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1.
Molecules ; 26(17)2021 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-34500777

RESUMO

Human neutrophil elastase (HNE) is a uniquely destructive serine protease with the ability to unleash a wave of proteolytic activity by destroying the inhibitors of other proteases. Although this phenomenon forms an important part of the innate immune response to invading pathogens, it is responsible for the collateral host tissue damage observed in chronic conditions such as chronic obstructive pulmonary disease (COPD), and in more acute disorders such as the lung injuries associated with COVID-19 infection. Previously, a combinatorially selected activity-based probe revealed an unexpected substrate preference for oxidised methionine, which suggests a link to oxidative pathogen clearance by neutrophils. Here we use oxidised model substrates and inhibitors to confirm this observation and to show that neutrophil elastase is specifically selective for the di-oxygenated methionine sulfone rather than the mono-oxygenated methionine sulfoxide. We also posit a critical role for ordered solvent in the mechanism of HNE discrimination between the two oxidised forms methionine residue. Preference for the sulfone form of oxidised methionine is especially significant. While both host and pathogens have the ability to reduce methionine sulfoxide back to methionine, a biological pathway to reduce methionine sulfone is not known. Taken together, these data suggest that the oxidative activity of neutrophils may create rapidly cleaved elastase "super substrates" that directly damage tissue, while initiating a cycle of neutrophil oxidation that increases elastase tissue damage and further neutrophil recruitment.


Assuntos
Imunidade Inata , Elastase de Leucócito/metabolismo , Metionina/análogos & derivados , Neutrófilos/imunologia , Biocatálise , COVID-19/imunologia , COVID-19/patologia , COVID-19/virologia , Domínio Catalítico/genética , Ensaios Enzimáticos , Interações Hospedeiro-Patógeno/imunologia , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/genética , Pulmão/imunologia , Pulmão/patologia , Pulmão/virologia , Metionina/metabolismo , Simulação de Dinâmica Molecular , Infiltração de Neutrófilos , Neutrófilos/enzimologia , Oxirredução/efeitos dos fármacos , Proteólise/efeitos dos fármacos , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , SARS-CoV-2/imunologia , Especificidade por Substrato/imunologia
2.
Sheng Wu Gong Cheng Xue Bao ; 37(8): 2623-2632, 2021 Aug 25.
Artigo em Chinês | MEDLINE | ID: mdl-34472283

RESUMO

α-L-rhamnosidase is a very important industrial enzyme that is widely distributed in a variety of organisms. α-L-rhamnosidase of different origins show functional diversity. For example, the optimal pH of α-L-rhamnosidase from bacteria is close to neutral or alkaline, while the optimal pH of α-L-rhamnosidase from fungi is in the acidic range. Furthermore, the enzymatic properties of α-L-rhamnosidases of different origins differ in terms of the optimal temperature, the thermal stability, and the substrate specificity, which determine the different applications of these enzymes. In this connection, it is crucial to elucidate the similarities and differences in the catalytic mechanism and substrate specificity of α-L-rhamnosidase of different origins through analyzing its enzymatic properties. Moreover, it is important to explore and understand the effects of aglycon and metal cations on enzyme activity and the competitive inhibition of L-rhamnose and glucose on enzymes. These knowledge can help discover α-L-rhamnosidase of industrial significance and promote its industrial application.


Assuntos
Glicosídeo Hidrolases , Ramnose , Glicosídeo Hidrolases/metabolismo , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura
3.
Appl Microbiol Biotechnol ; 105(18): 6793-6803, 2021 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-34477943

RESUMO

Some microbial-associated molecular patterns (MAMPs), like glucan oligosaccharides, can be recognized by pattern recognition receptors (PRRs) of plant to elicit further immunity response. In this study, a novel glycoside hydrolase family 55 ß-1,3-glucanase (AcGluA) from Archangium sp. strain AC19 was cloned and expressed in Escherichia coli. Among the reported ß-1, 3-glucanases from the glycoside hydrolase 55 family, the purified AcGluA exhibited the highest activity on laminarin at pH 6.0 and 60 °C with 112.3 U/mg. Activity of AcGluA was stable in the range of pH 4.0-9.0 and at temperatures below 60 °C. The Km and Vmax of AcGluA for laminarin were 3.5 mg/ml and 263.5 µmol/(ml·min). AcGluA hydrolyzed laminarin into a series of oligosaccharides, suggesting it was an endo-ß-1,3-glucanase. The high dose of oligosaccharides (1600 mg/l) had conspicuous biocontrol efficacy on the defense of rice seedlings to Magnaporthe oryzae, which provided a new idea for the development of green biopesticide.Key points• The AcGluA was determined bacteria-derived ß-1,3-glucanases in the GH55 family.• The AcGluA showed the highest activity towards laminarin among reported GH55 family.• The hydrolysates of laminarin showed conspicuous biocontrol efficacy to M. oryzae.


