia a Antineoplásicos , Neoplasias Encefálicas/patología , Células Madre Neoplásicas/patología , Proteínas Serina-Treonina Quinasas/metabolismoRESUMEN
Expanding the chemical space of evolvable non-natural genetic polymers (XNAs) to include functional groups that enhance protein target binding affinity offers a promising route to therapeutic aptamers with high biological stability. Here we describe the chemical synthesis and polymerase recognition of 10 chemically diverse functional groups introduced at the C-5 position of α-l-threofuranosyl uridine nucleoside triphosphate (tUTP). We show that the set of tUTP substrates is universally recognized by the laboratory-evolved polymerase Kod-RSGA. Insights into the mechanism of TNA synthesis were obtained from a high-resolution X-ray crystal structure of the postcatalytic complex bound to the primer-template duplex. A structural analysis reveals a large cavity in the enzyme active site that can accommodate the side chain of C-5-modified tUTP substrates. Our findings expand the chemical space of evolvable nucleic acid systems by providing a synthetic route to artificial genetic polymers that are uniformly modified with diversity-enhancing functional groups.
Asunto(s)
ADN Polimerasa Dirigida por ADN , Tetrosas , Uridina Trifosfato , Proteínas Arqueales/química , Proteínas Arqueales/metabolismo , Cristalografía por Rayos X , ADN Polimerasa Dirigida por ADN/química , ADN Polimerasa Dirigida por ADN/metabolismo , Nucleósidos/química , Unión Proteica , Tetrosas/síntesis química , Tetrosas/química , Tetrosas/metabolismo , Thermococcus/enzimología , Uridina Trifosfato/análogos & derivados , Uridina Trifosfato/síntesis química , Uridina Trifosfato/metabolismoRESUMEN
Threose nucleic acid (TNA) has been considered a potential RNA progenitor in evolution due to its chemical simplicity and base pairing property. Catalytic TNA sequences with RNA ligase activities might have facilitated the transition to the RNA world. Here we report the isolation of RNA ligase TNA enzymes by in vitro selection. The identified TNA enzyme T8-6 catalyzes the formation of a 2'-5' phosphoester bond between a 2',3'-diol and a 5'-triphosphate group, with a kobs of 1.1 × 10-2 min-1 (40 mM Mg2+, pH 9.0). For efficient reaction, T8-6 requires UA|GA at the ligation junction and tolerates variations at other substrate positions. Functional RNAs such as hammerhead ribozyme can be prepared by T8-6-catalyzed ligation, with site-specific introduction of a 2'-5' linkage. Together, this work provides experimental support for TNA as a plausible pre-RNA genetic polymer and also offers an alternative molecular tool for biotechnology.
Asunto(s)
Ácidos Nucleicos/metabolismo , ARN Ligasa (ATP)/metabolismo , Tetrosas/metabolismo , Conformación de Ácido Nucleico , Ácidos Nucleicos/química , ARN Ligasa (ATP)/química , Tetrosas/químicaRESUMEN
Exploring new multifunctional enzymes and understanding the mechanisms of catalytic promiscuity will be of enormous industrial and academic values. In the present study, we reported the discovery and characterization of a multifunctional enzyme BSGH13 from Bacillus subtilis BS-5. Remarkably, BSGH13 possessed α-amylase, endoglucanase, and xylanase activities. To our knowledge, this was the first report on an amylase from Bacillus species having additional endoglucanase and xylanase activities. Subsequently, we analyzed the effects of aromatic residues substitution at each site of the active site architecture on ligand-binding affinity and catalytic specificity of BSGH13 by a combination of virtual mutation and site-directed mutagenesis approaches. Our results indicated that the introduction of aromatic amino acids Phe or Trp at the positions L182 and L183 altered the local interaction network of BSGH13 towards different substrates, thus changing the multifunctional properties of BSGH13. Moreover, we provided an expanded perspective on studies of multifunctional enzymes.
