Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 99.533
Filtrar
1.
Nat Commun ; 13(1): 5242, 2022 Sep 06.
Artigo em Inglês | MEDLINE | ID: mdl-36068220

RESUMO

Enzymes with high activity are readily produced through protein engineering, but intentionally and efficiently engineering enzymes for an expanded substrate scope is a contemporary challenge. One approach to address this challenge is Substrate Multiplexed Screening (SUMS), where enzyme activity is measured on competing substrates. SUMS has long been used to rigorously quantitate native enzyme specificity, primarily for in vivo settings. SUMS has more recently found sporadic use as a protein engineering approach but has not been widely adopted by the field, despite its potential utility. Here, we develop principles of how to design and interpret SUMS assays to guide protein engineering. This rich information enables improving activity with multiple substrates simultaneously, identifies enzyme variants with altered scope, and indicates potential mutational hot-spots as sites for further engineering. These advances leverage common laboratory equipment and represent a highly accessible and customizable method for enzyme engineering.


Assuntos
Engenharia de Proteínas , Biocatálise , Engenharia de Proteínas/métodos , Especificidade por Substrato
2.
Acta Crystallogr D Struct Biol ; 78(Pt 9): 1180-1191, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-36048157

RESUMO

D-Allulose, a low-calorie rare sugar with various physiological functions, is mainly produced through the isomerization of D-fructose by ketose 3-epimerases (KEases), which exhibit various substrate specificities. A novel KEase from a Clostridia bacterium (CDAE) was identified to be a D-allulose 3-epimerase and was further characterized as thermostable and metal-dependent. In order to explore its structure-function relationship, the crystal structure of CDAE was determined using X-ray diffraction at 2.10 Šresolution, revealing a homodimeric D-allulose 3-epimerase structure with extensive interactions formed at the dimeric interface that contribute to structure stability. Structural analysis identified the structural features of CDAE, which displays a common (ß/α)8-TIM barrel and an ordered Mn2+-binding architecture at the active center, which may explain the positive effects of Mn2+ on the activity and stability of CDAE. Furthermore, comparison of CDAE and other KEase structures revealed several structural differences, highlighting the remarkable differences in enzyme-substrate binding at the O4, O5 and O6 sites of the bound substrate, which are mainly induced by distinct hydrophobic pockets in the active center. The shape and hydrophobicity of this pocket appear to produce the differences in specificity and affinity for substrates among KEase family enzymes. Exploration of the crystal structure of CDAE provides a better understanding of its structure-function relationship, which might provide a basis for molecular modification of CDAE and further provides a reference for other KEases.


Assuntos
Carboidratos Epimerases , Racemases e Epimerases , Carboidratos Epimerases/química , Frutose/química , Especificidade por Substrato
3.
Enzyme Microb Technol ; 161: 110117, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36049397

RESUMO

Cordyceps militaris, an entomopathogenic Cordyceps mushroom, is a crucial ethnopharmacological agricultural product with applications in traditional oriental remedies in East Asia. Since lipases are reported to serve as key enzymatic equipment for entomopathogenic fungi during the host infection, the presence of various lipases with different biochemical features in C. militaris was elucidated. Three lipases from C. militaris (CML) of 60-70 kDa were isolated according to protein hydrophobicity; isoform relationships were identified by peptide mapping using liquid chromatography-electrospray ionization-tandem mass spectrometry. The CML isoforms exhibited distinct substrate specificities, which were related to the hydrophobicity of each isoform. Furthermore, the integral stereoselectivity of each lipase towards trioleoylglycerol diverged into two classes (sn-1,3 and sn-2 regioselectivity) that are rare in canonical fungal lipases. Overall, our results demonstrate that C. militaris secretes lipase isoforms with cocktail-like enzyme functions that may contribute to the entomopathogenic life cycle of C. militaris. Each CML isoform has distinct advantages for biocatalyst applications in the food and oleochemical industries.


