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1.
Int J Biol Macromol ; 278(Pt 1): 134753, 2024 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-39147336

RESUMO

L-arabinose isomerase (L-AI) is a functional enzyme for the isomerizing of D-galactose to produce D-tagatose. In this study, L-AI-C6-encoding gene from the probiotic Lactobacillus fermentum C6 was cloned and expressed in Bacillus subtilis WB600 for investigating enzymatic characteristics and bioconverting D-tagatose by means of whole-cell catalysis. Results showed that the engineered B. subtilis WB600-pMA5-LAI achieved a maximum specific activity of L-AI-C6 (232.65 ± 15.54 U/mg protein) under cultivation in LB medium at 28 °C for 40 h. The recombinant L-AI-C6 was purified, and enzymatic characteristics test showed its optimum reaction temperature and pH at 60 °C and 8.0, respectively. In addition, L-AI-C6 exhibited good stability within the pH range of 5.5-9.0. By using B. subtilis WB600-pMA5-LAI cells as whole-cell catalyst, the highest D-tagatose yield reached 42.91 ± 0.28 % with D-galactose as substrate, which was 2.41 times that of L. fermentum C6 (17.79 ± 0.11 %). This suggested that the cloning and heterologous expression of L-AI-C6 was an effective strategy for improving D-tagatose conversion by whole-cell catalysis. In brief, the present study demonstrated that the reaction temperature, pH, and stability of L-AI-C6 from L. fermentum C6 meet the demands of industrial application, and the constructed B. subtilis WB600-pMA5-LAI shows promising potential for the whole-cell biotransformation of D-tagatose.


Assuntos
Aldose-Cetose Isomerases , Bacillus subtilis , Hexoses , Limosilactobacillus fermentum , Proteínas Recombinantes , Bacillus subtilis/genética , Bacillus subtilis/enzimologia , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo , Hexoses/biossíntese , Limosilactobacillus fermentum/enzimologia , Limosilactobacillus fermentum/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Concentração de Íons de Hidrogênio , Temperatura , Clonagem Molecular/métodos , Estabilidade Enzimática , Galactose/metabolismo , Cinética
2.
Biochem J ; 481(16): 1075-1096, 2024 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-39105673

RESUMO

Toxoplasma gondii is a widely distributed apicomplexan parasite causing toxoplasmosis, a critical health issue for immunocompromised individuals and for congenitally infected foetuses. Current treatment options are limited in number and associated with severe side effects. Thus, novel anti-toxoplasma agents need to be identified and developed. 1-Deoxy-d-xylulose 5-phosphate reductoisomerase (DXR) is considered the rate-limiting enzyme in the non-mevalonate pathway for the biosynthesis of the isoprenoid precursors isopentenyl pyrophosphate and dimethylallyl pyrophosphate in the parasite, and has been previously investigated for its key role as a novel drug target in some species, encompassing Plasmodia, Mycobacteria and Escherichia coli. In this study, we present the first crystal structure of T. gondii DXR (TgDXR) in a tertiary complex with the inhibitor fosmidomycin and the cofactor NADPH in dimeric conformation at 2.5 Šresolution revealing the inhibitor binding mode. In addition, we biologically characterize reverse α-phenyl-ß-thia and ß-oxa fosmidomycin analogues and show that some derivatives are strong inhibitors of TgDXR which also, in contrast with fosmidomycin, inhibit the growth of T. gondii in vitro. Here, ((3,4-dichlorophenyl)((2-(hydroxy(methyl)amino)-2-oxoethyl)thio)methyl)phosphonic acid was identified as the most potent anti T. gondii compound. These findings will enable the future design and development of more potent anti-toxoplasma DXR inhibitors.


