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1.
Microb Biotechnol ; 17(4): e14404, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38588312

RESUMO

Acid phosphatases are enzymes that play a crucial role in the hydrolysis of various organophosphorous molecules. A putative acid phosphatase called FS6 was identified using genetic profiles and sequences from different environments. FS6 showed high sequence similarity to type C acid phosphatases and retained more than 30% of consensus residues in its protein sequence. A histidine-tagged recombinant FS6 produced in Escherichia coli exhibited extremophile properties, functioning effectively in a broad pH range between 3.5 and 8.5. The enzyme demonstrated optimal activity at temperatures between 25 and 50°C, with a melting temperature of 51.6°C. Kinetic parameters were determined using various substrates, and the reaction catalysed by FS6 with physiological substrates was at least 100-fold more efficient than with p-nitrophenyl phosphate. Furthermore, FS6 was found to be a decamer in solution, unlike the dimeric forms of crystallized proteins in its family.


Assuntos
Fosfatase Ácida , Extremófilos , Fosfatase Ácida/metabolismo , Extremófilos/genética , Extremófilos/metabolismo , Hidrólise , Sequência de Aminoácidos , Especificidade por Substrato , Concentração de Íons de Hidrogênio
2.
Wiley Interdiscip Rev RNA ; 15(2): e1836, 2024.
Artigo em Inglês | MEDLINE | ID: mdl-38453211

RESUMO

Protein-only RNase P (PRORP) is an essential enzyme responsible for the 5' maturation of precursor tRNAs (pre-tRNAs). PRORPs are classified into three categories with unique molecular architectures, although all three classes of PRORPs share a mechanism and have similar active sites. Single subunit PRORPs, like those found in plants, have multiple isoforms with different localizations, substrate specificities, and temperature sensitivities. Most recently, Arabidopsis thaliana PRORP2 was shown to interact with TRM1A and B, highlighting a new potential role between these enzymes. Work with At PRORPs led to the development of a ribonuclease that is being used to protect against plant viruses. The mitochondrial RNase P complex, found in metazoans, consists of PRORP, TRMT10C, and SDR5C1, and has also been shown to have substrate specificity, although the cause is unknown. Mutations in mitochondrial tRNA and mitochondrial RNase P have been linked to human disease, highlighting the need to continue understanding this complex. The last class of PRORPs, homologs of Aquifex RNase P (HARPs), is found in thermophilic archaea and bacteria. This most recently discovered type of PRORP forms a large homo-oligomer complex. Although numerous structures of HARPs have been published, it is still unclear how HARPs bind pre-tRNAs and in what ratio. There is also little investigation into the substrate specificity and ideal conditions for HARPs. Moving forward, further work is required to fully characterize each of the three classes of PRORP, the pre-tRNA binding recognition mechanism, the rules of substrate specificity, and how these three distinct classes of PRORP evolved. This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics and Chemistry RNA Structure and Dynamics > Influence of RNA Structure in Biological Systems.


Assuntos
Arabidopsis , Ribonuclease P , Humanos , Ribonuclease P/genética , Ribonuclease P/química , Ribonuclease P/metabolismo , Precursores de RNA/genética , Precursores de RNA/metabolismo , Ribonucleases/metabolismo , Endonucleases/metabolismo , RNA de Transferência/genética , RNA de Transferência/metabolismo , RNA/metabolismo , Arabidopsis/genética , Especificidade por Substrato
3.
Carbohydr Polym ; 333: 121929, 2024 Jun 01.
Artigo em Inglês | MEDLINE | ID: mdl-38494211