Assuntos
Agaricales , Cordyceps , Lipase/metabolismo , Isoformas de Proteínas/metabolismo , Especificidade por Substrato
4.
J Cell Biol ; 221(10)2022 Oct 03.
Artigo em Inglês | MEDLINE | ID: mdl-36102907

RESUMO

Reversible protein phosphorylation by kinases controls a plethora of processes essential for the proper development and homeostasis of multicellular organisms. One main obstacle in studying the role of a defined kinase-substrate interaction is that kinases form complex signaling networks and most often phosphorylate multiple substrates involved in various cellular processes. In recent years, several new approaches have been developed to control the activity of a given kinase. However, most of them fail to regulate a single protein target, likely hiding the effect of a unique kinase-substrate interaction by pleiotropic effects. To overcome this limitation, we have created protein binder-based engineered kinases that permit a direct, robust, and tissue-specific phosphorylation of fluorescent fusion proteins in vivo. We show the detailed characterization of two engineered kinases based on Rho-associated protein kinase (ROCK) and Src. Expression of synthetic kinases in the developing fly embryo resulted in phosphorylation of their respective GFP-fusion targets, providing for the first time a means to direct the phosphorylation to a chosen and tagged target in vivo. We presume that after careful optimization, the novel approach we describe here can be adapted to other kinases and targets in various eukaryotic genetic systems to regulate specific downstream effectors.


Assuntos
Proteínas , Quinases Associadas a rho , Quinases da Família src , Animais , Drosophila , Fosforilação , Engenharia de Proteínas , Proteínas/metabolismo , Transdução de Sinais , Especificidade por Substrato , Quinases Associadas a rho/metabolismo , Quinases da Família src/metabolismo
5.
Biotechnol Lett ; 44(10): 1163-1173, 2022 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-36050605

RESUMO

PURPOSE: We screened nitrilases with significant nitrile hydratase activity to exploit their potential in benzylic amide biosynthesis. We also investigated the factors affecting their hydration activity to support further research on benzylic amide production by nitrilase. METHODS: A sequence-based screening method using previously reported crucial positions identified to be essential for amide-forming capacity of nitrilase (referred to as "amide-formation hotspots") as molecular probes to identify putative amide-forming nitrilases. RESULTS: Based on the previously reported "amide-formation hotspots," we identified a nitrilase NitPG from Paraburkholderia graminis DSM 17151 that could produce a significant amount of mandelamide toward mandelonitrile and exhibited general hydration activity toward various benzylic nitriles. The time-course experiment with NitPG demonstrated that amide was also a true reaction product of nitrilase, suggesting that the nitrile catalysis by amide-forming nitrilase could be a post-transition state bifurcation-mediated enzymatic reaction. Further research demonstrated that low temperature, metal ion addition, and specific substrate structure could profoundly improve the amide formation capability of nitrilase. CONCLUSIONS: NitPG with broad hydration activity is a potential candidate for the enzymatic synthesis of benzylic amides for biotechnological applications. Studying the effect of nitrilase hydration activity could promote our understanding of the factors that influence amide and acid distribution.


Assuntos
Aminoidrolases , Nitrilas , Amidas , Aminoidrolases/metabolismo , Hidroliases , Sondas Moleculares , Especificidade por Substrato
6.
Cells ; 11(17)2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36078095

RESUMO

Ectodomain shedding is an irreversible process to regulate inter- and intracellular signaling. Members of the a disintegrin and metalloprotease (ADAM) family are major mediators of ectodomain shedding. ADAM17 is involved in the processing of multiple substrates including tumor necrosis factor (TNF) α and EGF receptor ligands. Substrates of ADAM17 are selectively processed depending on stimulus and cellular context. However, it still remains largely elusive how substrate selectivity of ADAM17 is regulated. Tetraspanins (Tspan) are multi-membrane-passing proteins that are involved in the organization of plasma membrane micro-domains and diverse biological processes. Closely related members of the Tspan8 subfamily, including CD9, CD81 and Tspan8, are associated with cancer and metastasis. Here, we show that Tspan8 subfamily members use different strategies to regulate ADAM17 substrate selectivity. We demonstrate that in particular Tspan8 associates with both ADAM17 and TNF α and promotes ADAM17-mediated TNF α release through recruitment of ADAM17 into Tspan-enriched micro-domains. Yet, processing of other ADAM17 substrates is not altered by Tspan8. We, therefore, propose that Tspan8 contributes to tumorigenesis through enhanced ADAM17-mediated TNF α release and a resulting increase in tissue inflammation.