Assuntos
Ascomicetos , Glicosídeo Hidrolases , Ascomicetos/metabolismo , Clonagem Molecular , Escherichia coli/genética , Escherichia coli/metabolismo , Glicosídeo Hidrolases/genética , Glicosídeo Hidrolases/metabolismo , Hidrólise , Especificidade por Substrato
4.
Enzyme Microb Technol ; 150: 109882, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34489035

RESUMO

Glycogen branching enzymes (GBEs; 1,4-α-glucan branching enzyme; E.C. 2.4.1.18) have so far been described to be capable of both α-1,6-transglycosylation (branching) and α-1,4-hydrolytic activity. The aim of the present study was to elucidate the mode of action of three distantly related GBEs from the glycoside hydrolase family 13 by in depth analysis of the activity on a well-defined substrate. For this purpose, the GBEs from R. marinus (RmGBE), P. mobilis (PmGBE1), and B. fibrisolvens (BfGBE) were incubated with a highly pure fraction of a linear substrate of 18 anhydroglucose units. A well-known and characterized branching enzyme from E. coli (EcGBE) was also taken along. Analysis of the chain length distribution over time revealed that, next to hydrolytic and branching activity, all three GBEs were capable of generating chains longer than the substrate, clearly showing α-1,4-transglycosylation activity. Furthermore, the GBEs used those elongated chains for further branching. The sequential activity of elongation and branching enabled the GBEs to modify the substrate to a far larger extent than would have been possible with branching activity alone. Overall, the three GBEs acted ambiguous on the defined substrate. RmGBE appeared to have a strong preference towards transferring chains of nine anhydroglucose units, even during elongation, with a comparably low activity. BfGBE generated an array of elongated chains before using the chains for introducing branches while PmGBE1 exhibited a behaviour intermediate of the other two enzymes. On the basis of the mode of action revealed in this research, an updated model of the mechanism of GBEs was proposed now including the α-1,4-transglycosylation activity.


Assuntos
Enzima Ramificadora de 1,4-alfa-Glucana , Enzima Ramificadora de 1,4-alfa-Glucana/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Glucanos , Glicogênio , Especificidade por Substrato
5.
Sci Rep ; 11(1): 17810, 2021 09 08.
Artigo em Inglês | MEDLINE | ID: mdl-34497279

RESUMO

Transporters in the human liver play a major role in the clearance of endo- and xenobiotics. Apical (canalicular) transporters extrude compounds to the bile, while basolateral hepatocyte transporters promote the uptake of, or expel, various compounds from/into the venous blood stream. In the present work we have examined the in vitro interactions of some key repurposed drugs advocated to treat COVID-19 (lopinavir, ritonavir, ivermectin, remdesivir and favipiravir), with the key drug transporters of hepatocytes. These transporters included ABCB11/BSEP, ABCC2/MRP2, and SLC47A1/MATE1 in the canalicular membrane, as well as ABCC3/MRP3, ABCC4/MRP4, SLC22A1/OCT1, SLCO1B1/OATP1B1, SLCO1B3/OATP1B3, and SLC10A1/NTCP, residing in the basolateral membrane. Lopinavir and ritonavir in low micromolar concentrations inhibited BSEP and MATE1 exporters, as well as OATP1B1/1B3 uptake transporters. Ritonavir had a similar inhibitory pattern, also inhibiting OCT1. Remdesivir strongly inhibited MRP4, OATP1B1/1B3, MATE1 and OCT1. Favipiravir had no significant effect on any of these transporters. Since both general drug metabolism and drug-induced liver toxicity are strongly dependent on the functioning of these transporters, the various interactions reported here may have important clinical relevance in the drug treatment of this viral disease and the existing co-morbidities.