Asunto(s)
Bacillus subtilis/química , Proteínas Bacterianas/química , Celulasa/química , Endo-1,4-beta Xilanasas/química , alfa-Amilasas/química , Sustitución de Aminoácidos , Bacillus subtilis/enzimología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Celulasa/genética , Celulasa/metabolismo , Celulosa/análogos & derivados , Celulosa/química , Celulosa/metabolismo , Clonación Molecular , Endo-1,4-beta Xilanasas/genética , Endo-1,4-beta Xilanasas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Cinética , Maltosa/análogos & derivados , Maltosa/química , Maltosa/metabolismo , Modelos Moleculares , Mutación , Fenilalanina/química , Fenilalanina/genética , Fenilalanina/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homología Estructural de Proteína , Relación Estructura-Actividad , Especificidad por Sustrato , Tetrosas/química , Tetrosas/metabolismo , Triptófano/química , Triptófano/genética , Triptófano/metabolismo , Xilanos/química , Xilanos/metabolismo , alfa-Amilasas/genética , alfa-Amilasas/metabolismoRESUMEN
Proteins in the eye lens have negligible turnover and therefore progressively accumulate chemical modifications during aging. Carbonyls and oxidative stresses, which are intricately linked to one another, predominantly drive such modifications. Oxidative stress leads to the loss of glutathione (GSH) and ascorbate degradation; this in turn leads to the formation of highly reactive dicarbonyl compounds that react with proteins to form advanced glycation end products (AGEs). The formation of AGEs leads to the crosslinking and aggregation of proteins contributing to lens aging and cataract formation. To inhibit AGE formation, we developed a disulfide compound linking GSH diester and mercaptoethylguanidine, and we named it carboxitin. Bovine lens organ cultured with carboxitin showed higher levels of GSH and mercaptoethylguanidine in the lens nucleus. Carboxitin inhibited erythrulose-mediated mouse lens protein crosslinking, AGE formation and the formation of 3-deoxythreosone, a major ascorbate-derived AGE precursor in the human lens. Carboxitin inhibited the glycation-mediated increase in stiffness in organ-cultured mouse lenses measured using compressive mechanical strain. Delivery of carboxitin into the lens increases GSH levels, traps dicarbonyl compounds and inhibits AGE formation. These properties of carboxitin could be exploited to develop a therapy against the formation of AGEs and the increase in stiffness that causes presbyopia in aging lenses.
Asunto(s)
Glutatión/análogos & derivados , Glutatión/síntesis química , Cristalino/efectos de los fármacos , Animales , Bovinos , Productos Finales de Glicación Avanzada , Glicosilación , Cristalino/fisiología , Ratones , Ratones Endogámicos C57BL , Unión Proteica , Tetrosas/metabolismo , Células Tumorales CultivadasRESUMEN
In a recent paper, we showed the difference between the first stage of the one-substrate and the two-substrate transketolase reactions - the possibility of transfer of glycolaldehyde formed as a result of cleavage of the donor substrate from the thiazole ring of thiamine diphosphate to its aminopyrimidine ring through the tricycle formation stage, which is necessary for binding and splitting the second molecule of donor substrate [O.N. Solovjeva et al., The mechanism of a one-substrate transketolase reaction, Biosci. Rep. 40 (8) (2020) BSR20180246]. Here we show that under the action of the reducing agent a tricycle accumulates in a significant amount. Therefore, a significant decrease in the reaction rate of the one-substrate transketolase reaction compared to the two-substrate reaction is due to the stage of transferring the first glycolaldehyde molecule from the thiazole ring to the aminopyrimidine ring of thiamine diphosphate. Fragmentation of the four-carbon thiamine diphosphate derivatives showed that two glycolaldehyde molecules are bound to both coenzyme rings and the erythrulose molecule is bound to a thiazole ring. It was concluded that in the one-substrate reaction erythrulose is formed on the thiazole ring of thiamine diphosphate from two glycol aldehyde molecules linked to both thiamine diphosphate rings. The kinetic characteristics were determined for the two substrates, fructose 6-phosphate and glycolaldehyde.