Assuntos
Proteínas ADAM , Desintegrinas , Proteínas ADAM/genética , Proteínas ADAM/metabolismo , Proteínas de Membrana , Especificidade por Substrato , Tetraspaninas/genética , Fator de Necrose Tumoral alfa/metabolismo
7.
Molecules ; 27(16)2022 Aug 17.
Artigo em Inglês | MEDLINE | ID: mdl-36014501

RESUMO

Cleavage of DNA at noncanonical recognition sequences by restriction endonucleases (star activity) in bulk solution can be promoted by global experimental parameters, including enzyme or substrate concentration, temperature, pH, or buffer composition. To study the effect of nanoscale confinement on the noncanonical behaviour of BamHI, which cleaves a single unique sequence of 6 bp, we used AFM nanografting to generate laterally confined DNA monolayers (LCDM) at different densities, either in the form of small patches, several microns in width, or complete monolayers of thiol-modified DNA on a gold surface. We focused on two 44-bp DNAs, each containing a noncanonical BamHI site differing by 2 bp from the cognate recognition sequence. Topographic AFM imaging was used to monitor end-point reactions by measuring the decrease in the LCDM height with respect to the surrounding reference surface. At low DNA densities, BamHI efficiently cleaves only its cognate sequence while at intermediate DNA densities, noncanonical sequence cleavage occurs, and can be controlled in a stepwise (on/off) fashion by varying the DNA density and restriction site sequence. This study shows that endonuclease action on noncanonical sites in confined nanoarchitectures can be modulated by varying local physical parameters, independent of global chemical parameters.


Assuntos
Clivagem do DNA , DNA , Sequência de Bases , DNA/química , Enzimas de Restrição do DNA/metabolismo , Desoxirribonuclease BamHI/metabolismo , Especificidade por Substrato
8.
Planta ; 256(3): 58, 2022 Aug 18.
Artigo em Inglês | MEDLINE | ID: mdl-35980476

RESUMO

MAIN CONCLUSION: Two key amino acids of isomultiflorenol synthase, Y125 and M254, were first proposed. They could be associated with the production of isomultiflorenol. Oxidosqualene cyclases (OSCs) are the first committed enzymes in the triterpenoid biosynthesis by converting 2,3-oxidosqualene to specific triterpenoid backbones. Thus, these enzymes are potential targets for developing plant-active compounds through the study of triterpenoid biosynthesis. We applied transcriptome information and metabolite profiling from Trichosanthes cucumerina L. to define the diversity of triterpenoids in this plant through OSCs. Isomultiflorenol synthase and cucurbitadienol synthase were previously identified in this plant. Here, three new OSCs, TcBAS, TcLAS, and TcCAS, were cloned and functionally characterized as ß-amyrin synthase, lanosterol synthase, and cycloartenol synthase activities, respectively. We also took advantage of the multiple sequence alignment and molecular docking of OSCs exhibiting in this plant and other plant OSCs to identify key residues associated with isomultiflorenol synthase specificity. Two novel key amino acids, referred to the Y125 and M254, were first discovered. These results provide information on a possible catalytic mechanism for plant OSCs that produce specific products.


