Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 3.668
Filtrar
1.
Biochem Biophys Res Commun ; 625: 60-65, 2022 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-35947916

RESUMO

Glycoside hydrolase family 94 (GH94) contains enzymes that reversibly catalyze the phosphorolysis of ß-glycosides. We conducted this study to investigate a GH94 protein (PBOR_13355) encoded in the genome of Paenibacillus borealis DSM 13188 with low sequence identity to known phosphorylases. Screening of acceptor substrates for reverse phosphorolysis in the presence of α-d-glucose 1-phosphate as a donor substrate showed that PBOR_13355 utilized d-glucuronic acid and p-nitrophenyl ß-d-glucuronide as acceptors. In the reaction with d-glucuronic acid, 3-O-ß-d-glucopyranosyl-d-glucuronic acid was synthesized. PBOR_13355 showed a higher apparent catalytic efficiency to p-nitrophenyl ß-d-glucuronide than to d-glucuronic acid, and thus, PBOR_13355 was concluded to be a novel glycoside phosphorylase, 3-O-ß-d-glucopyranosyl ß-d-glucuronide phosphorylase. PBOR_13360, encoded by the gene immediately downstream of the PBOR_13355 gene, was shown to be ß-glucuronidase. Collectively, PBOR_13355 and PBOR_13360 are predicted to work together in the cytosol to metabolize oligosaccharides containing the 3-O-ß-d-glucopyranosyl ß-d-glucuronide structure released from bacterial and plant acidic carbohydrates.


Assuntos
Glucuronídeos , Glicosídeo Hidrolases , Glucosiltransferases/metabolismo , Ácido Glucurônico , Glicosídeo Hidrolases/química , Glicosídeos/metabolismo , Redes e Vias Metabólicas , Paenibacillus , Fosforilases/química , Fosforilases/genética , Fosforilases/metabolismo , Especificidade por Substrato
2.
J Comput Aided Mol Des ; 36(7): 507-520, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35809194

RESUMO

Enterococcus faecalis, a gram-positive bacterium, is among the most common nosocomial pathogens due to its limited susceptibility to antibiotics and its reservoir of the genes coding for virulence factors. Bacterial enzymes such as kinases and phosphorylases play important roles in diverse functions of a bacterial cell and, thus, are potential antibacterial drug targets. In Gram-positive bacteria, HPr Kinase/Phosphorylase (HPrK/P), a bifunctional enzyme is involved in the regulation of carbon catabolite repression by phosphorylating/dephosphorylating the histidine-containing phosphocarrier protein (HPr) at Ser46 residue. Deficiencies in HPrK/P function leads to severe defects in bacterial growth. This study aimed at identifying novel inhibitors of E. faecalis HPrK/P from a commercial compound library using structure-based virtual screening. The hit molecules were purchased and their effect on enzyme activity and growth of resistant E. faecalis was evaluated in vitro. Furthermore, docking and molecular dynamics simulations were performed to study the interactions of the hit compounds with HPrK/P. Among the identified hit molecules, two compounds inhibited the phosphorylation of HPr as well as significantly reduced the growth of resistant E. faecalis in vitro. These identified potential HPrK/P inhibitors open new research avenues towards the development of novel antimicrobials against resistant Gram-positive bacteria.


Assuntos
Anti-Infecciosos , Proteínas de Bactérias , Enterococcus faecalis , Anti-Infecciosos/farmacologia , Proteínas de Bactérias/antagonistas & inibidores , Enterococcus faecalis/efeitos dos fármacos , Enterococcus faecalis/enzimologia , Fosforilases/antagonistas & inibidores , Fosforilação , Proteínas Serina-Treonina Quinases/antagonistas & inibidores
3.
Int J Mol Sci ; 23(9)2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35562912

