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1.
J Am Chem Soc ; 143(21): 8154-8163, 2021 06 02.
Artículo en Inglés | MEDLINE | ID: mdl-34028252

RESUMEN

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/química
2.
J Am Chem Soc ; 143(42): 17761-17768, 2021 10 27.
Artículo en Inglés | MEDLINE | ID: mdl-34637287

RESUMEN

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/metabolismo
3.
Anal Biochem ; 613: 114022, 2021 01 15.
Artículo en Inglés | MEDLINE | ID: mdl-33217405

RESUMEN

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/metabolismo
4.
Glycoconj J ; 38(3): 347-359, 2021 06.
Artículo en Inglés | MEDLINE | ID: mdl-33245448

RESUMEN

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 Cultivadas
5.
J Biol Chem ; 294(44): 16095-16108, 2019 11 01.
Artículo en Inglés | MEDLINE | ID: mdl-31511322

RESUMEN

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/metabolismo
6.
Cell Biol Int ; 44(2): 651-660, 2020 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-31750586

RESUMEN

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 & desarrollo
7.
Appl Microbiol Biotechnol ; 103(11): 4393-4404, 2019 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-31001743

RESUMEN

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/deficiencia
8.
Biochem J ; 474(7): 1055-1070, 2017 03 08.
Artículo en Inglés | MEDLINE | ID: mdl-28108640

RESUMEN

Mixed-linkage glucan∶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 (1→3),(1→4)-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-(1→4)-bond following (towards the reducing terminus) a ß-(1→3)-bond. It then formed a ß-(1→4)-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.


Asunto(s)
Celulosa/análogos & derivados , Equisetum/enzimología , Glucanos/química , Glicosiltransferasas/metabolismo , Proteínas de Plantas/metabolismo , Tetrosas/química , Xilanos/química , beta-Glucanos/química , Biocatálisis , Secuencia de Carbohidratos , Pared Celular/química , Pared Celular/enzimología , Celulosa/química , Celulosa/metabolismo , Pruebas de Enzimas , Equisetum/química , Glucanos/metabolismo , Extractos Vegetales/química , Especificidad por Sustrato , Tetrosas/metabolismo , Xilanos/metabolismo , beta-Glucanos/metabolismo
9.
J Basic Microbiol ; 58(4): 310-321, 2018 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-29400405

RESUMEN

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/metabolismo
10.
Biochemistry ; 56(1): 167-178, 2017 Jan 10.
Artículo en Inglés | MEDLINE | ID: mdl-28026938

RESUMEN

Cellobiohydrolases (CBHs) make up an important group of enzymes for both natural carbon cycling and industrial deconstruction of lignocellulosic biomass. The consecutive hydrolysis of one cellulose strand relies on an intricate pattern of enzyme-substrate interactions in the long, tunnel-shaped binding site of the CBH. In this work, we have investigated the initial complexation mode with cellulose of the most thoroughly studied CBH, Cel7A from Hypocrea jecorina (HjCel7A). We found that HjCel7A predominantly produces glucose when it initiates a processive run on insoluble microcrystalline cellulose, confirming the validity of an even and odd product ratio as an estimate of processivity. Moreover, the glucose released from cellulose was predominantly α-glucose. A link between the initial binding mode of the enzyme and the reducing end configuration was investigated by inhibition studies with the two anomers of cellobiose. A clear preference for ß-cellobiose in product binding site +2 was observed for HjCel7A, but not the homologous endoglucanase, HjCe7B. Possible relationships between this anomeric preference in the product site and the prevalence of odd-numbered initial-cut products are discussed, and a correlation between processivity and anomer selectivity is proposed.