Assuntos
Transferases Intramoleculares , Trichosanthes , Triterpenos , Aminoácidos , Clonagem Molecular , Transferases Intramoleculares/genética , Transferases Intramoleculares/metabolismo , Simulação de Acoplamento Molecular , Esqualeno/análogos & derivados , Especificidade por Substrato , Trichosanthes/metabolismo , Triterpenos/metabolismo
9.
Mar Drugs ; 20(8)2022 Aug 05.
Artigo em Inglês | MEDLINE | ID: mdl-36005509

RESUMO

Alginate lyases with unique biochemical properties have irreplaceable value in food and biotechnology industries. Herein, the first new hybrid action mode Thalassotalea algicola-derived alginate lyase gene (TAPL7A) with both psychrophilic and cold-tolerance was cloned and expressed heterologously in E. coli. With the highest sequence identity (43%) to the exolytic alginate lyase AlyA5 obtained from Zobellia galactanivorans, TAPL7A was identified as a new polysaccharide lyases family 7 (PL7) alginate lyase. TAPL7A has broad substrate tolerance with specific activities of 4186.1 U/mg, 2494.8 U/mg, 2314.9 U/mg for polyM, polyG, and sodium alginate, respectively. Biochemical characterization of TAPL7A showed optimal activity at 15 °C, pH 8.0. Interestingly, TAPL7A exhibits both extreme psychrophilic and cold tolerance, which other cold-adapted alginate lyase do not possess. In a wide range of 5-30 °C, the activity can reach 80-100%, and the residual activity of more than 70% can still be maintained after 1 h of incubation. Product analysis showed that TAPL7A adopts a hybrid endo/exo-mode on all three substrates. FPLC and ESI-MS confirmed that the final products of TAPL7A are oligosaccharides with degrees of polymerization (Dps) of 1-2. This study provides excellent alginate lyase candidates for low-temperature environmental applications in food, agriculture, medicine and other industries.


Assuntos
Alginatos , Escherichia coli , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Oligossacarídeos/química , Polissacarídeo-Liases/metabolismo , Especificidade por Substrato
10.
Mar Drugs ; 20(8)2022 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-36005523

RESUMO

In order to discover a broad-specificity and high stability chitinase, a marine fungus, Aspergillus fumigatus df347, was identified in the sediments of mangrove wetlands in Qinzhou Bay, China. The chitinase gene (AfChi28) from A. fumigatus df347 was cloned and heterologously expressed in Escherichia coli, and the recombinant enzyme AfChi28 was purified and characterized. AfChi28 is an acido-halotolerant- and temperature-resistant bifunctional enzyme with both endo- and exo-cleavage functions. Its enzymatic products are mainly GlcNAc, (GlcNAc)2, (GlcNAc)3 and (GlcNAc)4. Na+, Mg2+, K+, Ca2+ and Tris at a concentration of 50 mM had a strong stimulatory effect on AfChi28. The crude enzyme and pure enzyme exhibited the highest specific activity of 0.737 mU/mg and 52.414 mU/mg towards colloidal chitin. The DxDxE motif at the end of strand ß5 and with Glu154 as the catalytic residue was verified by the AlphaFold2 prediction and sequence alignment of homologous proteins. Moreover, the results of molecular docking showed that molecular modeling of chitohexaose was shown to bind to AfChi28 in subsites -4 to +2 in the deep groove substrate-binding pocket. This study demonstrates that AfChi28 is a promising chitinase for the preparation of desirable chitin oligosaccharides, and provides a foundation for elucidating the catalytic mechanism of chitinases from marine fungi.


Assuntos
Quitinases , Aspergillus fumigatus/genética , Quitina/química , Quitinases/metabolismo , Escherichia coli/metabolismo , Fungos/metabolismo , Hidrólise , Simulação de Acoplamento Molecular , Especificidade por Substrato
11.
Int J Mol Sci ; 23(16)2022 Aug 12.
Artigo em Inglês | MEDLINE | ID: mdl-36012304

RESUMO

The set-up of highly sensitive detection tools to evaluate lipase activity remains a central goal in different fields. In this context, we proposed new chemiluminescent 1,2-dioxetane luminophores, sharing an octanoyl triggerable group, to monitor lipase activity. We herein report the synthesis and both the evaluation of their luminescence emission profile and their enzyme-substrate specificity, generated by three different commercial lipases (Candida cylindracea, Pseudomonas fluorescens, and Mucor miehei) and one esterase (porcine liver esterase, PLE, as a literature control). Remarkably, the present study confirmed the applicability of these 1,2-dioxetane luminophores as (i) highly efficient, broad-range, chemiluminescent probes for the detection and the enzymatic activity evaluation of lipases and as (ii) promising candidates for the future development of both flash- and glow-type luminescence assays.