RESUMO

Starch phosphorylase (PHO) is a multimeric enzyme with two distinct isoforms: plastidial starch phosphorylase (PHO1) and cytosolic starch phosphorylase (PHO2). PHO1 specifically resides in the plastid, while PHO2 is found in the cytosol. Both play a critical role in the synthesis and degradation of starch. This study aimed to report the detailed structure, function, and evolution of genes encoding PHO1 and PHO2 and their protein ligand-binding sites in eight monocots and four dicots. "True" orthologs of PHO1 and PHO2 of Oryza sativa were identified, and the structure of the enzyme at the protein level was studied. The genes controlling PHO2 were found to be more conserved than those controlling PHO1; the variations were mainly due to the variable sequence and length of introns. Cis-regulatory elements in the promoter region of both genes were identified, and the expression pattern was analyzed. The real-time quantitative polymerase chain reaction indicated that PHO2 was expressed in all tissues with a uniform pattern of transcripts, and the expression pattern of PHO1 indicates that it probably contributes to the starch biosynthesis during seed development in Zea mays. Under abscisic acid (ABA) treatment, PHO1 was found to be downregulated in Arabidopsis and Hordeum vulgare. However, we found that ABA could up-regulate the expression of both PHO1 and PHO2 within 12 h in Zea mays. In all monocots and dicots, the 3D structures were highly similar, and the ligand-binding sites were common yet fluctuating in the position of aa residues.


Assuntos
Arabidopsis , Magnoliopsida , Arabidopsis/metabolismo , Regulação da Expressão Gênica de Plantas , Ligantes , Magnoliopsida/metabolismo , Fosforilases/metabolismo , Plastídeos/metabolismo , Amido/genética , Amido/metabolismo , Amido Fosforilase/metabolismo , Zea mays/genética , Zea mays/metabolismo
4.
Biotechnol Bioeng ; 119(7): 1768-1780, 2022 07.
Artigo em Inglês | MEDLINE | ID: mdl-35383880

RESUMO

Currently, whole-cell catalysts face challenges due to the complexity of reaction systems, although they have a cost advantage over pure enzymes. In this study, cytarabine was synthesized by purified purine phosphorylase 1 (PNP1) and uracil phosphorylase (UP), and the conversion of cytarabine from adenine arabinoside reached 72.3 ± 4.3%. However, the synthesis was unsuccessful by whole-cell catalysis due to interference from unnecessary proteins (UNPs) in cells. Thus, we carried out a large-scale gene editing involving 377 genes in the genome of Escherichia coli to reduce the negative effect of UNPs on substrate conversion and cytarabine production. Finally, the PNP1 and UP activities of the obtained mutant were increased significantly compared with the parental strain, and more importantly, the conversion rate of cytarabine by whole-cell catalysis reached 67.4 ± 2.5%. The lack of 148 proteins and downregulation of 783 proteins caused by gene editing were equivalent to partial purification of the enzymes within cells, and thus, we provided inspiration to solve the problem caused by UNP interference, which is ubiquitous in the field of whole-cell catalysis.


Assuntos
Escherichia coli , Purina-Núcleosídeo Fosforilase , Citarabina/metabolismo , Escherichia coli/metabolismo , Fosforilases/metabolismo , Purina-Núcleosídeo Fosforilase/química , Purina-Núcleosídeo Fosforilase/genética , Purina-Núcleosídeo Fosforilase/metabolismo , Purinas/metabolismo , Uracila/metabolismo
5.
Eur J Appl Physiol ; 122(8): 1751-1772, 2022 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-35355125

RESUMO

Glycogen is a branched, glucose polymer and the storage form of glucose in cells. Glycogen has traditionally been viewed as a key substrate for muscle ATP production during conditions of high energy demand and considered to be limiting for work capacity and force generation under defined conditions. Glycogenolysis is catalyzed by phosphorylase, while glycogenesis is catalyzed by glycogen synthase. For many years, it was believed that a primer was required for de novo glycogen synthesis and the protein considered responsible for this process was ultimately discovered and named glycogenin. However, the subsequent observation of glycogen storage in the absence of functional glycogenin raises questions about the true role of the protein. In resting muscle, phosphorylase is generally considered to be present in two forms: non-phosphorylated and inactive (phosphorylase b) and phosphorylated and constitutively active (phosphorylase a). Initially, it was believed that activation of phosphorylase during intense muscle contraction was primarily accounted for by phosphorylation of phosphorylase b (activated by increases in AMP) to a, and that glycogen synthesis during recovery from exercise occurred solely through mechanisms controlled by glucose transport and glycogen synthase. However, it now appears that these views require modifications. Moreover, the traditional roles of glycogen in muscle function have been extended in recent years and in some instances, the original concepts have undergone revision. Thus, despite the extensive amount of knowledge accrued during the past 100 years, several critical questions remain regarding the regulation of glycogen metabolism and its role in living muscle.