Asunto(s)
Celobiosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/metabolismo , Proteínas Fúngicas/metabolismo , Hypocrea/enzimología , Algoritmos , Técnicas Biosensibles , Celobiosa/química , Celulosa/análogos & derivados , Celulosa/química , Celulosa/metabolismo , Celulosa 1,4-beta-Celobiosidasa/química , Cromatografía Liquida , Cristalografía por Rayos X , Proteínas Fúngicas/química , Glucosa/química , Glucosa/metabolismo , Hypocrea/metabolismo , Isoenzimas/química , Isoenzimas/metabolismo , Cinética , Espectrometría de Masas , Modelos Moleculares , Estructura Molecular , Unión Proteica , Dominios Proteicos , Especificidad por Sustrato , Tetrosas/química , Tetrosas/metabolismo
11.
J Biol Chem ; 291(18): 9596-609, 2016 Apr 29.
Artículo en Inglés | MEDLINE | ID: mdl-26941078

RESUMEN

Kynurenine pathway metabolites and ascorbate degradation products are present in human lenses. In this study, we showed that erythrulose, a major ascorbate degradation product, reacts spontaneously with 3-hydroxykynurenine to form a fluorescent product. Structural characterization of the product revealed it to be 2-amino-4-(2-hydroxy-3-(2-hydroxyethyl)-2H-benzo[b][1,4]oxazin-5-yl)-4-oxobutanoic acid, which we named kynoxazine. Unlike 3-hydroxykynurenine, 3-hydroxykynurenine glucoside and kynurenine were unable to form a kynoxazine-like compound, which suggested that the aminophenol moiety in 3-hydroxykynurenine is essential for the formation of kynoxazine. This reasoning was confirmed using a model compound, 1-(2-amino-3-hydroxyphenyl)ethan-1-one, which is an aminophenol lacking the amino acid moiety of 3-hydroxykynurenine. Ultra-performance liquid chromatography-tandem mass spectrometry analyses showed that kynoxazine is present in the human lens at levels ranging from 0 to 64 pmol/mg lens. Kynoxazine as well as erythrulose degraded under physiological conditions to generate 3-deoxythreosone, which modified and cross-linked proteins through the formation of an arginine adduct, 3-deoxythreosone-derived hydroimidazolone, and a lysine-arginine cross-linking adduct, 3-deoxythreosone-derived hydroimidazolimine cross-link. Ultra-performance liquid chromatography-tandem mass spectrometry quantification showed that 32-169 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolone and 1.1-11.2 pmol/mg protein of 3-deoxythreosone-derived hydroimidazolimine cross-link occurred in aging lenses. Taken together, these results demonstrate a novel biochemical mechanism by which ascorbate oxidation and the kynurenine pathway intertwine, which could promote protein modification and cross-linking in aging human lenses.


Asunto(s)
Envejecimiento/metabolismo , Proteínas del Ojo/metabolismo , Quinurenina/análogos & derivados , Cristalino/metabolismo , Procesamiento Proteico-Postraduccional , Tetrosas/metabolismo , Humanos , Quinurenina/metabolismo
12.
Appl Microbiol Biotechnol ; 101(17): 6587-6596, 2017 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-28608278

RESUMEN

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/metabolismo
13.
Appl Microbiol Biotechnol ; 101(5): 1919-1926, 2017 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-27822737

RESUMEN

Cytophaga hutchinsonii is a gram-negative bacterium that can efficiently degrade crystalline cellulose by a novel strategy without cell-free cellulases or cellulosomes. Genomic analysis implied that C. hutchinsonii had endoglucanases and ß-glucosidases but no exoglucanases which could processively digest cellulose and produce cellobiose. In this study, BglA was functionally expressed in Escherichia coli and found to be a ß-glucosidase with wide substrate specificity. It can hydrolyze pNPG, pNPC, cellobiose, and cellodextrins. Moreover, unlike most ß-glucosidases whose activity greatly decreases with increasing length of the substrate chains, BglA has similar activity on cellobiose and larger cellodextrins. The K m values of BglA on cellobiose, cellotriose, and cellotetraose were calculated to be 4.8 × 10-2, 5.6 × 10-2, and 5.3 × 10-2 mol/l, respectively. These properties give BglA a great advantage to cooperate with endoglucanases in C. hutchinsonii in cellulose degradation. We proposed that C. hutchinsonii could utilize a simple cellulase system which consists of endoglucanases and ß-glucosidases to completely digest amorphous cellulose into glucose. Moreover, BglA was also found to be highly tolerant to glucose as it retained 40 % activity when the concentration of glucose was 100 times higher than that of the substrate, showing potential application in the bioenergy industry.