Assuntos
Luminescência , Medições Luminescentes , Animais , Candida/metabolismo , Lipase/metabolismo , Especificidade por Substrato , Suínos
12.
Appl Microbiol Biotechnol ; 106(17): 5563-5574, 2022 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-35932295

RESUMO

Amine transaminases (ATA) convert ketones into optically active amines and are used to prepare active pharmaceutical ingredients and building blocks. Novel ATA can be identified in protein databases due to the extensive knowledge of sequence-function relationships. However, predicting thermo- and operational stability from the amino acid sequence is a persisting challenge and a vital step towards identifying efficient ATA biocatalysts for industrial applications. In this study, we performed a database mining and characterized selected putative enzymes of the ß-alanine:pyruvate transaminase cluster (3N5M) - a subfamily with so far only a few described members, whose tetrameric structure was suggested to positively affect operational stability. Four putative transaminases (TA-1: Bilophilia wadsworthia, TA-5: Halomonas elongata, TA-9: Burkholderia cepacia, and TA-10: Burkholderia multivorans) were obtained in a soluble form as tetramers in E. coli. During comparison of these tetrameric with known dimeric transaminases we found that indeed novel ATA with high operational stabilities can be identified in this protein subfamily, but we also found exceptions to the hypothesized correlation that a tetrameric assembly leads to increased stability. The discovered ATA from Burkholderia multivorans features a broad substrate specificity, including isopropylamine acceptance, is highly active (6 U/mg) in the conversion of 1-phenylethylamine with pyruvate and shows a thermostability of up to 70 °C under both, storage and operating conditions. In addition, 50% (v/v) of isopropanol or DMSO can be employed as co-solvents without a destabilizing effect on the enzyme during an incubation time of 16 h at 30 °C. KEY POINTS: • Database mining identified a thermostable amine transaminase in the ß-alanine:pyruvate transaminase subfamily. • The tetrameric transaminase tolerates 50% DMSO and isopropanol under operating conditions at 30 °C. • A tetrameric structure is not necessarily associated with a higher operational stability.


Assuntos
Aminas , Escherichia coli , 2-Propanol , Burkholderia , Dimetil Sulfóxido , Piruvatos , Especificidade por Substrato , Transaminases , beta-Alanina
13.
Commun Biol ; 5(1): 782, 2022 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-35918517

RESUMO

Alginate lyases can be used to produce well-defined alginate oligosaccharides (AOSs) because of their specificities for AOS products. A large number of alginate lyases have been recorded in the CAZy database; however, the majority are annotated-only alginate lyases that include little information on their products, thus limiting their applications. Here, we establish a simple and experiment-saving approach to predict product distributions for PL7 alginate lyases through extensive structural biology, bioinformatics and biochemical studies. Structural study on several PL7 alginate lyases reveals that two loops around the substrate binding cleft determine product distribution. Furthermore, a database containing the loop information of all annotated-only single-domain PL7 alginate lyases is constructed, enabling systematic exploration of the association between loop and product distribution. Based on these results, a simplified loop/product distribution relationship is proposed, giving us information on product distribution directly from the amino acid sequence.


Assuntos
Alginatos , Oligossacarídeos , Sequência de Aminoácidos , Oligossacarídeos/metabolismo , Especificidade por Substrato
14.
Commun Biol ; 5(1): 787, 2022 08 05.
Artigo em Inglês | MEDLINE | ID: mdl-35931745