Assuntos
Glicogenólise , Glucose/metabolismo , Glicogênio/metabolismo , Glicogênio Sintase/metabolismo , Humanos , Músculo Esquelético/metabolismo , Fosforilase b/metabolismo , Fosforilases/metabolismo
6.
New Phytol ; 234(5): 1782-1800, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35288947

RESUMO

Plant-derived Vitamin C (l-ascorbic acid (AsA)) is crucial for human health and wellbeing and thus increasing AsA content is of interest to plant breeders. In plants GDP-l-galactose phosphorylase (GGP) is a key biosynthetic control step and here evidence is presented for two new transcriptional activators of GGP. AsA measurement, transcriptomics, transient expression, hormone application, gene editing, yeast 1/2-hybrid, and electromobility shift assay (EMSA) methods were used to identify two positively regulating transcription factors. AceGGP3 was identified as the most highly expressed GGP in Actinidia eriantha fruit, which has high fruit AsA. A gene encoding a 1R-subtype myeloblastosis (MYB) protein, AceMYBS1, was found to bind the AceGGP3 promoter and activate its expression. Overexpression and gene-editing show AceMYBS1 effectively increases AsA accumulation. The bZIP transcription factor AceGBF3 (a G-box binding factor), also was shown to increase AsA content, and was confirmed to interact with AceMYBS1. Co-expression experiments showed that AceMYBS1 and AceGBF3 additively promoted AceGGP3 expression. Furthermore, AceMYBS1, but not GBF3, was repressed by abscisic acid, resulting in reduced AceGGP3 expression and accumulation of AsA. This study sheds new light on the roles of MYBS1 homologues and ABA in modulating AsA synthesis, and adds to the understanding of mechanisms underlying AsA accumulation.


Assuntos
Actinidia , Actinidia/genética , Actinidia/metabolismo , Ácido Ascórbico , Frutas/genética , Galactose/metabolismo , Regulação da Expressão Gênica de Plantas , Fosforilases/genética , Fosforilases/metabolismo , Fatores de Transcrição/metabolismo
7.
J Ind Microbiol Biotechnol ; 49(3)2022 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-35289917

RESUMO

Cellobiose, a natural disaccharide, attracts extensive attention as a potential functional food/feed additive. In this study, we present an inorganic phosphate (Pi) self-sufficient biotransformation system to produce cellobiose by co-expressing sucrose phosphorylase (SP) and cellobiose phosphorylase (CBP). The Bifidobacterium adolescentis SP (BASP) and Cellvibrio gilvus CBP (CGCBP) were co-expressed in Escherichia coli. Escherichia coli cells containing BASP and CGCBP were used as whole-cell catalysts to convert sucrose and glucose to cellobiose. The effects of reaction pH, temperature, Pi concentration, and substrate concentration were investigated. In the optimum biotransformation conditions, 800 mM cellobiose was produced from 1.0 M sucrose, 1.0 M glucose, and 50 mM Pi, within 12 hr. The by-product fructose and residual substrate (sucrose and glucose) were efficiently removed by treatment with yeast, to help purify the product cellobiose. The wider applicability of this Pi self-sufficiency strategy was demonstrated in the production of laminaribiose by co-expressing SP and laminaribiose phosphorylase. This study suggests that the Pi self-sufficiency strategy through co-expressing two phosphorylases has the advantage of great flexibility for enhanced production of cellobiose (or laminaribiose).


Assuntos
Celobiose , Fosfatos , Celobiose/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucose/metabolismo , Glucosiltransferases/metabolismo , Fosforilases/química , Fosforilases/genética , Sacarose
8.
Int J Mol Sci ; 23(6)2022 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-35328479

RESUMO

Among carbohydrate active enzymes, glycoside phosphorylases (GPs) are valuable catalysts for white biotechnologies, due to their exquisite capacity to efficiently re-modulate oligo- and poly-saccharides, without the need for costly activated sugars as substrates. The reversibility of the phosphorolysis reaction, indeed, makes them attractive tools for glycodiversification. However, discovery of new GP functions is hindered by the difficulty in identifying them in sequence databases, and, rather, relies on extensive and tedious biochemical characterization studies. Nevertheless, recent advances in automated tools have led to major improvements in GP mining, activity predictions, and functional screening. Implementation of GPs into innovative in vitro and in cellulo bioproduction strategies has also made substantial advances. Herein, we propose to discuss the latest developments in the strategies employed to efficiently discover GPs and make the best use of their exceptional catalytic properties for glycoside bioproduction.