Asunto(s)
Transcetolasa/química , Transcetolasa/metabolismo , Acetaldehído/análogos & derivados , Acetaldehído/química , Acetaldehído/metabolismo , Biocatálisis , Borohidruros/química , Coenzimas/metabolismo , Fructosafosfatos/química , Fructosafosfatos/metabolismo , Cinética , Saccharomyces cerevisiae/enzimología , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Espectrometría de Masa por Ionización de Electrospray , Especificidad por Sustrato , Tetrosas/metabolismo , Tiamina Pirofosfato/química , Tiamina Pirofosfato/metabolismoRESUMEN
Transketolase catalyzes the transfer of a glycolaldehyde residue from ketose (the donor substrate) to aldose (the acceptor substrate). In the absence of aldose, transketolase catalyzes a one-substrate reaction that involves only ketose. The mechanism of this reaction is unknown. Here, we show that hydroxypyruvate serves as a substrate for the one-substrate reaction and, as well as with the xylulose-5-phosphate, the reaction product is erythrulose rather than glycolaldehyde. The amount of erythrulose released into the medium is equimolar to a double amount of the transformed substrate. This could only be the case if the glycol aldehyde formed by conversion of the first ketose molecule (the product of the first half reaction) remains bound to the enzyme, waiting for condensation with the second molecule of glycol aldehyde. Using mass spectrometry of catalytic intermediates and their subsequent fragmentation, we show here that interaction of the holotransketolase with hydroxypyruvate results in the equiprobable binding of the active glycolaldehyde to the thiazole ring of thiamine diphosphate and to the amino group of its aminopyrimidine ring. We also show that these two loci can accommodate simultaneously two glycolaldehyde molecules. It explains well their condensation without release into the medium, which we have shown earlier.
Asunto(s)
Pentosafosfatos/metabolismo , Piruvatos/metabolismo , Proteínas de Saccharomyces cerevisiae/metabolismo , Saccharomyces cerevisiae/enzimología , Tetrosas/metabolismo , Transcetolasa/metabolismo , Sitios de Unión , Dominio Catalítico , Cinética , Simulación de Dinámica Molecular , Pentosafosfatos/química , Unión Proteica , Conformación Proteica , Piruvatos/química , Proteínas de Saccharomyces cerevisiae/química , Espectrometría de Masa por Ionización de Electrospray , Relación Estructura-Actividad , Especificidad por Sustrato , Espectrometría de Masas en Tándem , Tetrosas/química , Transcetolasa/químicaRESUMEN
In response to osmotic stress, the yeast Yarrowia lipolytica produces erythritol, a four-carbon sugar alcohol, from erythrose-P, an intermediate of the pentose phosphate pathway. Under non-stressing conditions (isotonic environment), the produced erythritol is subsequently recycled into erythrose-P that can feed the pentose phosphate pathway. Herein, gene YALI0F01584g was characterized as involved in the erythritol catabolic pathway. Several experimental evidences suggested that it encodes an erythrulose-1P isomerase that converts erythrulose-1P into erythrulose-4P. On the basis of our previous reports and results gathered in this study with genetically modified strains, including ΔYALI0F01584g and ΔYALI0F01628g disrupted mutants, the entire erythritol catabolic pathway has been characterized.