Assuntos
Aldose-Cetose Isomerases , Fosfomicina , Complexos Multienzimáticos , Toxoplasma , Toxoplasma/enzimologia , Toxoplasma/efeitos dos fármacos , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Fosfomicina/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Cristalografia por Raios X , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Proteínas de Protozoários/antagonistas & inibidores , Proteínas de Protozoários/química , Proteínas de Protozoários/metabolismo , Proteínas de Protozoários/genética , NADP/metabolismo , NADP/química , Humanos , Modelos Moleculares , Oxirredutases/antagonistas & inibidores , Oxirredutases/química , Oxirredutases/metabolismo
3.
Food Chem ; 457: 140127, 2024 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-38908252

RESUMO

The production of the sugars fructose and lactulose from lactose using the enzymes ß-galactosidase and glucose isomerase immobilized on bacterial cellulose (BC) membranes has been investigated. Lactose is hydrolyzed by ß-galactosidase at 30 °C to glucose and galactose at a high conversion rate, while at the same temperature, glucose isomerase is not effective in converting the produced glucose to fructose. The rate of the isomerization reaction of glucose to fructose at 70 °C has been studied. Two types of enzyme immobilization were investigated: immobilization in one stage and immobilization in two stages. The results showed that BC membrane increased three-fold the yield and the reaction rate of fructose and lactulose production from lactose. The noteworthy enhancement of BC membranes' impact on the isomerization reaction by immobilized enzymes grants permission for a novel research avenue within the context of white biotechnology development. Additionally, this effect amplifies the role of BC in sustainability and the circular economy.


Assuntos
Celulose , Enzimas Imobilizadas , Frutose , Lactose , Lactulose , beta-Galactosidase , Lactulose/química , Lactulose/metabolismo , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Lactose/química , Lactose/metabolismo , Celulose/química , Celulose/metabolismo , Frutose/química , Frutose/metabolismo , beta-Galactosidase/química , beta-Galactosidase/metabolismo , Isomerismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Proteínas de Bactérias/química , Proteínas de Bactérias/metabolismo , Biocatálise , Bactérias/enzimologia , Bactérias/química , Bactérias/metabolismo
4.
Appl Microbiol Biotechnol ; 108(1): 391, 2024 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-38910188

RESUMO

Metal cofactors are essential for catalysis and enable countless conversions in nature. Interestingly, the metal cofactor is not always static but mobile with movements of more than 4 Å. These movements of the metal can have different functions. In the case of the xylose isomerase and medium-chain dehydrogenases, it clearly serves a catalytic purpose. The metal cofactor moves during substrate activation and even during the catalytic turnover. On the other hand, in class II aldolases, the enzymes display resting states and active states depending on the movement of the catalytic metal cofactor. This movement is caused by substrate docking, causing the metal cofactor to take the position essential for catalysis. As these metal movements are found in structurally and mechanistically unrelated enzymes, it has to be expected that this metal movement is more common than currently perceived. KEY POINTS: • Metal ions are essential cofactors that can move during catalysis. • In class II aldolases, the metal cofactors can reside in a resting state and an active state. • In MDR, the movement of the metal cofactor is essential for substrate docking.


Assuntos
Coenzimas , Metais , Metais/metabolismo , Coenzimas/metabolismo , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/genética , Catálise , Oxirredutases/metabolismo , Oxirredutases/química
5.
EMBO J ; 43(13): 2636-2660, 2024 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-38778156

RESUMO

During infection viruses hijack host cell metabolism to promote their replication. Here, analysis of metabolite alterations in macrophages exposed to poly I:C recognises that the antiviral effector Protein Kinase RNA-activated (PKR) suppresses glucose breakdown within the pentose phosphate pathway (PPP). This pathway runs parallel to central glycolysis and is critical to producing NADPH and pentose precursors for nucleotides. Changes in metabolite levels between wild-type and PKR-ablated macrophages show that PKR controls the generation of ribose 5-phosphate, in a manner distinct from its established function in gene expression but dependent on its kinase activity. PKR phosphorylates and inhibits the Ribose 5-Phosphate Isomerase A (RPIA), thereby preventing interconversion of ribulose- to ribose 5-phosphate. This activity preserves redox control but decreases production of ribose 5-phosphate for nucleotide biosynthesis. Accordingly, the PKR-mediated immune response to RNA suppresses nucleic acid production. In line, pharmacological targeting of the PPP during infection decreases the replication of the Herpes simplex virus. These results identify an immune response-mediated control of host cell metabolism and suggest targeting the RPIA as a potential innovative antiviral treatment.