Assuntos
Glicosídeos Cardíacos , Glicosídeos , Biotecnologia , Catálise , Glicosídeo Hidrolases/química , Glicosídeos/química , Fosforilases/química
9.
Sci Rep ; 12(1): 259, 2022 01 07.
Artigo em Inglês | MEDLINE | ID: mdl-34997180

RESUMO

Glycoside phosphorylases (GPs), which catalyze the reversible phosphorolysis of glycosides, are promising enzymes for the efficient production of glycosides. Various GPs with new catalytic activities are discovered from uncharacterized proteins phylogenetically distant from known enzymes in the past decade. In this study, we characterized Paenibacillus borealis PBOR_28850 protein, belonging to glycoside hydrolase family 94. Screening of acceptor substrates for reverse phosphorolysis, in which α-D-glucose 1-phosphate was used as the donor substrate, revealed that the recombinant PBOR_28850 produced in Escherichia coli specifically utilized D-galactose as an acceptor and produced solabiose (ß-D-Glcp-(1 → 3)-D-Gal). This indicates that PBOR_28850 is a new GP, solabiose phosphorylase. PBOR_28850 catalyzed the phosphorolysis and synthesis of solabiose through a sequential bi-bi mechanism involving the formation of a ternary complex. The production of solabiose from lactose and sucrose has been established. Lactose was hydrolyzed to D-galactose and D-glucose by ß-galactosidase. Phosphorolysis of sucrose and synthesis of solabiose were then coupled by adding sucrose, sucrose phosphorylase, and PBOR_28850 to the reaction mixture. Using 210 mmol lactose and 280 mmol sucrose, 207 mmol of solabiose was produced. Yeast treatment degraded the remaining monosaccharides and sucrose without reducing solabiose. Solabiose with a purity of 93.7% was obtained without any chromatographic procedures.


Assuntos
Proteínas de Bactérias/metabolismo , Dissacarídeos/biossíntese , Lactose/metabolismo , Paenibacillus/enzimologia , Fosforilases/metabolismo , Sacarose/metabolismo , Proteínas de Bactérias/genética , Sítios de Ligação , Catálise , Domínio Catalítico , Hidrólise , Cinética , Paenibacillus/genética , Fosforilases/genética , Especificidade por Substrato
10.
Prep Biochem Biotechnol ; 52(6): 611-617, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-34550864

RESUMO

We previously reported an in vitro enzymatic pathway for conversion of nonfood cellulose to starch (PNAS,110 (18): 7182-7187, 2013), in which the two sequential enzymes cellobiose phosphorylase (CBP) from Clostridium thermocellum and potato alpha-glucan phosphorylase (PGP) from Solanum tuberosum were the two key enzymes responsible for the whole conversion rate. In this work CBP and PGP were fused to form a large enzyme and it turned out that the fusion protein could exhibit a good bifunctionality when PGP moiety was put at the N-terminus and CBP moiety at the C-terminus (designated as PGP-CBP). Although the coupled reaction rate of PGP-CBP was decreased by 23.0% compared with the free enzymes, substrate channeling between the two active sites in PGP-CBP was formed, demonstrated by the introduction of the competing enzyme of PGP to the reaction system. The potential of PGP-CBP fusion enzyme being applied to the conversion of cellulose to amylose was discussed.