Assuntos
Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/metabolismo , Antivirais/farmacologia , Transportador 1 de Ânion Orgânico Específico do Fígado/metabolismo , Fígado/efeitos dos fármacos , Proteínas de Transporte de Cátions Orgânicos/metabolismo , Membro 11 da Subfamília B de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Monofosfato de Adenosina/análogos & derivados , Monofosfato de Adenosina/química , Monofosfato de Adenosina/metabolismo , Monofosfato de Adenosina/farmacologia , Monofosfato de Adenosina/uso terapêutico , Alanina/análogos & derivados , Alanina/química , Alanina/metabolismo , Alanina/farmacologia , Alanina/uso terapêutico , Antivirais/química , Antivirais/metabolismo , Antivirais/uso terapêutico , COVID-19/tratamento farmacológico , Comorbidade , Reposicionamento de Medicamentos , Humanos , Fígado/metabolismo , Fígado/patologia , Transportador 1 de Ânion Orgânico Específico do Fígado/antagonistas & inibidores , Lopinavir/química , Lopinavir/metabolismo , Lopinavir/farmacologia , Lopinavir/uso terapêutico , Proteínas de Transporte de Cátions Orgânicos/antagonistas & inibidores , Ritonavir/química , Ritonavir/metabolismo , Ritonavir/farmacologia , Ritonavir/uso terapêutico , SARS-CoV-2/isolamento & purificação , Especificidade por Substrato
6.
Int J Mol Sci ; 22(15)2021 Jul 29.
Artigo em Inglês | MEDLINE | ID: mdl-34360902

RESUMO

Acyl-CoA:lysophosphatidylethanolamine acyltransferases (LPEATs) are known as enzymes utilizing acyl-CoAs and lysophospholipids to produce phosphatidylethanolamine. Recently, it has been discovered that they are also involved in the growth regulation of Arabidopsis thaliana. In our study we investigated expression of each Camelina sativa LPEAT isoform and their behavior in response to temperature changes. In order to conduct a more extensive biochemical evaluation we focused both on LPEAT enzymes present in microsomal fractions from C. sativa plant tissues, and on cloned CsLPEAT isoforms expressed in yeast system. Phylogenetic analyses revealed that CsLPEAT1c and CsLPEAT2c originated from Camelina hispida, whereas other isoforms originated from Camelina neglecta. The expression ratio of all CsLPEAT1 isoforms to all CsLPEAT2 isoforms was higher in seeds than in other tissues. The isoforms also displayed divergent substrate specificities in utilization of LPE; CsLPEAT1 preferred 18:1-LPE, whereas CsLPEAT2 preferred 18:2-LPE. Unlike CsLPEAT1, CsLPEAT2 isoforms were specific towards very-long-chain fatty acids. Above all, we discovered that temperature strongly regulates LPEATs activity and substrate specificity towards different acyl donors, making LPEATs sort of a sensor of external thermal changes. We observed the presented findings not only for LPEAT activity in plant-derived microsomal fractions, but also for yeast-expressed individual CsLPEAT isoforms.


Assuntos
Aciltransferases/metabolismo , Camellia/enzimologia , Camellia/genética , Fosfatidiletanolaminas/metabolismo , Proteínas de Plantas/metabolismo , Sementes/enzimologia , Temperatura , Acil Coenzima A/metabolismo , Aciltransferases/genética , Camellia/classificação , Camellia/crescimento & desenvolvimento , Resposta ao Choque Frio , DNA de Plantas/genética , Ativação Enzimática , Resposta ao Choque Térmico , Isoenzimas/genética , Microssomos/enzimologia , Filogenia , Proteínas de Plantas/genética , Sementes/crescimento & desenvolvimento , Especificidade por Substrato
7.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361767