RESUMO

Polymerized guluronates (polyG)-specific alginate lyase with lower polymerized mannuronates (polyM)-degrading activity, superior stability, and clear action mode is a powerful biotechnology tool for the preparation of AOSs rich in M blocks. In this study, we expressed and characterized a polyG-specific alginate lyase OUC-FaAly7 from Formosa agariphila KMM3901. OUC-FaAly7 belonging to polysaccharide lyase (PL) family 7 had highest activity (2743.7 ± 20.3 U/µmol) at 45 °C and pH 6.0. Surprisingly, its specific activity against polyG reached 8560.2 ± 76.7 U/µmol, whereas its polyM-degrading activity was nearly 0 within 10 min reaction. Suggesting that OUC-FaAly7 was a strict polyG-specific alginate lyase. Importantly, OUC-FaAly7 showed a wide range of temperature adaptations and remarkable temperature and pH stability. Its relative activity between 20 °C and 45 °C reached >90 % of the maximum activity. The minimum identifiable substrate of OUC-FaAly7 was guluronate tetrasaccharide (G4). Action process and mode showed that it was a novel alginate lyase digesting guluronate hexaose (G6), guluronate heptaose (G7), and polymerized guluronates, with the preferential generation of unsaturated guluronate pentasaccharide (UG5), although which could be further degraded into unsaturated guluronate disaccharide (UG3) and trisaccharide (UG2). This study contributes to illustrating the catalytic properties, substrate recognition, and action mode of novel polyG-specific alginate lyases.


Assuntos
Dissacarídeos , Oligossacarídeos , Especificidade por Substrato , Oligossacarídeos/metabolismo , Dissacarídeos/metabolismo , Polissacarídeo-Liases/metabolismo , Alginatos/metabolismo , Concentração de Íons de Hidrogênio , Proteínas de Bactérias/química
4.
J Agric Food Chem ; 72(11): 5805-5815, 2024 Mar 20.
Artigo em Inglês | MEDLINE | ID: mdl-38451212

RESUMO

Xylan is the main component of hemicellulose. Complete hydrolysis of xylan requires synergistically acting xylanases, such as ß-d-xylosidases. Salt-tolerant ß-d-xylosidases have significant application benefits, but few reports have explored the critical amino acids affecting the salt tolerance of xylosidases. Herein, the site-directed mutation was used to demonstrate that negative electrostatic potentials generated by 19 acidic residues in the loop regions of the structural surface positively correlated with the improved salt tolerance of GH39 ß-d-xylosidase JB13GH39P28. These mutants showed reduced negative potentials on structural surfaces as well as a 13-43% decrease in stability in 3.0-30.0% (w/v) NaCl. Six key residue sites, D201, D259, D297, D377, D395, and D474, were confirmed to influence both the stability and activity of GH39 ß-d-xylosidase. The activity of the GH39 ß-d-xylosidase was found promoting by SO42- and inhibiting by NO3-. Values of Km and Kcat/Km decreased aggravatedly in 30.0% (w/v) NaCl when mutation operated on residues E179 and D182 in the loop regions of the catalytic domain. Taken together, mutation on acidic residues in loop regions from catalytic and noncatalytic domains may cause the deformation of catalytic pocket and aggregation of protein particles then decrease the stability, binding affinity, and catalytic efficiency of the ß-d-xylosidase.


Assuntos
Tolerância ao Sal , Xilosidases , Xilanos/metabolismo , Cloreto de Sódio , Xilosidases/química , Especificidade por Substrato , Concentração de Íons de Hidrogênio
5.
Arch Microbiol ; 206(4): 181, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38502253

RESUMO

The α-L-arabinofuranosidase enzyme plays a crucial role in the degradation of ginsenosides. In this study, we successfully cloned and expressed a novel α-L-arabinofuranosidase bsafs gene (1503 bp, 501 amino acids, 55 kDa, and pI = 5.4) belonging to glycosyl hydrolase (GH) family 51 from Bacillus subtilis genome in Escherichia coli BL21 cells. The recombinant protein Bsafs was purified using Ni2+ sepharose fastflow affinity chromatography and exhibited a specific activity of 2.91 U/mg. Bsafs effectively hydrolyzed the α-L-arabinofuranoside at C20 site of ginsenoside Rc to produce Rd as the product. The Km values for hydrolysis of pNP-α-L-arabinofuranoside (pNPαAraf) and ginsenoside Rc were determined as 0.74 and 4.59 mmol/L, respectively; while the Vmax values for these substrates were found to be 24 and 164 µmol/min/mg, respectively; furthermore, the Kcat values for these enzymes were calculated as 22.3 and 1.58 S-1 correspondingly.