Assuntos
Celobiose , Solanum tuberosum , Celobiose/metabolismo , Celulose/metabolismo , Glucosiltransferases , Fosforilases/química , Fosforilases/genética , Solanum tuberosum/metabolismo , Amido
11.
Plant Sci ; 313: 111063, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34763857

RESUMO

Kiwifruit is known as 'the king of vitamin C' because of the high content of ascorbic acid (AsA) in the fruit. Deciphering the regulatory network and identification of the key regulators mediating AsA biosynthesis is vital for fruit nutrition and quality improvement. To date, however, the key transcription factors regulating AsA metabolism during kiwifruit developmental and ripening processes remains largely unknown. Here, we generated a putative transcriptional regulatory network mediating ascorbate metabolism by transcriptome co-expression analysis. Further studies identified an ethylene response factor AcERF91 from this regulatory network, which is highly co-expressed with a GDP-galactose phosphorylase encoding gene (AcGGP3) during fruit developmental and ripening processes. Through dual-luciferase reporter and yeast one-hybrid assays, it was shown that AcERF91 is able to bind and directly activate the activity of the AcGGP3 promoter. Furthermore, transient expression of AcERF91 in kiwifruit fruits resulted in a significant increase in AsA content and AcGGP3 transcript level, indicating a positive role of AcERF91 in controlling AsA accumulation via regulation of the expression of AcGGP3. Overall, our results provide a new insight into the regulation of AsA metabolism in kiwifruit.


Assuntos
Actinidia/genética , Actinidia/metabolismo , Ácido Ascórbico/metabolismo , Etilenos/metabolismo , Galactose/metabolismo , Guanosina Difosfato/metabolismo , Fosforilases/metabolismo , Ácido Ascórbico/genética , China , Produtos Agrícolas/genética , Produtos Agrícolas/metabolismo , Frutas/genética , Frutas/metabolismo , Galactose/genética , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Guanosina Difosfato/genética , Fosforilases/genética
12.
Molecules ; 26(20)2021 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-34684901

RESUMO

The Glycoside Hydrolase Family 65 (GH65) is an enzyme family of inverting α-glucoside phosphorylases and hydrolases that currently contains 10 characterized enzyme specificities. However, its sequence diversity has never been studied in detail. Here, an in-silico analysis of correlated mutations was performed, revealing specificity-determining positions that facilitate annotation of the family's phylogenetic tree. By searching these positions for amino acid motifs that do not match those found in previously characterized enzymes from GH65, several clades that may harbor new functions could be identified. Three enzymes from across these regions were expressed in E. coli and their substrate profile was mapped. One of those enzymes, originating from the bacterium Mucilaginibacter mallensis, was found to hydrolyze kojibiose and α-1,2-oligoglucans with high specificity. We propose kojibiose glucohydrolase as the systematic name and kojibiose hydrolase or kojibiase as the short name for this new enzyme. This work illustrates a convenient strategy for mapping the natural diversity of enzyme families and smartly mining the ever-growing number of available sequences in the quest for novel specificities.


Assuntos
Dissacarídeos/metabolismo , Glicosídeo Hidrolases/metabolismo , Motivos de Aminoácidos/fisiologia , Bacteroidetes/metabolismo , Escherichia coli/metabolismo , Fosforilases/metabolismo , Filogenia , Especificidade por Substrato
13.
Biochem Biophys Res Commun ; 579: 54-61, 2021 11 19.
Artigo em Inglês | MEDLINE | ID: mdl-34587555

RESUMO

1,2-ß-Mannobiose phosphorylases (1,2-ß-MBPs) from glycoside hydrolase 130 (GH130) family are important bio-catalysts in glycochemistry applications owing to their ability in synthesizing oligomannans. Here, we report the crystal structure of a thermostable 1,2-ß-MBP from Thermoanaerobacter sp. X-514 termed Teth514_1789 to reveal the molecular basis of its higher thermostability and mechanism of action. We also solved the enzyme complexes of mannose, mannose-1-phosphate (M1P) and 1,4-ß-mannobiose to manifest the enzyme-substrate interaction networks of three main subsites. Notably, a Zn ion that should be derived from crystallization buffer was found in the active site and coordinates the phosphate moiety of M1P. Nonetheless, this Zn-coordination should reflect an inhibitory status as supplementing Zn severely impairs the enzyme activity. These results indicate that the effects of metal ions should be taken into consideration when applying Teth514_1789 and other related enzymes. Based on the structure, a reliable model of Teth514_1788 that shares 61.7% sequence identity to Teth514_1789 but displays a different substrate preference was built. Analyzing the structural features of these two closely related enzymes, we hypothesized that the length of a loop fragment that covers the entrance of the catalytic center might regulate the substrate selectivity. In conclusion, these information provide in-depth understanding of GH130 1,2-ß-MBPs and should serve as an important guidance for enzyme engineering for further applications.