Asunto(s)
Celulosa/metabolismo , Cytophaga/enzimología , Escherichia coli/metabolismo , beta-Glucosidasa/genética , beta-Glucosidasa/metabolismo , Celobiosa/biosíntesis , Celulosa/análogos & derivados , Cytophaga/metabolismo , Dextrinas/metabolismo , Escherichia coli/genética , Glucosa/metabolismo , Ingeniería de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por Sustrato , Tetrosas/metabolismo
14.
J Sci Food Agric ; 97(3): 743-752, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27145288

RESUMEN

BACKGROUND: The structure of ß-glucan influences its use in cereal-based foods and feed. The objective of this study was to determine the effect of environment (E) and genotype (G) on ß-glucan fine structure and its genetic control in two-row spring barley with normal starch characteristics. RESULTS: A population of 89 recombinant inbred lines, derived from the cross of two-row spring barley genotypes Merit × H93174006 (H92076F1 × TR238), was characterized for concentration and structure of grain ß-glucan in two environments. Results showed that concentrations of ß-glucan, DP3, DP4 and DP3 + DP4 were positively correlated with each other, suggesting no preference for DP3 or DP4 subunit production in high- or low-ß-glucan lines. The concentrations of ß-glucan, DP3, DP4 and DP3:DP4 ratios were significantly influenced by genotype and environment. However, only DP3:DP4 ratio showed a significant effect of G × E interaction. Association mapping of candidate markers in 119 barley genotypes showed that marker CSLF6_4105 was associated with ß-glucan concentration, whereas Bmac504 and Bmac211 were associated with DP3:DP4 ratio. Bmac273e was associated with both ß-glucan concentration and DP3:DP4 ratio. CONCLUSION: The grain ß-glucan concentration and DP3:DP4 ratio are strongly affected by genotype and environment. Single-marker analyses suggested that the genetic control of ß-glucan concentration and DP3:DP4 ratio was linked to separate chromosomal regions on barley genome. © 2016 Society of Chemical Industry.


Asunto(s)
Carbohidratos de la Dieta/análisis , Interacción Gen-Ambiente , Glucosiltransferasas/metabolismo , Hordeum/química , Proteínas de Plantas/metabolismo , Semillas/química , beta-Glucanos/análisis , Alberta , Altitud , Alimentación Animal/análisis , Animales , Secuencia de Carbohidratos , Celulosa/genética , Celulosa/metabolismo , Clima , Cruzamientos Genéticos , Carbohidratos de la Dieta/metabolismo , Marcadores Genéticos , Glucosiltransferasas/genética , Hordeum/genética , Hordeum/crecimiento & desarrollo , Hordeum/metabolismo , Humanos , Valor Nutritivo , Fitomejoramiento , Proteínas de Plantas/genética , Semillas/genética , Semillas/crecimiento & desarrollo , Semillas/metabolismo , Especificidad por Sustrato , Tetrosas/metabolismo , Triosas/metabolismo , beta-Glucanos/química , beta-Glucanos/metabolismo
15.
Chembiochem ; 17(19): 1804-1808, 2016 10 04.
Artículo en Inglés | MEDLINE | ID: mdl-27383648

RESUMEN

Recent advances in polymerase engineering have enabled the replication of xenonucleic acid (XNA) polymers with backbone structures distinct from those found in nature. By introducing a selective amplification step into the replication cycle, functional XNA molecules have been isolated by in vitro selection with binding and catalytic activity. Despite these successes, coding and decoding genetic information in XNA polymers remains limited by the fidelity and catalytic efficiency of engineered XNA polymerases. In particular, the process of reverse transcribing XNA back into DNA for amplification by PCR has been problematic. Here, we show that Geobacillus stearothermophilus (Bst) DNA polymerase I functions as an efficient and faithful threose nucleic acid (TNA)-dependent DNA polymerase. Bst DNA polymerase generates ∼twofold more cDNA with threefold fewer mutations than Superscript II (SSII), which was previously the best TNA reverse transcriptase. Notably, Bst also functions under standard magnesium-dependent conditions, whereas SSII requires manganese ions to relax the enzyme's substrate specificity. We further demonstrate that Bst DNA polymerase can support the in vitro selection of TNA aptamers by evolving a TNA aptamer to human α-thrombin.