RESUMO

Human spermine oxidase (hSMOX) plays a central role in polyamine catabolism. Due to its association with several pathological processes, including inflammation and cancer, hSMOX has garnered interest as a possible therapeutic target. Therefore, determination of the structure of hSMOX is an important step to enable drug discovery and validate hSMOX as a drug target. Using insights from hydrogen/deuterium exchange mass spectrometry (HDX-MS), we engineered a hSMOX construct to obtain the first crystal structure of hSMOX bound to the known polyamine oxidase inhibitor MDL72527 at 2.4 Å resolution. While the overall fold of hSMOX is similar to its homolog, murine N1-acetylpolyamine oxidase (mPAOX), the two structures contain significant differences, notably in their substrate-binding domains and active site pockets. Subsequently, we employed a sensitive biochemical assay to conduct a high-throughput screen that identified a potent and selective hSMOX inhibitor, JNJ-1289. The co-crystal structure of hSMOX with JNJ-1289 was determined at 2.1 Å resolution, revealing that JNJ-1289 binds to an allosteric site, providing JNJ-1289 with a high degree of selectivity towards hSMOX. These results provide crucial insights into understanding the substrate specificity and enzymatic mechanism of hSMOX, and for the design of highly selective inhibitors.


Assuntos
Oxirredutases atuantes sobre Doadores de Grupo CH-NH , Animais , Domínio Catalítico , Humanos , Camundongos , Oxirredutases/metabolismo , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/química , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/genética , Oxirredutases atuantes sobre Doadores de Grupo CH-NH/metabolismo , Especificidade por Substrato
15.
PLoS One ; 17(8): e0269684, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35921328

RESUMO

Canonical aminoglycosides are a large group of antibiotics, where the part of chemical diversity stems from the substitution of the neamine ring system on positions 5 and 6. Certain aminoglycoside modifying enzymes can modify a broad range of 4,5- and 4,6-disubstituted aminoglycosides, with some as many as 15. This study presents the structural and kinetic results describing a promiscuous aminoglycoside acetyltransferase AAC(3)-IIIa. This enzyme has been crystallized in ternary complex with coenzyme A and 4,5- and 4,6-disubstituted aminoglycosides. We have followed up this work with kinetic characterization utilizing a panel of diverse aminoglycosides, including a next-generation aminoglycoside, plazomicin. Lastly, we observed an alternative binding mode of gentamicin in the aminoglycoside binding site, which was proven to be a crystallographic artifact based on mutagenesis.


Assuntos
Acetiltransferases , Aminoglicosídeos , Acetiltransferases/metabolismo , Aminoglicosídeos/química , Antibacterianos/química , Especificidade por Substrato
16.
Nat Commun ; 13(1): 5120, 2022 Aug 31.
Artigo em Inglês | MEDLINE | ID: mdl-36045135

RESUMO

Binding of precursor tRNAs (ptRNAs) by bacterial ribonuclease P (RNase P) involves an encounter complex (ES) that isomerizes to a catalytic conformation (ES*). However, the structures of intermediates and the conformational changes that occur during binding are poorly understood. Here, we show that pairing between the 5' leader and 3'RCCA extending the acceptor stem of ptRNA inhibits ES* formation. Cryo-electron microscopy single particle analysis reveals a dynamic enzyme that becomes ordered upon formation of ES* in which extended acceptor stem pairing is unwound. Comparisons of structures with alternative ptRNAs reveals that once unwinding is completed RNase P primarily uses stacking interactions and shape complementarity to accommodate alternative sequences at its cleavage site. Our study reveals active site interactions and conformational changes that drive molecular recognition by RNase P and lays the foundation for understanding how binding interactions are linked to helix unwinding and catalysis.


Assuntos
RNA Catalítico , Ribonuclease P , Catálise , Microscopia Crioeletrônica , Conformação de Ácido Nucleico , Precursores de RNA/metabolismo , RNA Catalítico/metabolismo , RNA de Transferência/metabolismo , Ribonuclease P/metabolismo , Especificidade por Substrato
17.
Sci Rep ; 12(1): 14685, 2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36038587