Assuntos
Macrófagos , Via de Pentose Fosfato , Ribosemonofosfatos , eIF-2 Quinase , Animais , Ribosemonofosfatos/metabolismo , Camundongos , eIF-2 Quinase/metabolismo , eIF-2 Quinase/genética , Macrófagos/imunologia , Macrófagos/metabolismo , Macrófagos/virologia , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , RNA/metabolismo , RNA/genética , Poli I-C/farmacologia , Ácidos Nucleicos/metabolismo , Ácidos Nucleicos/imunologia , Replicação Viral , Fosforilação
6.
Biomolecules ; 14(4)2024 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-38672412

RESUMO

Alzheimer's disease (AD) is a neurodegenerative olfactory disorder affecting millions of people worldwide. Alterations in the hexosamine- or glucose-related pathways have been described through AD progression. Specifically, an alteration in glucosamine 6 phosphate isomerase 2 (GNPDA2) protein levels has been observed in olfactory areas of AD subjects. However, the biological role of GNPDA2 in neurodegeneration remains unknown. Using mass spectrometry, multiple GNPDA2 interactors were identified in human nasal epithelial cells (NECs) mainly involved in intraciliary transport. Moreover, GNPDA2 overexpression induced an increment in NEC proliferation rates, accompanied by transcriptomic alterations in Type II interferon signaling or cellular stress responses. In contrast, the presence of beta-amyloid or mutated Tau-P301L in GNPDA2-overexpressing NECs induced a slowdown in the proliferative capacity in parallel with a disruption in protein processing. The proteomic characterization of Tau-P301L transgenic zebrafish embryos demonstrated that GNPDA2 overexpression interfered with collagen biosynthesis and RNA/protein processing, without inducing additional changes in axonal outgrowth defects or neuronal cell death. In humans, a significant increase in serum GNPDA2 levels was observed across multiple neurological proteinopathies (AD, Lewy body dementia, progressive supranuclear palsy, mixed dementia and amyotrophic lateral sclerosis) (n = 215). These data shed new light on GNPDA2-dependent mechanisms associated with the neurodegenerative process beyond the hexosamine route.


Assuntos
Aldose-Cetose Isomerases , Doença de Alzheimer , Peptídeos beta-Amiloides , Peixe-Zebra , Proteínas tau , Animais , Humanos , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/genética , Doença de Alzheimer/metabolismo , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/metabolismo , Animais Geneticamente Modificados , Proliferação de Células , Células Epiteliais/metabolismo , Proteômica , Proteínas tau/metabolismo , Proteínas tau/genética , Peixe-Zebra/metabolismo
7.
ACS Infect Dis ; 10(5): 1739-1752, 2024 05 10.
Artigo em Inglês | MEDLINE | ID: mdl-38647213

RESUMO

Reverse analogs of the phosphonohydroxamic acid antibiotic fosmidomycin are potent inhibitors of the nonmevalonate isoprenoid biosynthesis enzyme 1-deoxy-d-xylulose 5-phosphate reductoisomerase (DXR, IspC) of Plasmodium falciparum. Some novel analogs with large phenylalkyl substituents at the hydroxamic acid nitrogen exhibit nanomolar PfDXR inhibition and potent in vitro growth inhibition of P. falciparum parasites coupled with good parasite selectivity. X-ray crystallographic studies demonstrated that the N-phenylpropyl substituent of the newly developed lead compound 13e is accommodated in a subpocket within the DXR catalytic domain but does not reach the NADPH binding pocket of the N-terminal domain. As shown for reverse carba and thia analogs, PfDXR selectively binds the S-enantiomer of the new lead compound. In addition, some representatives of the novel inhibitor subclass are nanomolar Escherichia coli DXR inhibitors, whereas the inhibition of Mycobacterium tuberculosis DXR is considerably weaker.


Assuntos
Aldose-Cetose Isomerases , Antimaláricos , Fosfomicina , Ácidos Hidroxâmicos , Complexos Multienzimáticos , Plasmodium falciparum , Fosfomicina/farmacologia , Fosfomicina/análogos & derivados , Fosfomicina/química , Aldose-Cetose Isomerases/antagonistas & inibidores , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/química , Plasmodium falciparum/efeitos dos fármacos , Plasmodium falciparum/enzimologia , Ácidos Hidroxâmicos/farmacologia , Ácidos Hidroxâmicos/química , Antimaláricos/farmacologia , Antimaláricos/química , Complexos Multienzimáticos/antagonistas & inibidores , Complexos Multienzimáticos/metabolismo , Complexos Multienzimáticos/química , Cristalografia por Raios X , Inibidores Enzimáticos/farmacologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/síntese química , Relação Estrutura-Atividade , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Escherichia coli/enzimologia , Modelos Moleculares , Mycobacterium tuberculosis/efeitos dos fármacos , Mycobacterium tuberculosis/enzimologia , Domínio Catalítico , Oxirredutases/antagonistas & inibidores , Oxirredutases/metabolismo
8.
J Agric Food Chem ; 72(14): 7684-7693, 2024 Apr 10.
Artigo em Inglês | MEDLINE | ID: mdl-38532701