Asunto(s)
Eritritol/metabolismo , Proteínas Fúngicas/metabolismo , Fosfatos/metabolismo , Tetrosas/metabolismo , Yarrowia/metabolismo , Secuencia de Aminoácidos , Proteínas Fúngicas/genética , Homología de Secuencia , Yarrowia/genética , Yarrowia/crecimiento & desarrolloRESUMEN
The low-calorie sweetener erythritol is endogenously produced from glucose through the pentose phosphate pathway in humans. Erythritol is of medical interest because elevated plasma levels of this polyol are predictive for visceral adiposity gain and development of type 2 diabetes. However, the mechanisms behind these associations remain unknown because the erythritol biosynthesis pathway, particularly the enzyme catalyzing the final step of erythritol synthesis (reduction of erythrose to erythritol), is not characterized. In this study, we purified two enzymes from rabbit liver capable of catalyzing the conversion of erythrose to erythritol: alcohol dehydrogenase 1 (ADH1) and sorbitol dehydrogenase (SORD). Both recombinant human ADH1 and SORD reduce erythrose to erythritol, using NADPH as a co-factor, and cell culture studies indicate that this activity is primarily NADPH-dependent. We found that ADH1 variants vary markedly in both their affinity for erythrose and their catalytic capacity (turnover number). Interestingly, the recombinant protein produced from the ADH1B2 variant, common in Asian populations, is not active when NADPH is used as a co-factor in vitro We also confirmed SORD contributes to intracellular erythritol production in human A549 lung cancer cells, where ADH1 is minimally expressed. In summary, human ADH1 and SORD catalyze the conversion of erythrose to erythritol, pointing to novel roles for two dehydrogenase proteins in human glucose metabolism that may contribute to individual responses to diet. Proteomics data are available via ProteomeXchange with identifier PXD015178.
Asunto(s)
Alcohol Deshidrogenasa/metabolismo , Eritritol/biosíntesis , L-Iditol 2-Deshidrogenasa/metabolismo , Células A549 , Animales , Humanos , Hígado/enzimología , Hígado/metabolismo , Conejos , Tetrosas/metabolismoRESUMEN
Many ketoses or organic acids can be produced by membrane-associated oxidation with Gluconobacter oxydans. In this study, the oxidation of meso-erythritol to L-erythrulose was investigated with the strain G. oxydans 621HΔupp BP.8, a multideletion strain lacking the genes for eight membrane-bound dehydrogenases. First batch biotransformations with growing cells showed re-consumption of L-erythrulose by G. oxydans 621HΔupp BP.8 in contrast to resting cells. The batch biotransformation with 2.8 g L-1 resting cells of G. oxydans 621HΔupp BP.8 in a DO-controlled stirred-tank bioreactor resulted in 242 g L-1 L-erythrulose with a product yield of 99% (w/w) and a space-time yield of 10 g L-1 h-1. Reaction engineering studies showed substrate excess inhibition as well as product inhibition of G. oxydans 621HΔupp BP.8 in batch biotransformations. In order to overcome substrate inhibition, a continuous membrane bioreactor with full cell retention was applied for meso-erythritol oxidation with resting cells of G. oxydans 621HΔupp BP.8. At a mean hydraulic residence time of 2 h, a space-time yield of 27 g L-1 h-1 L-erythrulose was achieved without changing the product yield of 99% (w/w) resulting in a cell-specific product yield of up to 4.4 gP gX-1 in the steady state. The product concentration (54 g L-1 L-erythrulose) was reduced in the continuous biotransformation process compared with the batch process to avoid product inhibition.
Asunto(s)
Eritritol/metabolismo , Eliminación de Gen , Gluconobacter oxydans/genética , Gluconobacter oxydans/metabolismo , Ingeniería Metabólica/métodos , Tetrosas/metabolismo , Biotransformación , Gluconobacter oxydans/enzimología , Gluconobacter oxydans/crecimiento & desarrollo , Oxidación-Reducción , Oxidorreductasas/deficienciaRESUMEN
Enzymatic conversion of the most abundant renewable source of organic compounds, cellulose to fermentable sugars is attractive for production of green fuels and chemicals. The major component of industrial enzyme systems, cellobiohydrolase I from Hypocrea jecorina (Trichoderma reesei) (HjCel7A) processively splits disaccharide units from the reducing ends of tightly packed cellulose chains. HjCel7A consists of a catalytic domain (CD) and a carbohydrate-binding module (CBM) separated by a linker peptide. A tunnel-shaped substrate-binding site in the CD includes nine subsites for ß-d-glucose units, seven of which (-7 to -1) precede the catalytic center. Low catalytic activity of Cel7A is the bottleneck and the primary target for improvement. Here it is shown for the first time that, in spite