RESUMO

8-Oxoguanine DNA glycosylase (OGG1) initiates base excision repair of the oxidative DNA damage product 8-oxoguanine. OGG1 is bifunctional; catalyzing glycosyl bond cleavage, followed by phosphodiester backbone incision via a ß-elimination apurinic lyase reaction. The product from the glycosylase reaction, 8-oxoguanine, and its analogues, 8-bromoguanine and 8-aminoguanine, trigger the rate-limiting AP lyase reaction. The precise activation mechanism remains unclear. The product-assisted catalysis hypothesis suggests that 8-oxoguanine and analogues bind at the product recognition (PR) pocket to enhance strand cleavage as catalytic bases. Alternatively, they may allosterically activate OGG1 by binding outside of the PR pocket to induce an active-site conformational change to accelerate apurinic lyase. Herein, steady-state kinetic analyses demonstrated random binding of substrate and activator. 9-Deazaguanine, which can't function as a substrate-competent base, activated OGG1, albeit with a lower Emax value than 8-bromoguanine and 8-aminoguanine. Random compound screening identified small molecules with Emax values similar to 8-bromoguanine. Paraquat-induced mitochondrial dysfunction was attenuated by several small molecule OGG1 activators; benefits included enhanced mitochondrial membrane and DNA integrity, less cytochrome c translocation, ATP preservation, and mitochondrial membrane dynamics. Our results support an allosteric mechanism of OGG1 and not product-assisted catalysis. OGG1 small molecule activators may improve mitochondrial function in oxidative stress-related diseases.


Assuntos
DNA Glicosilases , Regulação Alostérica , DNA Glicosilases/metabolismo , Reparo do DNA , Guanina/análogos & derivados , Mitocôndrias/metabolismo , Especificidade por Substrato
18.
Microb Cell Fact ; 21(1): 175, 2022 Aug 29.
Artigo em Inglês | MEDLINE | ID: mdl-36038906

RESUMO

BACKGROUND: The regioselective hydroxylation of phenolic compounds, especially flavonoids, is still a bottleneck of classical organic chemistry that could be solved using enzymes with high activity and specificity. Yeast Rhodotorula glutinis KCh735 in known to catalyze the C-8 hydroxylation of flavones and flavanones. The enzyme F8H (flavonoid C8-hydroxylase) is involved in the reaction, but the specific gene has not yet been identified. In this work, we present identification, heterologous expression and characterization of the first F8H ortho-hydroxylase from yeast. RESULTS: Differential transcriptome analysis and homology to bacterial monooxygenases, including also a FAD-dependent motif and a GD motif characteristic for flavin-dependent monooxygenases, provided a set of coding sequences among which RgF8H was identified. Phylogenetic analysis suggests that RgF8H is a member of the flavin monooxygenase group active on flavonoid substrates. Analysis of recombinant protein showed that the enzyme catalyzes the C8-hydroxylation of naringenin, hesperetin, eriodyctiol, pinocembrin, apigenin, luteolin, chrysin, diosmetin and 7,4'-dihydroxyflavone. The presence of the C7-OH group is necessary for enzymatic activity indicating ortho-hydroxylation mechanism. The enzyme requires the NADPH coenzyme for regeneration prosthetic group, displays very low hydroxyperoxyflavin decupling rate, and addition of FAD significantly increases its activity. CONCLUSIONS: This study presents identification of the first yeast hydroxylase responsible for regioselective C8-hydroxylation of flavonoids (F8H). The enzyme was biochemically characterized and applied in in vitro cascade with Bacillus megaterium glucose dehydrogenase reactions. High in vivo activity in Escherichia coli enable further synthetic biology application towards production of rare highly antioxidant compounds.


Assuntos
Flavina-Adenina Dinucleotídeo , Oxigenases de Função Mista , Flavina-Adenina Dinucleotídeo/metabolismo , Flavinas/metabolismo , Flavonoides/metabolismo , Hidroxilação , Oxigenases de Função Mista/genética , Oxigenases de Função Mista/metabolismo , Filogenia , Rhodotorula , Especificidade por Substrato
19.
mBio ; 13(4): e0093522, 2022 Aug 30.
Artigo em Inglês | MEDLINE | ID: mdl-35913158