RESUMO

Fosmidomycin (FOS) is a natural product inhibiting the DXR enzyme in the MEP pathway and has stimulated interest for finding more suitable FOS analogues. Herein, two series of FOS analogue hydroxamate-containing bisphosphonates as proherbicides were designed, with bisphosphonate replacing the phosphonic unit in FOS while retaining the hydroxamate (BPF series) or replacing it with retro-hydroxamate (BPRF series). The BPF series were synthesized through a three-step reaction sequence including Michael addition of vinylidenebisphosphonate, N-acylation, and deprotection, and the BPRF series were synthesized with a retro-Claisen condensation incorporated into the reaction sequence. Evaluation on model plants demonstrated several compounds having considerable herbicidal activities, and in particular, compound 8m exhibited multifold activity enhancement as compared to the control FOS. The proherbicide properties were comparatively validated. Furthermore, DXR enzyme assay, dimethylallyl pyrophosphate rescue, and molecular docking verified 8m to be a promising proherbicide candidate targeting the DXR enzyme. In addition, 8m also displayed good antimalarial activities.


Assuntos
Aldose-Cetose Isomerases , Antimaláricos , Fosfomicina , Fosfomicina/análogos & derivados , Difosfonatos , Simulação de Acoplamento Molecular , Fosfomicina/farmacologia , Aldose-Cetose Isomerases/metabolismo
9.
Prep Biochem Biotechnol ; 54(8): 1058-1067, 2024 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-38349751

RESUMO

Saccharomyces cerevisiae cannot assimilate xylose, second to glucose derived from lignocellulosic biomass. Here, the engineered S. cerevisiae strains INVSc-XI and INVSc-XI/XT were constructed using xylA and Xltr1p to co-utilize xylose and glucose, achieving economic viability and sustainable production of fuels. The xylose utilization rate of INVSc-XI/XT was 2.3-fold higher than that of INVSc-XI, indicating that overexpressing Xltr1p could further enhance xylose utilization. In mixed sugar media, a small amount of glucose enhanced the consumption of xylose by INVSc-XI/XT. Transcriptome analysis showed that glucose increased the upregulation of acetate of coenzyme A synthetase (ACS), alcohol dehydrogenase (ADH), and transketolase (TKL) gene expression in INVSc-XI/XT, further promoting xylose utilization and ethanol yield. The highest ethanol titer of 2.91 g/L with a yield of 0.29 g/g at 96 h by INVSc-XI/XT was 56.9% and 63.0% of the theoretical ethanol yield from glucose and xylose, respectively. These results showed overexpression of xylA and Xltr1p is a promising strategy for improving xylose and glucose conversion to ethanol. Although the ability of strain INVSc-XI/XT to produce ethanol was not very satisfactory, glucose was discovered to influence xylose utilization in strain INVSc-XI/XT. Altering the glucose concentration is a promising strategy to improve the xylose and glucose co-utilization.


INVSc-XI and INVSc-XI/XT strains were newly constructed to utilize xylose and glucose.XylA, in combination with xylose transporter Xltr1p, enhances xylose consumption.A small amount of glucose enhanced xylose utilization in INVSc-XI/XT strain.The expression of ACS, ADH, and TKL genes is upregulated in the media containing mixed sugars.The highest ethanol yield of 0.29 g/g was produced in a 2-L scale-up fermenter.