of much lower apparent kcat of HjCel7A at the hydrolysis of ß-1,4-glucosidic linkages in the fluorogenic cellotetra- and -pentaose compared to the structurally related endoglucanase I (HjCel7B), the specificity constants (catalytic efficiency) kcat /Km for both enzymes are almost equal in these reactions. The observed activity difference appears from strong nonproductive substrate binding by HjCel7A, particularly significant for MU-ß-cellotetraose (MUG4 ). Interaction of substrates with the subsites -6 and -5 proximal to the nonconserved Gln101 residue in HjCel7A decreases Km,ap by >1500 times. HjCel7A can be nonproductively bound onto cellulose surface with Kd ≈2-9 nM via CBM and CD that captures six terminal glucose units of cellulose chain. Decomposition of this nonproductive complex can determine the rate of cellulose conversion. MUG4 is a promising substrate to select active cellobiohydrolase I variants with reduced nonproductive substrate binding.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/metabolismo , Proteínas Fúngicas/metabolismo , Sitios de Unión , Catálisis , Dominio Catalítico/fisiología , Celulasa/metabolismo , Celulosa/análogos & derivados , Celulosa/metabolismo , Hidrólisis , Cinética , Unión Proteica , Tetrosas/metabolismo , Trichoderma/enzimologíaRESUMEN
Endo-ß-1,4-glucanase AkEG21 belonging to glycosyl hydrolase family 45 (GHF45) is the most abundant cellulase in the digestive fluid of sea hare (Aplysia kurodai). The specific activity of this 21-kDa enzyme is considerably lower than those of other endo ß-1,4-glucanases in the digestive fluid of A. kurodai, therefore its role in whole cellulose hydrolysis by sea hare is still uncertain. Although AkEG21 has a catalytic domain without a cellulose binding domain, it demonstrated stable binding to cellulose fibers, similar to that of fungal cellobiohydrolase (CBH) 1 and CBH 2, which is strongly inhibited by cellohexaose, suggesting the involvement of the catalytic site in cellulose binding. Cellulose-bound AkEG21 hydrolyzed cellulose to cellobiose, cellotriose and cellotetraose, but could not digest an external substrate, azo-carboxymethyl cellulose. Cellulose hydrolysis was considerably stimulated by the synergistic action of cellulose-bound AkEG21 and AkEG45, another ß-1,4-endoglucanase present in the digestive fluid of sea hare; however no synergy in carboxymethylcellulose hydrolysis was observed. When AkEG21 was removed from the digestive fluid by immunoprecipitation, the cellulose hydrolyzing activity of the fluid was significantly reduced, indicating a critical role of AkEG21 in cellulose hydrolysis by A. kurodai. These findings suggest that AkEG21 is a processive endoglucanase functionally equivalent to the CBH, which provides a CBH-independent mechanism for the mollusk to digest seaweed cellulose to glucose.
Asunto(s)
Aplysia/enzimología , Celulasa/química , Celulosa/química , Digestión/genética , Animales , Aplysia/genética , Dominio Catalítico/genética , Celobiosa/química , Celulasa/genética , Celulosa/análogos & derivados , Celulosa/genética , Celulosa/metabolismo , Digestión/fisiología , Glucosa/química , Glucosa/metabolismo , Hidrólisis , Cinética , Oligosacáridos/química , Oligosacáridos/metabolismo , Unión Proteica , Dominios Proteicos/genética , Tetrosas/química , Tetrosas/metabolismoRESUMEN
The sco6546 gene of Streptomyces coelicolor A3(2) was annotated as a putative glycosyl hydrolase belonging to family 48. It is predicted to encode a 973-amino acid polypeptide (103.4 kDa) with a 39-amino acid secretion signal. Here, the SCO6546 protein was overexpressed in Streptomyces lividans TK24, and the purified protein showed the expected molecular weight of the mature secreted form (934 aa, 99.4 kDa) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis. SCO6546 showed high activity toward Avicel and carboxymethyl cellulose, but low activity toward filter paper and ß-glucan. SCO6546 showed maximum cellulase activity toward Avicel at pH 5.0 and 50 °C, which is similar to the conditions for maximum activity toward cellotetraose and cellopentaose substrates. The kinetic parameters kcat and KM , for cellotetraose at pH 5.0 and 50 °C were 13.3 s-1 and 2.7 mM, respectively. Thin layer chromatography (TLC) of the Avicel hydrolyzed products generated by SCO6546 showed cellobiose only, which was confirmed by mass spectral analysis. TLC analysis of the cello-oligosaccharide and chromogenic substrate hydrolysates generated by SCO6546 revealed that it can hydrolyze cellodextrins mainly from the non-reducing end into cellobiose. These data clearly demonstrated that SCO6546 is an exo-ß-1,4-cellobiohydrolase (EC 3.2.1.91), acting on nonreducing end of cellulose.