RESUMO

This study describes the catalytic properties of a GH30_7 xylanase produced by the fungus Talaromyces leycettanus. The enzyme is an ando-ß-1,4-xylanase, showing similar specific activity towards glucuronoxylan, arabinoxylan, and rhodymenan (linear ß-1,3-ß-1,4-xylan). The heteroxylans are hydrolyzed to a mixture of linear as well as branched ß-1,4-xylooligosaccharides that are shorter than the products generated by GH10 and GH11 xylanases. In the rhodymenan hydrolyzate, the linear ß-1,4-xylooligosaccharides are accompanied with a series of mixed linkage homologues. Initial hydrolysis of glucuronoxylan resembles the action of other GH30_7 and GH30_8 glucuronoxylanases, resulting in a series of aldouronic acids of a general formula MeGlcA2Xyln. Due to the significant non-specific endoxylanase activity of the enzyme, these acidic products are further attacked in the unbranched regions, finally yielding MeGlcA2Xyl2-3. The accommodation of a substituted xylosyl residue in the -2 subsite also applies in arabinoxylan depolymerization. Moreover, the xylose residue may be arabinosylated at both positions 2 and 3, without negatively affecting the main chain cleavage. The catalytic properties of the enzyme, particularly the great tolerance of the side-chain substituents, make the enzyme attractive for biotechnological applications. The enzyme is also another example of extraordinarily great catalytic diversity among eukaryotic GH30_7 xylanases.


Assuntos
Endo-1,4-beta-Xilanases/metabolismo , Proteínas Fúngicas/metabolismo , Talaromyces/enzimologia , Xilanos/metabolismo , Sequência de Aminoácidos , Arabinose/química , Arabinose/metabolismo , Sequência de Carboidratos , Endo-1,4-beta-Xilanases/genética , Proteínas Fúngicas/genética , Expressão Gênica , Glucuronatos/química , Glucuronatos/metabolismo , Hidrólise , Oligossacarídeos/química , Oligossacarídeos/metabolismo , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Talaromyces/química , Talaromyces/genética , Xilanos/química
8.
Molecules ; 26(15)2021 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-34361778

RESUMO

Protein-ligand interaction analysis is important for drug discovery and rational protein design. The existing online tools adopt only a single conformation of the complex structure for calculating and displaying the interactions, whereas both protein residues and ligand molecules are flexible to some extent. The interactions evolved with time in the trajectories are of greater interest. MolADI is a user-friendly online tool which analyzes the protein-ligand interactions in detail for either a single structure or a trajectory. Interactions can be viewed easily with both 2D graphs and 3D representations. MolADI is available as a web application.


Assuntos
Receptor A2A de Adenosina/química , Bibliotecas de Moléculas Pequenas/química , Software , Sítios de Ligação , Humanos , Cinética , Ligantes , Modelos Moleculares , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Proteínas , Receptor A2A de Adenosina/metabolismo , Bibliotecas de Moléculas Pequenas/metabolismo , Especificidade por Substrato , Termodinâmica
9.
FASEB J ; 35(9): e21825, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34383978

RESUMO

Ubiquitination is an essential post-translational modification that regulates protein stability or function. Its substrate specificity is dictated by various E3 ligases. The human C-terminal to LisH (CTLH) complex is a newly discovered multi-subunit really interesting new gene (RING) E3 ligase with only a few known ubiquitination targets. Here, we used mass spectrometry-based proteomic techniques to gain insight into CTLH complex function and ubiquitination substrates in HeLa cells. First, global proteomics determined proteins that were significantly increased, and thus may be substrates targeted for degradation, in cells depleted of CTLH complex member RanBPM. RanBPM-dependent ubiquitination determined using diGLY-enriched proteomics and the endogenous RanBPM interactome further revealed candidate ubiquitination targets. Three glycolysis enzymes alpha-enolase, L-lactate dehydrogenase A chain (LDHA), and pyruvate kinase M1/2 (PKM) had decreased ubiquitin sites in shRanBPM cells and were found associated with RanBPM in the interactome. Reduced polyubiquitination was validated for PKM2 and LDHA in cells depleted of RanBPM and CTLH complex RING domain subunit RMND5A. PKM2 and LDHA protein levels were unchanged, yet their activity was increased in extracts of cells with downregulated RanBPM. Finally, RanBPM deficient cells displayed enhanced glycolysis and deregulated central carbon metabolism. Overall, this study identifies potential CTLH complex ubiquitination substrates and uncovers that the CTLH complex inhibits glycolysis via non-degradative ubiquitination of PKM2 and LDHA.