Assuntos
Thermoanaerobacter/enzimologia , beta-Manosidase/química , Sítios de Ligação , Catálise , Domínio Catalítico , Glicosídeo Hidrolases/química , Íons , Ligantes , Mananas/química , Manose/química , Manosefosfatos/química , Fosforilases/química , Plasmídeos/metabolismo , Conformação Proteica , Reprodutibilidade dos Testes , Eletricidade Estática , Temperatura , Zinco/química
14.
Carbohydr Res ; 508: 108411, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34392134

RESUMO

Bottom-up synthesis of ß-glucans such as callose, fungal ß-(1,3)(1,6)-glucan and cellulose, can create the defined compounds that are needed to perform fundamental studies on glucan properties and develop applications. With the importance of ß-glucans and cellulose in high-profile fields such as nutrition, renewables-based biotechnology and materials science, the enzymatic synthesis of such relevant carbohydrates and their derivatives has attracted much attention. Here we review recent developments in enzymatic synthesis of ß-glucans and cellulose, with a focus on progress made over the last five years. We cover the different types of biocatalysts employed, their incorporation in cascades, the exploitation of enzyme promiscuity and their engineering, and reaction conditions affecting the production as well as in situ self-assembly of (non)functionalised glucans. The recent achievements in the application of glycosyl transferases and ß-1,4- and ß-1,3-glucan phosphorylases demonstrate the high potential and versatility of these biocatalysts in glucan synthesis in both industrial and academic contexts.


Assuntos
Celulose , beta-Glucanas , Fosforilases
15.
Sci Rep ; 11(1): 16880, 2021 08 19.
Artigo em Inglês | MEDLINE | ID: mdl-34413335

RESUMO

Catalytically active inclusion bodies (CatIBs) produced in Escherichia coli are an interesting but currently underexplored strategy for enzyme immobilization. They can be purified easily and used directly as stable and reusable heterogenous catalysts. However, very few examples of CatIBs that are naturally formed during heterologous expression have been reported so far. Previous studies have revealed that the adenosine 5'-monophosphate phosphorylase of Thermococcus kodakarensis (TkAMPpase) forms large soluble multimers with high thermal stability. Herein, we show that heat treatment of soluble protein from crude extract induces aggregation of active protein which phosphorolyse all natural 5'-mononucleotides. Additionally, inclusion bodies formed during the expression in E. coli were found to be similarly active with 2-6 folds higher specific activity compared to these heat-induced aggregates. Interestingly, differences in the substrate preference were observed. These results show that the recombinant thermostable TkAMPpase is one of rare examples of naturally formed CatIBs.


Assuntos
Monofosfato de Adenosina/metabolismo , Biocatálise , Fosforilases/metabolismo , Thermococcus/enzimologia , Monofosfato de Adenosina/química , Citidina Monofosfato , Estabilidade Enzimática , Corpos de Inclusão/metabolismo , Agregados Proteicos , Solubilidade , Especificidade por Substrato , Temperatura
16.
Appl Biochem Biotechnol ; 193(11): 3719-3731, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34379312

RESUMO

Phosphorylase is a type of enzyme-producing sugar phosphates through the reversible phosphorolysis reactions of glycosides, which makes it an important starting enzyme in multi-enzyme systems for rare sugar biomanufacturing. To investigate its application in D-tagatose biosynthesis from maltodextrin using in vitro multi-enzyme cascade biosystem, the α-glucan phosphorylase (αGP; EC 2.4.1.1) from the thermophile D. turgidum DSM 6724 was prepared and characterized. It exhibited the specific activity of 30.28 U/mg at its optimal temperature of 70 °C. Thermostability results revealed that DituαGP could maintain more than 25% of initial activity for 4 h, even at 90 °C. The highest activity was observed at pH 5.5, and most divalent metal ions deactivated the enzyme. DituαGP exhibited great application potential in the multi-enzyme system that about 3.919 g/L of D-tagatose was produced from 150 g/L of maltodextrin within 36 h. DituαGP has played an important role in this biosystem and will also be applied in the synthesis of other rare sugars from maltodextrin.