Assuntos
Ginsenosídeos , Ginsenosídeos/metabolismo , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Clonagem Molecular , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato
6.
Toxins (Basel) ; 16(3)2024 Mar 05.
Artigo em Inglês | MEDLINE | ID: mdl-38535802

RESUMO

DepA, a pyrroloquinoline quinone (PQQ)-dependent enzyme isolated from Devosia mutans 17-2-E-8, exhibits versatility in oxidizing deoxynivalenol (DON) and its derivatives. This study explored DepA's substrate specificity and enzyme kinetics, focusing on DON and 15-acetyl-DON. Besides efficiently oxidizing DON, DepA also transforms 15-acetyl-DON into 15-acetyl-3-keto-DON, as identified via LC-MS/MS and NMR analysis. The kinetic parameters, including the maximum reaction rate, turnover number, and catalytic efficiency, were thoroughly evaluated. DepA-PQQ complex docking was deployed to rationalize the substrate specificity of DepA. This study further delves into the reduced toxicity of the transformation products, as demonstrated via enzyme homology modeling and in silico docking analysis with yeast 80S ribosomes, indicating a potential decrease in toxicity due to lower binding affinity. Utilizing the response surface methodology and central composite rotational design, mathematical models were developed to elucidate the relationship between the enzyme and cofactor concentrations, guiding the future development of detoxification systems for liquid feeds and grain processing. This comprehensive analysis underscores DepA's potential for use in mycotoxin detoxification, offering insights for future applications.


Assuntos
Micotoxinas , Tricotecenos , Especificidade por Substrato , Cromatografia Líquida , Espectrometria de Massas em Tandem
7.
Int J Biol Macromol ; 263(Pt 1): 130688, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38458294

RESUMO

This study reports the rational engineering of the S1' substrate-binding pocket of a thermally-stable keratinase from Pseudomonas aeruginosa 4-3 (4-3Ker) to improve substrate specificity to typical keratinase (K/C > 0.5) and catalytic activity without compromising thermal stability for efficient keratin degradation. Of 10 chosen mutation hotspots in the S1' substrate-binding pocket, the top three mutations M128R, A138V, and V142I showing the best catalytic activity and substrate specificity were identified. Their double and triple combinatorial mutants synergistically overcame limitations of single mutants, fabricating an excellent M128R/A138V/V142I triple mutant which displayed a 1.21-fold increase in keratin catalytic activity, 1.10-fold enhancement in keratin/casein activity ratio, and a 3.13 °C increase in half-inactivation temperature compared to 4-3Ker. Molecular dynamics simulations revealed enhanced flexibility of critical amino acid residues at the substrate access tunnel, improved global protein rigidity, and heightened hydrophobicity within the active site likely underpinned the increased catalytic activity and substrate specificity. Additionally, the triple mutant improved the feather degradation rate by 32.86 % over the wild-type, far exceeding commercial keratinase in substrate specificity and thermal stability. This study exemplified engineering a typical keratinase with enhanced substrate specificity, catalytic activity, and thermal stability from thermally-stable 4-3Ker, providing a more robust tool for feather degradation.


Assuntos
Queratinas , Peptídeo Hidrolases , Queratinas/metabolismo , Especificidade por Substrato , Peptídeo Hidrolases/metabolismo , Temperatura , Concentração de Íons de Hidrogênio
8.
Biomolecules ; 14(3)2024 Mar 08.
Artigo em Inglês | MEDLINE | ID: mdl-38540745

RESUMO

Enzymes of the carbohydrate esterase family 4 (CE4) deacetylate a broad range of substrates, including linear, branched and mesh-like polysaccharides. Although they are enzymes of variable amino acid sequence length, they all comprise the conserved catalytic domain NodB. NodB carries the metal binding and active site residues and is characterized by a set of conserved sequence motifs, which are linked to the deacetylation activity. Besides a non-structured, flexible peptide of variable length that precedes NodB, several members of the CE4 family contain additional domains whose function or contribution to substrate specificity are not efficiently characterized. Evidence suggests that CE4 family members comprising solely the NodB domain have developed features linked to a variety of substrate specificities. To understand the NodB-based substrate diversity within the CE4 family, we perform a comparative analysis of all NodB domains structurally characterized so far. We show that amino acid sequence variations, topology diversities and excursions away from the framework structure give rise to different NodB domain classes associated with different substrate specificities and particular functions within and beyond the CE4 family. Our work reveals a link between specific NodB domain characteristics and substrate recognition. Thus, the details of the fold are clarified, and the structural basis of its variations is deciphered and associated with function. The conclusions of this work are also used to make predictions and propose specific functions for biochemically/enzymatically uncharacterized NodB-containing proteins, which have generally been considered as putative CE4 deacetylases. We show that some of them probably belong to different enzymatic families.