Assuntos
Glicólise/fisiologia , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitinação/fisiologia , Animais , Linhagem Celular Tumoral , Células HeLa , Humanos , L-Lactato Desidrogenase/metabolismo , Camundongos , Proteômica/métodos , Especificidade por Substrato , Ubiquitina/metabolismo
10.
Molecules ; 26(15)2021 Aug 03.
Artigo em Inglês | MEDLINE | ID: mdl-34361849

RESUMO

Proteases play a central role in various biochemical pathways catalyzing and regulating key biological events. Proteases catalyze an irreversible post-translational modification called proteolysis by hydrolyzing peptide bonds in proteins. Given the destructive potential of proteolysis, protease activity is tightly regulated. Dysregulation of protease activity has been reported in numerous disease conditions, including cancers, neurodegenerative diseases, inflammatory conditions, cardiovascular diseases, and viral infections. The proteolytic profile of a cell, tissue, or organ is governed by protease activation, activity, and substrate specificity. Thus, identifying protease substrates and proteolytic events under physiological conditions can provide crucial information about how the change in protease regulation can alter the cellular proteolytic landscape. In recent years, mass spectrometry-based techniques called N-terminomics have become instrumental in identifying protease substrates from complex biological mixtures. N-terminomics employs the labeling and enrichment of native and neo-N-termini peptides, generated upon proteolysis followed by mass spectrometry analysis allowing protease substrate profiling directly from biological samples. In this review, we provide a brief overview of N-terminomics techniques, focusing on their strengths, weaknesses, limitations, and providing specific examples where they were successfully employed to identify protease substrates in vivo and under physiological conditions. In addition, we explore the current trends in the protease field and the potential for future developments.


Assuntos
Peptídeo Hidrolases/química , Peptídeos/química , Proteólise , Proteômica , Humanos , Espectrometria de Massas , Especificidade por Substrato
11.
Bioresour Technol ; 340: 125732, 2021 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-34426240

RESUMO

The ancestor of ß-1,3-xylanases (AncXyl09) were reconstructed by the optimized ancestral sequences reconstruction strategy to solve the poor catalytic performances of existing ß-1,3-xylanases. The results showed that the half-life at 50 °C was 65.08 h, indicating good thermostability. The large number of hydrogen bonds and the disulfide bonds were the major attributes related with the thermal stability of Anxyl09. Interestingly, AncXyl09 could hydrolyze lichen besides the original substrate of ß-1, 3-xylan, which is the first reported ß-1,3-xylanase with substrate promiscuity. Moreover, the hydrolytic products are mainly disaccharides, the content of ß-1,3-xylobiose and lichoridiose more than 70% as determined by high performance liquid chromatography (HPLC), which could significantly facilitate the separation and purification of oligosaccharides. The successful design of AncXyl09 was the representative of the semi-rationally engineered ß-1, 3-xylanase, which will shield a new light on the ß-1,3-xylanase engineering, active oligosaccharide preparation and marine algae resource utilization.


Assuntos
Endo-1,4-beta-Xilanases , Xilanos , Endo-1,4-beta-Xilanases/metabolismo , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Especificidade por Substrato , Temperatura
12.
J Enzyme Inhib Med Chem ; 36(1): 1783-1797, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34340630

RESUMO

Carbonic Anhydrase Activators (CAAs) could represent a novel approach for the treatment of Alzheimer's disease, ageing, and other conditions that require remedial achievement of spatial learning and memory therapy. Within a research project aimed at developing novel CAAs selective for certain isoforms, three series of indole-based derivatives were investigated. Enzyme activation assay on human CA I, II, VA, and VII isoforms revealed several effective micromolar activators, with promising selectivity profiles towards the brain-associated cytosolic isoform hCA VII. Molecular modelling studies suggested a theoretical model of the complex between hCA VII and the new activators and provide a possible explanation for their modulating as well as selectivity properties. Preliminary biological evaluations demonstrated that one of the most potent CAA 7 is not cytotoxic and is able to increase the release of the brain-derived neurotrophic factor (BDNF) from human microglial cells, highlighting its possible application in the treatment of CNS-related disorders.