Assuntos
Bactérias/enzimologia , Proteínas de Bactérias/química , Hexoses/síntese química , Fosforilases/química , Hexoses/química
17.
Int J Mol Sci ; 22(13)2021 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-34281256

RESUMO

Plants are often challenged by an array of unfavorable environmental conditions. During cold exposure, many changes occur that include, for example, the stabilization of cell membranes, alterations in gene expression and enzyme activities, as well as the accumulation of metabolites. In the presented study, the carbohydrate metabolism was analyzed in the very early response of plants to a low temperature (2 °C) in the leaves of 5-week-old potato plants of the Russet Burbank cultivar during the first 12 h of cold treatment (2 h dark and 10 h light). First, some plant stress indicators were examined and it was shown that short-term cold exposure did not significantly affect the relative water content and chlorophyll content (only after 12 h), but caused an increase in malondialdehyde concentration and a decrease in the expression of NDA1, a homolog of the NADH dehydrogenase gene. In addition, it was shown that the content of transitory starch increased transiently in the very early phase of the plant response (3-6 h) to cold treatment, and then its decrease was observed after 12 h. In contrast, soluble sugars such as glucose and fructose were significantly increased only at the end of the light period, where a decrease in sucrose content was observed. The availability of the monosaccharides at constitutively high levels, regardless of the temperature, may delay the response to cold, involving amylolytic starch degradation in chloroplasts. The decrease in starch content, observed in leaves after 12 h of cold exposure, was preceded by a dramatic increase in the transcript levels of the key enzymes of starch degradation initiation, the α-glucan, water dikinase (GWD-EC 2.7.9.4) and the phosphoglucan, water dikinase (PWD-EC 2.7.9.5). The gene expression of both dikinases peaked at 9 h of cold exposure, as analyzed by real-time PCR. Moreover, enhanced activities of the acid invertase as well as of both glucan phosphorylases during exposure to a chilling temperature were observed. However, it was also noticed that during the light phase, there was a general increase in glucan phosphorylase activities for both control and cold-stressed plants irrespective of the temperature. In conclusion, a short-term cold treatment alters the carbohydrate metabolism in the leaves of potato, which leads to an increase in the content of soluble sugars.


Assuntos
Metabolismo dos Carboidratos , Resposta ao Choque Frio/fisiologia , Solanum tuberosum/metabolismo , Amilases/metabolismo , Metabolismo dos Carboidratos/genética , Clorofila/metabolismo , Temperatura Baixa/efeitos adversos , Resposta ao Choque Frio/genética , Complexo I de Transporte de Elétrons/genética , Regulação Enzimológica da Expressão Gênica , Regulação da Expressão Gênica de Plantas , Genes de Plantas , Sistema da Enzima Desramificadora do Glicogênio/metabolismo , Malondialdeído/metabolismo , Fosforilases/metabolismo , Fosfotransferases (Aceptores Pareados)/genética , Folhas de Planta/metabolismo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Solanum tuberosum/genética , Amido/metabolismo , Água/metabolismo , beta-Frutofuranosidase/metabolismo
18.
Biochemistry ; 60(20): 1573-1577, 2021 05 25.
Artigo em Inglês | MEDLINE | ID: mdl-33955225

RESUMO

Enzyme-catalyzed reactions sometimes display curvature in their Eyring plots in the absence of denaturation, indicative of a change in activation heat capacity. However, the effects of pH and (de)protonation on this phenomenon have remained unexplored. Herein, we report a kinetic characterization of the thermophilic pyrimidine nucleoside phosphorylase from Geobacillus thermoglucosidasius across a two-dimensional working space covering 35 °C and 3 pH units with two substrates displaying different pKa values. Our analysis revealed the presence of a measurable activation heat capacity change ΔCp⧧ in this reaction system, which showed no significant dependence on medium pH or substrate charge. Our results further describe the remarkable effects of a single halide substitution that has a minor influence on ΔCp⧧ but conveys a significant kinetic effect by decreasing the activation enthalpy, causing a >10-fold rate increase. Collectively, our results present an important piece in the understanding of enzymatic systems across multidimensional working spaces where the choice of reaction conditions can affect the rate, affinity, and thermodynamic phenomena independently of one another.