Assuntos
Aldose-Cetose Isomerases , Etanol , Fermentação , Glucose , Engenharia Metabólica , Saccharomyces cerevisiae , Xilose , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo , Etanol/metabolismo , Glucose/metabolismo , Engenharia Metabólica/métodos , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Monossacarídeos/genética , Proteínas de Transporte de Monossacarídeos/metabolismo
10.
Enzyme Microb Technol ; 173: 110355, 2024 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-38041880

RESUMO

Due to the increasing demand for health-conscious and environmentally friendly products, D-mannose has gained significant attention as a natural, low-calorie sweetener. The use of D-mannose isomerases (D-MIases) for D-mannose production has emerged as a prominent area of research, offering superior advantages compared with conventional methods such as plant extraction and chemical synthesis. In this study, a gene encoding D-MIase was cloned from Bifidobacterium and expressed in E. coli BL21 (DE3). The heterologously expressed enzyme, Bifi-mannose, formed a trimer with a molecular weight of 146.3 kDa and a melting temperature (Tm) of 63.39 ± 1.3 °C. Bifi-mannose exhibited optimal catalytic activity at pH 7.5 and 55 °C, and retained more than 80% of its activity after a 3-hour incubation at 55 °C, demonstrating excellent thermal stability. The Km, Vmax, and kcat/Km values of Bifi-mannose for D-fructose isomerization were determined as 538.7 ± 62.5 mM, 11.7 ± 0.9 µmol·mg1·s1, and 1.02 ± 0.3 mM1·s1, respectively. Notably, under optimized conditions, catalytic yields of 29.4, 87.1, and 148.5 mg·mL1 were achieved when using 100, 300, and 500 mg·mL1 of D-fructose as substrates, resulting in a high conversion rate (29%). Furthermore, kinetic parameters and molecular docking studies revealed that His387 residue primarily participates in the opening of the pyranose ring, while His253 acts as a basic catalyst in the isomerization process.


Assuntos
Aldose-Cetose Isomerases , Bifidobacterium bifidum , Manose , Escherichia coli/metabolismo , Bifidobacterium bifidum/genética , Bifidobacterium bifidum/metabolismo , Simulação de Acoplamento Molecular , Aldose-Cetose Isomerases/metabolismo , Frutose , Temperatura , Concentração de Íons de Hidrogênio , Cinética , Proteínas Recombinantes/genética , Proteínas Recombinantes/química , Clonagem Molecular
11.
Enzyme Microb Technol ; 168: 110259, 2023 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-37245327

RESUMO

D-Allose is a rare cis-caprose with a wide range of physiological functions, which has a wide range of applications in medicine, food, and other industries. L-Rhamnose isomerase (L-Rhi) is the earliest enzyme found to catalyze the production of D-allose from D-psicose. This catalyst has a high conversion rate, but its specificity for substrates is limited; thus, it cannot fulfill the requirements of industrial production of D-allose. In this study, L-Rhi derived from Bacillus subtilis was employed as the research subject, and D-psicose as the conversion substrate. Two mutant libraries were constructed through alanine scanning, saturation mutation, and rational design based on the analysis of the secondary structure, tertiary structure, and interactions with ligands of the enzyme. The yield of D-allose produced by these mutants was assessed; it was found that the conversion rate of mutant D325M to D-allose was increased by 55.73 %, and the D325S improved by 15.34 %, while mutant W184H increased by 10.37 % at 55 °C, respectively. According to modeling analysis, manganese (Mn2+) had no significant effect on the production of D-psicose from D-psicose by L-Rhi. The results of molecular dynamics simulation demonstrated that the mutants W184H, D325M, and D325S had more stable protein structures while binding with the substrate D-psicose, as evidenced by its root mean square deviation (RMSD), root mean square fluctuation (RMSF), and binding free energy values. It was more conducive to binding D-psicose and facilitating its conversion to D-allose, providing the basis for the production of D-allose.


Assuntos
Aldose-Cetose Isomerases , Glucose , Glucose/metabolismo , Frutose/metabolismo , Aldose-Cetose Isomerases/metabolismo , Mutação
12.
Methods Enzymol ; 685: 279-318, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37245905

RESUMO

Methylthio-d-ribose-1-phosphate (MTR1P) isomerase (MtnA) catalyzes the reversible isomerization of the aldose MTR1P into the ketose methylthio-d-ribulose 1-phosphate. It serves as a member of the methionine salvage pathway that many organisms require for recycling methylthio-d-adenosine, a byproduct of S-adenosylmethionine metabolism, back to methionine. MtnA is of mechanistic interest because unlike most other aldose-ketose isomerases, its substrate exists as an anomeric phosphate ester and therefore cannot equilibrate with a ring-opened aldehyde that is otherwise required to promote isomerization. To investigate the mechanism of MtnA, it is necessary to establish reliable methods for determining the concentration of MTR1P and to measure enzyme activity in a continuous assay. This chapter describes several such protocols needed to perform steady-state kinetics measurements. It additionally outlines the preparation of [32P]MTR1P, its use in radioactively labeling the enzyme, and the characterization of the resulting phosphoryl adduct.