Assuntos
Anidrases Carbônicas/efeitos dos fármacos , Ativadores de Enzimas/farmacologia , Indóis/farmacologia , Isoenzimas/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Espectroscopia de Ressonância Magnética Nuclear de Carbono-13 , Anidrases Carbônicas/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Ativação Enzimática , Ativadores de Enzimas/química , Ensaio de Imunoadsorção Enzimática/métodos , Humanos , Indóis/química , Isoenzimas/metabolismo , Microglia/citologia , Microglia/efeitos dos fármacos , Modelos Moleculares , Espectroscopia de Prótons por Ressonância Magnética , Especificidade por Substrato
13.
Int J Mol Sci ; 22(16)2021 Aug 16.
Artigo em Inglês | MEDLINE | ID: mdl-34445469

RESUMO

Abasic (apurinic/apyrimidinic, AP) sites are ubiquitous DNA lesions arising from spontaneous base loss and excision of damaged bases. They may be processed either by AP endonucleases or AP lyases, but the relative roles of these two classes of enzymes are not well understood. We hypothesized that endonucleases and lyases may be differentially influenced by the sequence surrounding the AP site and/or the identity of the orphan base. To test this idea, we analysed the activity of plant and human AP endonucleases and AP lyases on DNA substrates containing an abasic site opposite either G or C in different sequence contexts. AP sites opposite G are common intermediates during the repair of deaminated cytosines, whereas AP sites opposite C frequently arise from oxidized guanines. We found that the major Arabidopsis AP endonuclease (ARP) exhibited a higher efficiency on AP sites opposite G. In contrast, the main plant AP lyase (FPG) showed a greater preference for AP sites opposite C. The major human AP endonuclease (APE1) preferred G as the orphan base, but only in some sequence contexts. We propose that plant AP endonucleases and AP lyases play complementary DNA repair functions on abasic sites arising at C:G pairs, neutralizing the potential mutagenic consequences of C deamination and G oxidation, respectively.


Assuntos
Arabidopsis/enzimologia , Pareamento de Bases , Dano ao DNA , Reparo do DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Endonucleases/metabolismo , Arabidopsis/genética , Sítios de Ligação , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/genética , Endonucleases/genética , Humanos , Especificidade por Substrato
14.
Int J Mol Sci ; 22(16)2021 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-34445525

RESUMO

Carotenoids are natural lipophilic pigments mainly found in plants, but also found in some animals and can be synthesized by fungi, some bacteria, algae, and aphids. These pigments are used in food industries as natural replacements for artificial colors. Carotenoids are also known for their benefits to human health as antioxidants and some compounds have provitamin A activity. The production of carotenoids by biotechnological approaches might exceed yields obtained by extraction from plants or chemical synthesis. Many microorganisms are carotenoid producers; however, not all are industrially feasible. Therefore, in this review, we provide an overview regarding fungi that are potentially interesting to industry because of their capacity to produce carotenoids in response to stresses on the cultivation medium, focusing on low-cost substrates.


Assuntos
Antioxidantes/metabolismo , Biotecnologia/métodos , Carotenoides/metabolismo , Engenharia Genética , Animais , Humanos , Especificidade por Substrato
15.
Int J Mol Sci ; 22(16)2021 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-34445579

RESUMO

Apurinic/apyrimidinic (AP) endonucleases Nfo (Escherichia coli) and APE1 (human) represent two conserved structural families of enzymes that cleave AP-site-containing DNA in base excision repair. Nfo and APE1 have completely different structures of the DNA-binding site, catalytically active amino acid residues and catalytic metal ions. Nonetheless, both enzymes induce DNA bending, AP-site backbone eversion into the active-site pocket and extrusion of the nucleotide located opposite the damage. All these stages may depend on local stability of the DNA duplex near the lesion. Here, we analysed effects of natural nucleotides located opposite a lesion on catalytic-complex formation stages and DNA cleavage efficacy. Several model DNA substrates that contain an AP-site analogue [F-site, i.e., (2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] opposite G, A, T or C were used to monitor real-time conformational changes of the tested enzymes during interaction with DNA using changes in the enzymes' intrinsic fluorescence intensity mainly caused by Trp fluorescence. The extrusion of the nucleotide located opposite F-site was recorded via fluorescence intensity changes of two base analogues. The catalytic rate constant slightly depended on the opposite-nucleotide nature. Thus, structurally different AP endonucleases Nfo and APE1 utilise a common strategy of damage recognition controlled by enzyme conformational transitions after initial DNA binding.