Asunto(s)
ADN Polimerasa I/metabolismo , Ácidos Nucleicos/genética , Transcripción Reversa , Tetrosas/metabolismo , Geobacillus stearothermophilus/enzimología , Humanos , Ácidos Nucleicos/metabolismo
16.
J Am Chem Soc ; 137(12): 4014-7, 2015 Apr 01.
Artículo en Inglés | MEDLINE | ID: mdl-25785966

RESUMEN

Threose nucleic acid (TNA) is an unnatural genetic polymer capable of undergoing Darwinian evolution to generate folded molecules with ligand-binding activity. This property, coupled with a nuclease-resistant backbone, makes TNA an attractive candidate for future applications in biotechnology. Previously, we have shown that an engineered form of the Archaean replicative DNA polymerase 9°N, known commercially as Therminator DNA polymerase, can copy a three-letter genetic alphabet (A,T,C) from DNA into TNA. However, our ability to transcribe four-nucleotide libraries has been limited by chain termination events that prevent the synthesis of full-length TNA products. Here, we show that chain termination is caused by tG:dG mispairing in the enzyme active site. We demonstrate that the unnatural base analogue 7-deazaguanine (7dG) will suppress tGTP misincorporation by inhibiting the formation of Hoogsteen tG:dG base pairs. DNA templates that contain 7dG in place of natural dG residues replicate with high efficiency and >99% overall fidelity. Pre-steady-state kinetic measurements indicate that the rate of tCTP incorporation is 5-fold higher opposite 7dG than dG and only slightly lower than dCTP incorporation opposite either 7dG or dG. These results provide a chemical solution to the problem of how to synthesize large, unbiased pools of TNA molecules by polymerase-mediated synthesis.


Asunto(s)
Archaea/enzimología , ADN Polimerasa Dirigida por ADN/metabolismo , Guanina/análogos & derivados , Ácidos Nucleicos/química , Tetrosas/química , Emparejamiento Base , Secuencia de Bases , Guanina/química , Guanina/metabolismo , Ácidos Nucleicos/metabolismo , Tetrosas/metabolismo
17.
Proc Natl Acad Sci U S A ; 109(16): 6012-7, 2012 Apr 17.
Artículo en Inglés | MEDLINE | ID: mdl-22474347

RESUMEN

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular ß-glucosidase enzymes and one intracellular ß-glucosidase lacks ß-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple ß-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.


Asunto(s)
Celulasa/genética , Celulasas/genética , Celulosa/análogos & derivados , Dextrinas/farmacología , Proteínas Fúngicas/genética , Neurospora crassa/genética , Celobiosa/metabolismo , Celobiosa/farmacología , Celulasa/metabolismo , Celulasas/clasificación , Celulasas/metabolismo , Celulosa/metabolismo , Celulosa/farmacología , Análisis por Conglomerados , Dextrinas/metabolismo , Proteínas Fúngicas/metabolismo , Eliminación de Gen , Perfilación de la Expresión Génica , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Regulación Fúngica de la Expresión Génica/efectos de los fármacos , Lignina/metabolismo , Lignina/farmacología , Espectrometría de Masas , Mutación , Neurospora crassa/metabolismo , Filogenia , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Tetrosas/metabolismo , Tetrosas/farmacología , Triosas/metabolismo , Triosas/farmacología
18.
Biochem J ; 451(2): 289-300, 2013 Apr 15.
Artículo en Inglés | MEDLINE | ID: mdl-23356867