Assuntos
Carboidratos , Esterases , Humanos , Esterases/metabolismo , Carboidratos/química , Sequência de Aminoácidos , Polissacarídeos , Domínio Catalítico , Especificidade por Substrato
9.
Int J Mol Sci ; 25(6)2024 Mar 09.
Artigo em Inglês | MEDLINE | ID: mdl-38542148

RESUMO

Bifidobacteria are probiotic microorganisms commonly found in the gastrointestinal tract, some of which are known to utilize linear arabino-oligosaccharides (AOS) as prebiotic carbohydrates. In general, the synergistic actions of exo-type α-l-arabinofuranosidases (ABFs) and endo-α-1,5-l-arabinanases (ABNs) are required for efficient arabinan degradation. In this study, the putative gene cluster for arabinan degradation was discovered in the genome of Bifidobacterium longum subsp. suis. It consists of a variety of genes encoding exo- and endo-hydrolases, sugar-binding proteins, ABC-binding cassettes, and transcriptional regulators. Among them, two endo-ABNs GH43 (BflsABN43A and BflsABN43B), two exo-ABFs GH43 (BflsABF43A and BflsABF43B), and an exo-ABF GH51 (BflsABF51) were predicted to be the key hydrolases for arabinan degradation. These hydrolase genes were functionally expressed in Escherichia coli, and their enzymatic properties were characterized. Their synergism in arabinan degradation has been proposed from the detailed modes of action. Extracellular endo-BflsABN43A hydrolyzes sugar beet and debranched arabinans into the short-chain branched and linear AOS. Intracellularly, AOS can be further degraded into l-arabinose via the cooperative actions of endo-BflsABN43B, exo-BflsABF43A with debranching activity, α-1,5-linkage-specific exo-BflsABF43B, and exo-BflsABF51 with dual activities. The resulting l-arabinose is expected to be metabolized into energy through the pentose phosphate pathway by three enzymes expressed from the ara operon of bifidobacteria. It is anticipated that uncovering arabinan utilization gene clusters and their detailed functions in the genomes of diverse microorganisms will facilitate the development of customized synbiotics.


Assuntos
Arabinose , Bifidobacterium , Polissacarídeos , Polissacarídeos/metabolismo , Família Multigênica , Oligossacarídeos , Glicosídeo Hidrolases/metabolismo , Especificidade por Substrato
10.
Molecules ; 29(6)2024 Mar 17.
Artigo em Inglês | MEDLINE | ID: mdl-38542970

RESUMO

Currently, little is known about the characteristics of polyphenol oxidase from wheat bran, which is closely linked to the browning of wheat product. The wheat PPO was purified by ammonium sulfate precipitation, DEAE-Sepharose ion-exchange column, and Superdex G-75 chromatography column. Purified wheat PPO activity was 11.05-fold higher, its specific activity was 1365.12 U/mg, and its yield was 8.46%. SDS-PAGE showed that the molecular weight of wheat PPO was approximately 21 kDa. Its optimal pH and temperature were 6.5 and 35 °C for catechol as substrate, respectively. Twelve phenolic substrates from wheat and green tea were used for analyzing the substrate specificity. Wheat PPO showed the highest affinity to catechol due to its maximum Vmax (517.55 U·mL-1·min-1) and low Km (6.36 mM) values. Docking analysis revealed strong affinities between catechol, gallic acid, EGCG, and EC with binding energies of -5.28 kcal/mol, -4.65 kcal/mol, -4.21 kcal/mol, and -5.62 kcal/mol, respectively, for PPO. Sodium sulfite, ascorbic acid, and sodium bisulfite dramatically inhibited wheat PPO activity. Cu2+ and Ca2+ at 10 mM were considered potent activators and inhibitors for wheat PPO, respectively. This report provides a theoretical basis for controlling the enzymatic browning of wheat products fortified with green tea.