Assuntos
Clivagem do DNA , Dano ao DNA , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/química , DNA Liase (Sítios Apurínicos ou Apirimidínicos)/metabolismo , Nucleotídeos/metabolismo , Sítios de Ligação , Domínio Catalítico , Reparo do DNA , Escherichia coli , Humanos , Cinética , Simulação de Dinâmica Molecular , Conformação de Ácido Nucleico , Nucleotídeos/química , Conformação Proteica , Especificidade por Substrato
16.
Molecules ; 26(15)2021 Aug 02.
Artigo em Inglês | MEDLINE | ID: mdl-34361831

RESUMO

The interaction of small organic molecules such as drugs, agrochemicals, and cosmetics with cytochrome P450 enzymes (CYPs) can lead to substantial changes in the bioavailability of active substances and hence consequences with respect to pharmacological efficacy and toxicity. Therefore, efficient means of predicting the interactions of small organic molecules with CYPs are of high importance to a host of different industries. In this work, we present a new set of machine learning models for the classification of xenobiotics into substrates and non-substrates of nine human CYP isozymes: CYPs 1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1, and 3A4. The models are trained on an extended, high-quality collection of known substrates and non-substrates and have been subjected to thorough validation. Our results show that the models yield competitive performance and are favorable for the detection of CYP substrates. In particular, a new consensus model reached high performance, with Matthews correlation coefficients (MCCs) between 0.45 (CYP2C8) and 0.85 (CYP3A4), although at the cost of coverage. The best models presented in this work are accessible free of charge via the "CYPstrate" module of the New E-Resource for Drug Discovery (NERDD).


Assuntos
Sistema Enzimático do Citocromo P-450/metabolismo , Aprendizado de Máquina , Xenobióticos/classificação , Xenobióticos/metabolismo , Animais , Humanos , Especificidade por Substrato
17.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360577

RESUMO

Cytochrome P450 monooxygenase CYP51 (sterol 14α-demethylase) is a well-known target of the azole drug fluconazole for treating cryptococcosis, a life-threatening fungal infection in immune-compromised patients in poor countries. Studies indicate that mutations in CYP51 confer fluconazole resistance on cryptococcal species. Despite the importance of CYP51 in these species, few studies on the structural analysis of CYP51 and its interactions with different azole drugs have been reported. We therefore performed in silico structural analysis of 11 CYP51s from cryptococcal species and other Tremellomycetes. Interactions of 11 CYP51s with nine ligands (three substrates and six azoles) performed by Rosetta docking using 10,000 combinations for each of the CYP51-ligand complex (11 CYP51s × 9 ligands = 99 complexes) and hierarchical agglomerative clustering were used for selecting the complexes. A web application for visualization of CYP51s' interactions with ligands was developed (http://bioshell.pl/azoledocking/). The study results indicated that Tremellomycetes CYP51s have a high preference for itraconazole, corroborating the in vitro effectiveness of itraconazole compared to fluconazole. Amino acids interacting with different ligands were found to be conserved across CYP51s, indicating that the procedure employed in this study is accurate and can be automated for studying P450-ligand interactions to cater for the growing number of P450s.


Assuntos
Aminoácidos/metabolismo , Azóis/metabolismo , Basidiomycota/enzimologia , Sistema Enzimático do Citocromo P-450/metabolismo , Fluconazol/metabolismo , Proteínas Fúngicas/metabolismo , Itraconazol/metabolismo , Aminoácidos/química , Antifúngicos/química , Antifúngicos/metabolismo , Azóis/química , Simulação por Computador , Sistema Enzimático do Citocromo P-450/química , Fluconazol/química , Proteínas Fúngicas/química , Itraconazol/química , Ligantes , Modelos Moleculares , Filogenia , Ligação Proteica , Conformação Proteica , Especificidade por Substrato
18.
Int J Mol Sci ; 22(15)2021 Jul 22.
Artigo em Inglês | MEDLINE | ID: mdl-34360597

RESUMO

Toxoplasma gondii is a protozoan parasite that causes toxoplasmosis and infects almost one-third of the global human population. A lack of effective drugs and vaccines and the emergence of drug resistant parasites highlight the need for the development of new drugs. The mitochondrial electron transport chain (ETC) is an essential pathway for energy metabolism and the survival of T. gondii. In apicomplexan parasites, malate:quinone oxidoreductase (MQO) is a monotopic membrane protein belonging to the ETC and a key member of the tricarboxylic acid cycle, and has recently been suggested to play a role in the fumarate cycle, which is required for the cytosolic purine salvage pathway. In T. gondii, a putative MQO (TgMQO) is expressed in tachyzoite and bradyzoite stages and is considered to be a potential drug target since its orthologue is not conserved in mammalian hosts. As a first step towards the evaluation of TgMQO as a drug target candidate, in this study, we developed a new expression system for TgMQO in FN102(DE3)TAO, a strain deficient in respiratory cytochromes and dependent on an alternative oxidase. This system allowed, for the first time, the expression and purification of a mitochondrial MQO family enzyme, which was used for steady-state kinetics and substrate specificity analyses. Ferulenol, the only known MQO inhibitor, also inhibited TgMQO at IC50 of 0.822 µM, and displayed different inhibition kinetics compared to Plasmodium falciparum MQO. Furthermore, our analysis indicated the presence of a third binding site for ferulenol that is distinct from the ubiquinone and malate sites.