Assuntos
Aldose-Cetose Isomerases , Ribose , Cinética , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo
13.
J Agric Food Chem ; 71(10): 4228-4234, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36867179

RESUMO

The valorization of galactose derived from acid whey to low-calorie tagatose has gained increasing attention. Enzymatic isomerization is of great interest but faces several challenges, such as poor thermal stability of enzymes and a long processing time. In this work, non-enzymatic (supercritical fluids, triethylamine, arginine, boronate affinity, hydrotalcite, Sn-ß zeolite, and calcium hydroxide) pathways for galactose to tagatose isomerization were critically discussed. Unfortunately, most of these chemicals showed poor tagatose yields (<30%), except for calcium hydroxide (>70%). The latter is able to form a tagatose-calcium hydroxide-water complex, which stimulates the equilibrium toward tagatose and prevents sugar degradation. Nevertheless, the excessive use of calcium hydroxide may pose challenges in terms of economic and environmental feasibility. Moreover, the proposed mechanisms for the base (enediol intermediate) and Lewis acid (hydride shift between C-2 and C-1) catalysis of galactose were elucidated. Overall, it is crucial to explore novel and effective catalysts as well as integrated systems for isomerizing of galactose to tagatose.


Assuntos
Aldose-Cetose Isomerases , Galactose , Galactose/metabolismo , Isomerismo , Hidróxido de Cálcio , Aldose-Cetose Isomerases/metabolismo , Hexoses/metabolismo
14.
ChemMedChem ; 18(11): e202200590, 2023 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-36896721

RESUMO

In this work, we demonstrate how important it is to investigate not only on-target activity but to keep antibiotic activity against critical pathogens in mind. Since antimicrobial resistance is spreading in bacteria such as Mycobacterium tuberculosis, investigations into new targets are urgently needed. One promising new target is 1-deoxy-d-xylulose 5-phosphate synthase (DXPS) of the 2-C-methyl-d-erythritol 4-phosphate (MEP) pathway. We have recently solved the crystal structure of truncated M. tuberculosis DXPS and used it to perform a virtual screening in collaboration with Atomwise Inc. using their deep convolutional neural network-based AtomNet® platform. Of 94 virtual hit compounds only one showed interesting results in binding and activity studies. We synthesized 30 close derivatives using a straightforward synthetic route that allowed for easy derivatization. However, no improvement in activity was observed for any of the derivatives. Therefore, we tested them against a variety of pathogens and found them to be good inhibitors against Escherichia coli.


Assuntos
Aldose-Cetose Isomerases , Mycobacterium tuberculosis , Fosfatos Açúcares , Antibacterianos/farmacologia , Antibacterianos/metabolismo , Óxido Nítrico Sintase/metabolismo , Escherichia coli/metabolismo , Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo
15.
Int J Biol Macromol ; 231: 123406, 2023 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-36702217

RESUMO

Aim of present study was to develop biological catalysts of L-arabinose isomerase (L-AI) by immobilizing on four different supports such as multiwalled carbon nanotube (MWCNT), graphene oxide (GOx), Santa Barbara Amorphous (SBA-15) and mobile composite matter (MCM-41). Also, comparative analysis of the developed catalysts was performed to evolve the best in terms of transformation efficiency for D-tagatose production. The developed nano-enzyme conjugates (NECs) were characterized using the high resolution transmission electron microscopy (HR-TEM) and elemental analysis was performed by energy dispersive X-ray spectroscopy (EDS). The functional groups were investigated by Fourier transform infra red spectroscopy. Also, the thermo gravimetric analysis (TGA) was employed to plot a thermal degradation weight loss profile of NECs. The conjugated L-AI with MWCNT and GOx were found to be more promising immobilized catalysts due to their ability to provide more surface area. Conversion of D-Galactose to D-Tagatose at moderate temperature and pH was observed to attain the equilibrium level of transformation (~50%). On the contrary, NECs prepared using SBA-15 and MCM-41 as support matrix were unable to reach the equilibrium level of conversion. Additionally, the developed NECs were suitable for reuse in multiple batch cycles. Thus, promising nanotechnology coupled with biocatalysis made the transformation of D-Galactose into D-tagatose more economically sustainable.