Assuntos
Catecol Oxidase , Fibras na Dieta , Catecol Oxidase/química , Fibras na Dieta/análise , Concentração de Íons de Hidrogênio , Cinética , Proteínas de Plantas/metabolismo , Catecóis/análise , Especificidade por Substrato , Chá
11.
Int J Biol Macromol ; 264(Pt 1): 130427, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38428763

RESUMO

The asymmetric reduction of α, ß-unsaturated compounds conjugated with electron-withdrawing group by ene-reductases (ERs) is a valuable method for the synthesis of enantiopure chiral compounds. This study introduced an ER from Corynebacterium casei (CcER) which was heterologously expressed in Escherichia coli BL21(DE3), and the purified recombinant CcER was characterized for its biocatalytic properties. CcER exhibited the highest specific activity at 40 °C and pH 6.5, and showcased appreciable stability below 40 °C over a pH range of 6.0-7.0. The enzyme displayed high resistance to methanol. CcER accepted NADH or NADPH as a cofactor and exhibited a broad substrate spectrum towards α, ß-unsaturated compounds. It achieved complete conversion of 2-cyclohexen-1-one and good performance for stereoselective reduction of (R)-carvone (conversion 98 %, diastereoselectivity 96 %). This study highlights the robustness and potential of CcER.


Assuntos
Corynebacterium , Oxirredutases , Oxirredutases/metabolismo , NADP/metabolismo , Especificidade por Substrato
12.
Int J Biol Macromol ; 264(Pt 2): 130484, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38431002

RESUMO

The oxidative reaction of Fusarium mycotoxin deoxynivalenol (DON) using the dehydrogenase is a desirable strategy and environmentally friendly to mitigate its toxicity. However, a critical issue for these dehydrogenases shows widespread substrate promiscuity. In this study, we conducted pocket reshaping of Devosia strain A6-243 pyrroloquinoline quinone (PQQ)-dependent dehydrogenase (DADH) on the basis of protein structure and kinetic analysis of substrate libraries to improve preference for particular substrate DON (10a). The variant presented an increased preference for substrate 10a and enhanced catalytic efficiency. A 4.7-fold increase in preference for substrate 10a was observed. Kinetic profiling and molecular dynamics (MD) simulations provided insights into the enhanced substrate specificity and activity. Moreover, the variant exhibited stronger conversion of substrate 10a to 3-keto-DON compared to the wild DADH. Overall, this study provides a feasible protocol for the redesign of PQQ-dependent dehydrogenases with favourable substrate specificity and catalytic activity, which is desperately needed for DON antidote development.


Assuntos
Acetamidas , Quinonas , Tricotecenos , Especificidade por Substrato , Cinética
13.
Int J Biol Macromol ; 264(Pt 2): 130739, 2024 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-38460639

RESUMO

Extradiol dioxygenases (EDOs) catalyzing meta-cleavage of catecholic compounds promise an effective way to detoxify aromatic pollutants. This work reported a novel scenario to engineer our recently identified Type I EDO from Tcu3516 for a broader substrate scope and enhanced activity, which was based on 2,3-dihydroxybiphenyl (2,3-DHB)-liganded molecular docking of Tcu3516 and multiple sequence alignment with other 22 Type I EDOs. 11 non-conservative residues of Tcu3516 within 6 Å distance to the 2,3-DHB ligand center were selected as potential hotspots and subjected to semi-rational design using 6 catecholic analogues as substrates; the mutants V186L and V212N returned with progressive evolution in substrate scope and catalytic activity. Both mutants were combined with D285A for construction of double mutants and final triple mutant V186L/V212N/D285A. Except for 2,3-DHB (the mutant V186L/D285A gave the best catalytic performance), the triple mutant prevailed all other 5 catecholic compounds for their degradation; affording the catalytic efficiency kcat/Km value increase by 10-30 folds, protein Tm (structural rigidity) increase by 15 °C and the half-life time enhancement by 10 times compared to the wild type Tcu3516. The molecular dynamic simulation suggested that a stabler core and a more flexible entrance are likely accounting for enhanced catalytic activity and stability of enzymes.