RESUMO

Cellulose being the most abundant polysaccharide on earth, beta-glucosidases hydrolyzing cello-oligosaccharides are key enzymes to fuel glycolysis in microorganisms developing on plant material. In Streptomyces scabiei, the causative agent of common scab in root and tuber crops, a genetic compensation phenomenon safeguards the loss of the gene encoding the cello-oligosaccharide hydrolase BglC by awakening the expression of alternative beta-glucosidases. Here, we revealed that the BglC compensating enzyme BcpE2 was the GH3-family beta-glucosidase that displayed the highest reported substrate promiscuity and was able to release the glucose moiety of all tested types of plant-derived heterosides (aryl ß-glucosides, monolignol glucosides, cyanogenic glucosides, anthocyanosides, and coumarin heterosides). BcpE2 structure analysis highlighted a large cavity in the PA14 domain that covered the active site, and the high flexibility of this domain would allow proper adjustment of this cavity for disparate heterosides. The exceptional substrate promiscuity of BcpE2 provides microorganisms a versatile tool for scavenging glucose from plant-derived nutrients that widely vary in size and structure. Importantly, scopolin was the only substrate commonly hydrolyzed by both BglC and BcpE2, thereby generating the potent virulence inhibitor scopoletin. Next to fueling glycolysis, both enzymes would also fine-tune the strength of virulence. IMPORTANCE Plant decaying biomass is the most abundant provider of carbon sources for soil-dwelling microorganisms. To optimally evolve in such environmental niches, microorganisms possess an arsenal of hydrolytic enzymatic complexes to feed on the various types of polysaccharides, oligosaccharides, and monosaccharides. In this work, structural, enzymatic, and expression studies revealed the existence of a "swiss-army knife" enzyme, BcpE2, that was able to retrieve the glucose moiety of a multitude of plant-derived substrates that vary in size, structure, and origin. This enzyme would provide the microorganisms with a tool that would allow them to find nutrients from any type of plant-derived material.


Assuntos
Glucose , beta-Glucosidase , Glucose/metabolismo , Glucosídeos/metabolismo , Hidrólise , Oligossacarídeos/metabolismo , Polissacarídeos/metabolismo , Especificidade por Substrato , beta-Glucosidase/genética , beta-Glucosidase/metabolismo
20.
J Am Chem Soc ; 144(32): 14578-14589, 2022 08 17.
Artigo em Inglês | MEDLINE | ID: mdl-35917336

RESUMO

A-to-I RNA editing is widespread in human cells but is uncommon in the coding regions of proteins outside the nervous system. An unusual target for recoding by the adenosine deaminase ADAR1 is the pre-mRNA of the base excision DNA repair enzyme NEIL1 that results in the conversion of a lysine (K) to arginine (R) within the lesion recognition loop and alters substrate specificity. Differences in base removal by unedited (UE, K242) vs edited (Ed, R242) NEIL1 were evaluated using a series of oxidatively modified DNA bases to provide insight into the chemical and structural features of the lesion base that impact isoform-specific repair. We find that UE NEIL1 exhibits higher activity than Ed NEIL1 toward the removal of oxidized pyrimidines, such as thymine glycol, uracil glycol, 5-hydroxyuracil, and 5-hydroxymethyluracil. Gas-phase calculations indicate that the relative rates in excision track with the more stable lactim tautomer and the proton affinity of N3 of the base lesion. These trends support the contribution of tautomerization and N3 protonation in NEIL1 excision catalysis of these pyrimidine base lesions. Structurally similar but distinct substrate lesions, 5-hydroxycytosine and guanidinohydantoin, are more efficiently removed by the Ed NEIL1 isoform, consistent with the inherent differences in tautomerization, proton affinities, and lability. We also observed biphasic kinetic profiles and lack of complete base removal with specific combinations of the lesion and NEIL1 isoform, suggestive of multiple lesion binding modes. The complexity of NEIL1 isoform activity implies multiple roles for NEIL1 in safeguarding accurate repair and as an epigenetic regulator.


Assuntos
DNA Glicosilases , Edição de RNA , DNA/metabolismo , DNA Glicosilases/metabolismo , Reparo do DNA , Humanos , Prótons , Especificidade por Substrato
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...