Assuntos
Aldose-Cetose Isomerases , Galactose , Galactose/química , Açúcares , Hexoses/química , Aldose-Cetose Isomerases/metabolismo
16.
J Agric Food Chem ; 70(43): 13959-13968, 2022 Nov 02.
Artigo em Inglês | MEDLINE | ID: mdl-36264233

RESUMO

The development of a suitable immobilization strategy to improve the performance of immobilized glucose isomerase for the isomerization of glucose to fructose is crucial to promoting the industrial production of high-fructose syrup. In this work, a novel recyclable upper critical solution temperature (UCST)-type mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst (PVAA@GI) was prepared, and the immobilized glucose isomerase could capture the glucose substrate through the affinity of 4-vinylbenzeneboronic acid (4-VBA) and the glucose substrate, which led to the enhanced substrate affinity and catalytic efficiency of the PVAA@GI. The biocatalyst exhibited excellent stability in pH, thermal, storage, and recycling compared to the free enzyme. The mVBA-b-P(AAm-co-AN)@glucose isomerase biocatalyst displayed reversibly soluble-insoluble characteristics with temperature change, which was in the soluble state during the enzyme reaction process but could be recovered in an insoluble form by lowering the temperature after the reaction. The highest fructose production rate reached 62.79%, which would have potential application in the industrial production of high-fructose syrup.


Assuntos
Aldose-Cetose Isomerases , Frutose , Glucose , Temperatura , Isomerismo , Enzimas Imobilizadas/metabolismo , Aldose-Cetose Isomerases/metabolismo
17.
J Biotechnol ; 358: 9-16, 2022 Nov 10.
Artigo em Inglês | MEDLINE | ID: mdl-36030895

RESUMO

l-Rhamnose isomerase (l-RhI) catalyzes rare sugar isomerization between aldoses and ketoses. In an attempt to alter the substrate specificity of Thermoanaerobacterium saccharolyticus NTOU1 l-RhI (TsRhI), residue Ile102 was changed to other polar or charged amino acid residues by site-directed mutagenesis. The results of activity-screening using different substrates indicate that I102N, I102Q, and I102R TsRhIs can increase the preference against d-allose in comparison with the wild-type enzyme. The catalytic efficiencies of the purified I102N, I102Q, and I102R TsRhIs against d-allose are 148 %, 277 %, and 191 %, respectively, of that of wild-type enzyme, while those against l-rhamnose are 100 %, 167 % and 87 %, respectively. Mutant I102N, I102Q, and I102R TsRhIs were noted to have the altered substrate specificity, and I102Q TsRhI has the highest catalytic efficiency against d-allose presumably through the formation of an additional hydrogen bond with d-allose. The purified wild-type and mutant TsRhIs were further used to produce d-allose from 100 g/L d-fructose in the presence of d-allulose 3-epimerase, and the yields can reach as high as 22 % d-allulose and 12 % d-allose upon equilibrium. I102Q TsRhI takes only around half of the time to reach the same 12 % d-allose yield, suggesting that this mutant enzyme has a potential to be applied in d-allose production.


Assuntos
Aldose-Cetose Isomerases , Thermoanaerobacterium , Aldose-Cetose Isomerases/metabolismo , Aminoácidos , Frutose/metabolismo , Glucose/metabolismo , Cetoses , Racemases e Epimerases/metabolismo , Ramnose/metabolismo , Especificidade por Substrato , Thermoanaerobacterium/genética , Thermoanaerobacterium/metabolismo
18.
Appl Biochem Biotechnol ; 194(10): 4852-4866, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-35670905