RESUMEN

Non-catalytic cellulosomal CBMs (carbohydrate-binding modules) are responsible for increasing the catalytic efficiency of cellulosic enzymes by selectively putting the substrate (a wide range of poly- and oligo-saccharides) and enzyme into close contact. In the present study we carried out an atomistic rationalization of the molecular determinants of ligand specificity for a family 11 CBM from thermophilic Clostridium thermocellum [CtCBM11 (C. thermocellum CBM11)], based on a NMR and molecular modelling approach. We have determined the NMR solution structure of CtCBM11 at 25°C and 50°C and derived information on the residues of the protein that are involved in ligand recognition and on the influence of the length of the saccharide chain on binding. We obtained models of the CtCBM11-cellohexaose and CtCBM11-cellotetraose complexes by docking in accordance with the NMR experimental data. Specific ligand-protein CH-π and Van der Waals interactions were found to be determinant for the stability of the complexes and for defining specificity. Using the order parameters derived from backbone dynamics analysis in the presence and absence of ligand and at 25°C and 50°C, we determined that the protein's backbone conformational entropy is slightly positive. This data in combination with the negative binding entropy calculated from ITC (isothermal titration calorimetry) studies supports a selection mechanism where a rigid protein selects a defined oligosaccharide conformation.


Asunto(s)
Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Metabolismo de los Hidratos de Carbono , Clostridium thermocellum/metabolismo , Oligosacáridos/química , Proteínas Bacterianas/genética , Sitios de Unión , Calorimetría , Celulosa/análogos & derivados , Celulosa/química , Celulosa/metabolismo , Entropía , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Simulación del Acoplamiento Molecular , Simulación de Dinámica Molecular , Oligosacáridos/metabolismo , Conformación Proteica , Tetrosas/química , Tetrosas/metabolismo
19.
Int J Biol Macromol ; 277(Pt 4): 134311, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39094869

RESUMEN

Nature has developed extremozymes that catalyze complex reaction processes in extreme environmental conditions. Accordingly, a combined approach consisting of extremozyme screening, ancestral sequence resurrection (ASR), and molecular dynamic simulation was utilized to construct a developed endoglucanase. The primary experimental and in-silico data led to the prediction of a hypothetical sequence of endoglucanase (EG5-G131) using Bacillus sp. G131 confirmed by amplification and sequencing. EG5-G131 exhibited noticeable stability in a broad-pH range, several detergents, organic solvents, and temperatures up to 80 °C. The molecular weight, Vmax, and Km of the purified endoglucanase were estimated to be 36 kDa, 4.32 µmol/min, and 23.62 mg/ml, respectively. The calculated thermodynamic parameters for EG5-G131 confirmed its intrinsic thermostability. Computational analysis revealed Glu142 and Glu230 as active-site residues of the enzyme. Furthermore, the enzyme remained bound to cellotetraose at 298 K, 333 K, 343 K, and 353 K for 300 ns, consistent with our experimental data. ASR of EG5-G131 led to the introduction of ancestral ANC204 and ANC205, which show similar thermodynamic characteristics with the last Firmicute common ancestor. Finally, truncating loops from the N-terminal of two sequences created two variants with desirable thermal stability, suggesting the evolutionary deciphering of the functional domain of the GH5 family in Bacillus sp. G131.


Asunto(s)
Bacillus , Celulasa , Estabilidad de Enzimas , Evolución Molecular , Simulación de Dinámica Molecular , Bacillus/enzimología , Bacillus/genética , Celulasa/química , Celulasa/genética , Celulasa/metabolismo , Termodinámica , Concentración de Iones de Hidrógeno , Dominio Catalítico , Secuencia de Aminoácidos , Tetrosas/metabolismo , Tetrosas/química , Temperatura , Filogenia , Cinética , Extremófilos/enzimología , Extremófilos/genética , Celulosa/análogos & derivados
20.
Int J Biol Macromol ; 273(Pt 2): 133212, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38897502

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 & derivados
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