Asunto(s)
Celulosa 1,4-beta-Celobiosidasa/genética , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Streptomyces coelicolor/enzimología , Streptomyces lividans/genética , Celulosa/análogos & derivados , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/aislamiento & purificación , Cromatografía en Capa Delgada , Clonación Molecular , Dextrinas/metabolismo , Escherichia coli/genética , Expresión Génica , Concentración de Iones de Hidrógeno , Hidrólisis , Cinética , Peso Molecular , Streptomyces coelicolor/genética , Especificidad por Sustrato , Tetrosas/metabolismoRESUMEN
L-erythrose, a rare aldotetrose, possesses various pharmacological activities. However, efficient L-erythrose production is challenging. Currently, L-erythrose is produced by a two-step fermentation process from erythritol. Here, we describe a novel strategy for the production of L-erythrose in Gluconobacter oxydans (G. oxydans) by localizing the assembly of L-ribose isomerase (L-RI) to membrane-bound sorbitol dehydrogenase (SDH) via the protein-peptide interactions of the PDZ domain and PDZ ligand. To demonstrate this self-assembly, green fluorescent protein (GFP) replaced L-RI and its movement to membrane-bound SDH was observed by fluorescence microscopy. The final L-erythrose production was improved to 23.5 g/L with the stepwise metabolic engineering of G. oxydans, which was 1.4-fold higher than that obtained using coexpression of SDH and L-RI in G. oxydans. This self-assembly strategy shows remarkable potential for further improvement of L-erythrose production.
Asunto(s)
Isomerasas Aldosa-Cetosa/metabolismo , Proteínas Bacterianas/metabolismo , Gluconobacter oxydans/metabolismo , L-Iditol 2-Deshidrogenasa/metabolismo , Tetrosas/metabolismo , Isomerasas Aldosa-Cetosa/genética , Proteínas Bacterianas/genética , Gluconobacter oxydans/enzimología , Gluconobacter oxydans/genética , L-Iditol 2-Deshidrogenasa/genética , Ingeniería MetabólicaRESUMEN
A hypothetic gene (THA_1941) encoding a putative cellobiose phosphorylase (CBP) from Thermosipho africanus TCF52B has very low amino acid identities (less than 12%) to all known GH94 enzymes. This gene was cloned and over-expressed in Escherichia coli BL21(DE3). The recombinant protein was hypothesized to be a CBP enzyme and it showed an optimum temperature of 75 °C and an optimum pH of 7.5. Beyond its CBP activity, this enzyme can use cellobiose and long-chain cellodextrins with a degree of polymerization of greater than two as a glucose acceptor, releasing phosphate from glucose 1-phosphate. The catalytic efficiencies (k cat/K m) indicated that cellotetraose and cellopentaose were the best substrates for the phosphorolytic and reverse synthetic reactions, respectively. These results suggested that this enzyme was the first enzyme having both cellodextrin and cellobiose phosphorylases activities. Because it preferred cellobiose and cellodextrins to glucose in the synthetic direction, it was categorized as a cellodextrin phosphorylase (CDP). Due to its unique ability of the reverse synthetic reaction, this enzyme could be a potential catalyst for the synthesis of various oligosaccharides. The speculative function of this CDP in the carbohydrate metabolism of T. africanus TCF52B was also discussed.