RESUMO

L-ribulose, a kind of high-value rare sugar, could be utilized to manufacture L-form sugars and antiviral drugs, generally produced from L-arabinose as a substrate. However, the production of L-ribulose from L-arabinose is limited by the equilibrium ratio of the catalytic reaction, hence, it is necessary to explore a new biological enzymatic method to produce L-ribulose. Ribose-5-phosphate isomerase (Rpi) is an enzyme that can catalyze the reversible isomerization between L-ribose and L-ribulose, which is of great significance for the preparation of L-ribulose. In order to obtain highly active ribose-5-phosphate isomerase to manufacture L-ribulose, ribose-5-phosphate isomerase A (OsRpiA) from Ochrobactrum sp. CSL1 was engineered based on structural and sequence analyses. Through a rational design strategy, a triple-mutant strain A10T/T32S/G101N with 160% activity was acquired. The enzymatic properties of the mutant were systematically investigated, and the optimum conditions were characterized to achieve the maximum yield of L-ribulose. Kinetic analysis clarified that the A10T/T32S/G101N mutant had a stronger affinity for the substrate and increased catalytic efficiency. Furthermore, molecular dynamics simulations indicated that the binding of the substrate to A10T/T32S/G101N was more stable than that of wild type. The shorter distance between the catalytic residues of A10T/T32S/G101N and L-ribose illuminated the increased activity. Overall, the present study provided a solid basis for demonstrating the complex functions of crucial residues in RpiAs as well as in rare sugar preparation.


Assuntos
Aldose-Cetose Isomerases , Ochrobactrum , Aldose-Cetose Isomerases/metabolismo , Antivirais , Arabinose/metabolismo , Cinética , Ochrobactrum/genética , Ochrobactrum/metabolismo , Pentoses , Ribose
19.
Lett Appl Microbiol ; 74(6): 941-948, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35239207

RESUMO

The current climate crisis demands replacement of fossil energy sources with sustainable alternatives. In this scenario, second-generation bioethanol, a product of lignocellulosic biomass fermentation, represents a more sustainable alternative. However, Saccharomyces cerevisiae cannot metabolize pentoses, such as xylose, present as a major component of lignocellulosic biomass. Xylose isomerase (XI) is an enzyme that allows xylose consumption by yeasts, because it converts xylose into xylulose, which is further converted to ethanol by the pentose-phosphate pathway. Only a few XI were successfully expressed in S. cerevisiae strains. This work presents a new bacterial XI, named GR-XI 1, obtained from a Brazilian goat rumen metagenomic library. Phylogenetic analysis confirmed the bacterial origin of the gene, which is related to Firmicutes XIs. After codon optimization, this enzyme, renamed XySC1, was functionally expressed in S. cerevisiae, allowing growth in media with xylose as sole carbon source. Overexpression of XySC1 in S. cerevisiae allowed the recombinant strain to efficiently consume and metabolize xylose under aerobic conditions.


Assuntos
Aldose-Cetose Isomerases , Cabras , Microbiota , Rúmen , Saccharomyces cerevisiae , Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Animais , Fermentação , Cabras/microbiologia , Filogenia , Rúmen/enzimologia , Rúmen/microbiologia , Saccharomyces cerevisiae/metabolismo , Xilose/metabolismo
20.
Proc Natl Acad Sci U S A ; 119(7)2022 02 15.
Artigo em Inglês | MEDLINE | ID: mdl-35101915

RESUMO

Protein crystallization is an astounding feat of nature. Even though proteins are large, anisotropic molecules with complex, heterogeneous surfaces, they can spontaneously group into two- and three-dimensional arrays with high precision. And yet, the biggest hurdle in this assembly process, the formation of a nucleus, is still poorly understood. In recent years, the two-step nucleation model has emerged as the consensus on the subject, but it still awaits extensive experimental verification. Here, we set out to reconstruct the nucleation pathway of the candidate protein glucose isomerase (GI), for which there have been indications that it may follow a two-step nucleation pathway under certain conditions. We find that the precursor phase present during the early stages of the reaction process is nanoscopic crystallites that have lattice symmetry equivalent to the mature crystals found at the end of a crystallization experiment. Our observations underscore the need for experimental data at a lattice-resolving resolution on other proteins so that a general picture of protein crystal nucleation can be formed.


Assuntos
Aldose-Cetose Isomerases/química , Aldose-Cetose Isomerases/metabolismo , Cristalização , Microscopia Crioeletrônica , Modelos Químicos
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