Assuntos
Cumarínicos/metabolismo , Malatos/metabolismo , Proteínas Mitocondriais/metabolismo , Oxirredutases/metabolismo , Proteínas de Protozoários/metabolismo , Toxoplasma/enzimologia , Ubiquinona/metabolismo , Animais , Humanos , Proteínas Mitocondriais/genética , Oxirredutases/genética , Proteínas de Protozoários/genética , Especificidade por Substrato
19.
Appl Microbiol Biotechnol ; 105(16-17): 6215-6228, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34410440

RESUMO

Deoxyribose-5-phosphate aldolases (DERAs, EC 4.1.2.4) are acetaldehyde-dependent, Class I aldolases catalyzing in nature a reversible aldol reaction between an acetaldehyde donor (C2 compound) and glyceraldehyde-3-phosphate acceptor (C3 compound, C3P) to generate deoxyribose-5-phosphate (C5 compound, DR5P). DERA enzymes have been found to accept also other types of aldehydes as their donor, and in particular as acceptor molecules. Consequently, DERA enzymes can be applied in C-C bond formation reactions to produce novel compounds, thus offering a versatile biocatalytic alternative for synthesis. DERA enzymes, found in all kingdoms of life, share a common TIM barrel fold despite the low overall sequence identity. The catalytic mechanism is well-studied and involves formation of a covalent enzyme-substrate intermediate. A number of protein engineering studies to optimize substrate specificity, enzyme efficiency, and stability of DERA aldolases have been published. These have employed various engineering strategies including structure-based design, directed evolution, and recently also machine learning-guided protein engineering. For application purposes, enzyme immobilization and usage of whole cell catalysis are preferred methods as they improve the overall performance of the biocatalytic processes, including often also the stability of the enzyme. Besides single-step enzymatic reactions, DERA aldolases have also been applied in multi-enzyme cascade reactions both in vitro and in vivo. The DERA-based applications range from synthesis of commodity chemicals and flavours to more complicated and high-value pharmaceutical compounds. KEY POINTS: • DERA aldolases are versatile biocatalysts able to make new C-C bonds. • Synthetic utility of DERAs has been improved by protein engineering approaches. • Computational methods are expected to speed up the future DERA engineering efforts.


Assuntos
Aldeído Liases , Ribosemonofosfatos , Aldeído Liases/genética , Aldeído Liases/metabolismo , Frutose-Bifosfato Aldolase , Especificidade por Substrato
20.
Front Cell Infect Microbiol ; 11: 725615, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34422684

RESUMO

Protein phosphatase 2A (PP2A) is one of the most ubiquitous cellular proteins and is responsible for the vast majority of Ser/Thr phosphatase activity in eukaryotes. PP2A is a heterotrimer, and its assembly, intracellular localization, enzymatic activity, and substrate specificity are subject to dynamic regulation. Each of its subunits can be targeted by viral proteins to hijack and modulate its activity and downstream signaling to the advantage of the virus. Binding to PP2A is known to be essential to the life cycle of many viruses and seems to play a particularly crucial role for oncogenic viruses, which utilize PP2A to transform infected cells through controlling the cell cycle and apoptosis. Here we summarise the latest developments in the field of PP2A viral targeting; in particular recent discoveries of PP2A hijacking through molecular mimicry of a B56-specific motif by several different viruses. We also discuss the potential as well as shortcomings for therapeutic intervention in the face of our current understanding of viral PP2A targeting.


Assuntos
Proteína Fosfatase 2 , Vírus , Apoptose , Proteína Fosfatase 2/metabolismo , Transdução de Sinais , Especificidade por Substrato
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