Asunto(s)
Bacterias/enzimología , Celobiosa/metabolismo , Celulosa/análogos & derivados , Dextrinas/metabolismo , Glucosiltransferasas/metabolismo , Bacterias/genética , Celulosa/metabolismo , Clonación Molecular , Expresión Génica , Glucosa/metabolismo , Glucosiltransferasas/genética , Concentración de Iones de Hidrógeno , Cinética , Oligosacáridos/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Temperatura , Tetrosas/metabolismoRESUMEN
Erythritol is a four-carbon sugar alcohol synthesized by osmophilic yeasts, such as Yarrowia lipolytica, in response to osmotic stress. This metabolite has application as food additive due to its sweetening properties. Although Y. lipolytica can produce erythritol at a high level from glycerol, it is also able to consume it as carbon source. This ability negatively affects erythritol productivity and represents a serious drawback for the development of an efficient erythritol production process. In this study, we have isolated by insertion mutagenesis a Y. lipolytica mutant unable to grow on erythritol. Genomic characterization of the latter highlighted that the mutant phenotype is directly related to the disruption of the YALI0F01606g gene. Several experimental evidences suggested that the identified gene, renamed EYK1, encodes an erythrulose kinase. The mutant strain showed an enhanced capacity to produce erythritol as compared to the wild-type strain. Moreover, in specific experimental conditions, it is also able to convert erythritol to erythrulose, another compound of biotechnological interest.
Asunto(s)
Eritritol/metabolismo , Genes Fúngicos/genética , Yarrowia/genética , Eritritol/biosíntesis , Eritritol/farmacología , Glicerol/metabolismo , Mutagénesis Insercional , Mutación , Presión Osmótica , Fosfotransferasas/genética , Tetrosas/metabolismo , Yarrowia/efectos de los fármacos , Yarrowia/crecimiento & desarrollo , Yarrowia/metabolismoRESUMEN
We report here on EYK1, encoding erythrulose kinase, as an efficient catabolic selectable marker for genome editing in Y. lipolytica. Compared to auxotrophic markers, EYK1 increases the growth rate of transformants and allows improved efficiency of transformation. The utility of the marker EYK1 in a replicative vector was also demonstrated.
Asunto(s)
Edición Génica , Fosfotransferasas/genética , Tetrosas/metabolismo , Yarrowia/enzimología , Yarrowia/genética , Clonación Molecular , ADN de Hongos/genética , Regulación Fúngica de la Expresión Génica , Marcadores Genéticos , Fosfotransferasas/metabolismo , Saccharomyces cerevisiae/genética , Transformación Genética , Yarrowia/crecimiento & desarrolloRESUMEN
Mixed-linkage glucanâ¶xyloglucan endotransglucosylase (MXE) is one of the three activities of the recently characterised hetero-trans-ß-glucanase (HTG), which among land plants is known only from Equisetum species. The biochemical details of the MXE reaction were incompletely understood - details that would promote understanding of MXE's role in vivo and enable its full technological exploitation. We investigated HTG's site of attack on one of its donor substrates, mixed-linkage (1â3),(1â4)-ß-d-glucan (MLG), with radioactive oligosaccharides of xyloglucan as the acceptor substrate. Comparing three different MLG preparations, we showed that the enzyme favours those with a high content of cellotetraose blocks. The reaction products were analysed by enzymic digestion, thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC) and gel-permeation chromatography (GPC). Equisetum HTG consistently cleaved the MLG at the third consecutive ß-(1â4)-bond following (towards the reducing terminus) a ß-(1â3)-bond. It then formed a ß-(1â4)-bond between the MLG and the non-reducing terminal glucose residue of the xyloglucan oligosaccharide, consistent with its xyloglucan endotransglucosylase/hydrolase subfamily membership. Using size-homogeneous barley MLG as the donor substrate, we showed that HTG does not favour any particular region of the MLG chain relative to the polysaccharide's reducing and non-reducing termini; rather, it selects its target cellotetraosyl unit stochastically along the MLG molecule. This work improves our understanding of how enzymes can exhibit promiscuous substrate specificities and provides the foundations to explore strategies for engineering novel substrate specificities into transglycanases.