Assuntos
Bacillaceae/metabolismo , Fosforilases/metabolismo , Purina-Núcleosídeo Fosforilase/química , Catálise , Temperatura Alta , Concentração de Íons de Hidrogênio , Cinética , Pentosiltransferases/química , Fosforilases/fisiologia , Pirimidina Fosforilases/química , Especificidade por Substrato , Condutividade Térmica , Termodinâmica
19.
Appl Microbiol Biotechnol ; 105(10): 4073-4087, 2021 May.
Artigo em Inglês | MEDLINE | ID: mdl-33970317

RESUMO

ß-Glucan phosphorylases are carbohydrate-active enzymes that catalyze the reversible degradation of ß-linked glucose polymers, with outstanding potential for the biocatalytic bottom-up synthesis of ß-glucans as major bioactive compounds. Their preference for sugar phosphates (rather than nucleotide sugars) as donor substrates further underlines their significance for the carbohydrate industry. Presently, they are classified in the glycoside hydrolase families 94, 149, and 161 ( www.cazy.org ). Since the discovery of ß-1,3-oligoglucan phosphorylase in 1963, several other specificities have been reported that differ in linkage type and/or degree of polymerization. Here, we present an overview of the progress that has been made in our understanding of ß-glucan and associated ß-glucobiose phosphorylases, with a special focus on their application in the synthesis of carbohydrates and related molecules. KEY POINTS: • Discovery, characteristics, and applications of ß-glucan phosphorylases. • ß-Glucan phosphorylases in the production of functional carbohydrates.


Assuntos
beta-Glucanas , Biocatálise , Metabolismo dos Carboidratos , Glicosídeo Hidrolases/metabolismo , Humanos , Fosforilases/metabolismo
20.
Plant Physiol ; 185(4): 1574-1594, 2021 04 23.
Artigo em Inglês | MEDLINE | ID: mdl-33793952

RESUMO

The enzymes involved in l-ascorbate biosynthesis in photosynthetic organisms (the Smirnoff-Wheeler [SW] pathway) are well established. Here, we analyzed their subcellular localizations and potential physical interactions and assessed their role in the control of ascorbate synthesis. Transient expression of C terminal-tagged fusions of SW genes in Nicotiana benthamiana and Arabidopsis thaliana mutants complemented with genomic constructs showed that while GDP-d-mannose epimerase is cytosolic, all the enzymes from GDP-d-mannose pyrophosphorylase (GMP) to l-galactose dehydrogenase (l-GalDH) show a dual cytosolic/nuclear localization. All transgenic lines expressing functional SW protein green fluorescent protein fusions driven by their endogenous promoters showed a high accumulation of the fusion proteins, with the exception of those lines expressing GDP-l-galactose phosphorylase (GGP) protein, which had very low abundance. Transient expression of individual or combinations of SW pathway enzymes in N. benthamiana only increased ascorbate concentration if GGP was included. Although we did not detect direct interaction between the different enzymes of the pathway using yeast-two hybrid analysis, consecutive SW enzymes, as well as the first and last enzymes (GMP and l-GalDH) associated in coimmunoprecipitation studies. This association was supported by gel filtration chromatography, showing the presence of SW proteins in high-molecular weight fractions. Finally, metabolic control analysis incorporating known kinetic characteristics showed that previously reported feedback repression at the GGP step, combined with its relatively low abundance, confers a high-flux control coefficient and rationalizes why manipulation of other enzymes has little effect on ascorbate concentration.


Assuntos
Arabidopsis/genética , Arabidopsis/metabolismo , Ácido Ascórbico/biossíntese , Galactose/metabolismo , Guanosina Difosfato/metabolismo , Fosforilases/metabolismo , Tabaco/genética , Tabaco/metabolismo , Ácido Ascórbico/genética , Galactose/genética , Regulação da Expressão Gênica de Plantas , Variação Genética , Genótipo , Guanosina Difosfato/genética , Mutação , Fosforilases/genética , Plantas Geneticamente Modificadas/genética , Plantas Geneticamente Modificadas/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...