Assuntos
Compostos Orgânicos , Oxigenases , Simulação de Acoplamento Molecular , Oxigenases/química , Alinhamento de Sequência , Especificidade por Substrato
14.
Appl Microbiol Biotechnol ; 108(1): 275, 2024 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-38530470

RESUMO

Prenylation plays a pivotal role in the diversification and biological activities of natural products. This study presents the functional characterization of TolF, a multiple prenyltransferase from Tolypocladium inflatum. The heterologous expression of tolF in Aspergillus oryzae, coupled with feeding the transformed strain with paxilline, resulted in the production of 20- and 22-prenylpaxilline. Additionally, TolF demonstrated the ability to prenylated the reduced form of paxilline, ß-paxitriol. A related prenyltransferase TerF from Chaunopycnis alba, exhibited similar substrate tolerance and regioselectivity. In vitro enzyme assays using purified recombinant enzymes TolF and TerF confirmed their capacity to catalyze prenylation of paxilline, ß-paxitriol, and terpendole I. Based on previous reports, terpendole I should be considered a native substrate. This work not only enhances our understanding of the molecular basis and product diversity of prenylation reactions in indole diterpene biosynthesis, but also provides insights into the potential of fungal indole diterpene prenyltransferase to alter their position specificities for prenylation. This could be applicable for the synthesis of industrially useful compounds, including bioactive compounds, thereby opening up new avenues for the development of novel biosynthetic strategies and pharmaceuticals. KEY POINTS: • The study characterizes TolF as a multiple prenyltransferase from Tolypocladium inflatum. • TerF from Chaunopycnis alba shows similar substrate tolerance and regioselectivity compared to TolF. • The research offers insights into the potential applications of fungal indole diterpene prenyltransferases.


Assuntos
Dimetilaliltranstransferase , Diterpenos , Hypocreales , Dimetilaliltranstransferase/metabolismo , Prenilação , Indóis/metabolismo , Diterpenos/metabolismo , Especificidade por Substrato
15.
Molecules ; 29(5)2024 Feb 26.
Artigo em Inglês | MEDLINE | ID: mdl-38474529

RESUMO

As a crucial enzyme for cellulose degradation, ß-glucosidase finds extensive applications in food, feed, and bioethanol production; however, its potential is often limited by inadequate thermal stability and glucose tolerance. In this study, a functional gene (lq-bg5) for a GH1 family ß-glucosidase was obtained from the metagenomic DNA of a hot spring sediment sample and heterologously expressed in E. coli and the recombinant enzyme was purified and characterized. The optimal temperature and pH of LQ-BG5 were 55 °C and 4.6, respectively. The relative residual activity of LQ-BG5 exceeded 90% at 55 °C for 9 h and 60 °C for 6 h and remained above 100% after incubation at pH 5.0-10.0 for 12 h. More importantly, LQ-BG5 demonstrated exceptional glucose tolerance with more than 40% activity remaining even at high glucose concentrations of 3000 mM. Thus, LQ-BG5 represents a thermophilic ß-glucosidase exhibiting excellent thermal stability and remarkable glucose tolerance, making it highly promising for lignocellulose development and utilization.


Assuntos
Glucose , Fontes Termais , Glucose/metabolismo , beta-Glucosidase/metabolismo , Escherichia coli/metabolismo , Temperatura , Concentração de Íons de Hidrogênio , Estabilidade Enzimática , Especificidade por Substrato
16.
J Struct Biol ; 216(1): 108065, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38310992

RESUMO

Bacteria use the fatty acid composition of membrane lipids to maintain homeostasis of the bilayer. ß-Ketoacyl-ACP synthase III (FabH) initiates fatty acid biosynthesis and is the primary determinant of the fatty acid composition. FabH condenses malonyl-acyl carrier protein with an acyl-Coenzyme A primer to form ß -ketoacyl-acyl carrier protein which is used to make substrates for lipid synthesis. The acyl-Coenzyme A primer determines whether an acyl chain in the membrane has iso, anteiso, or no branching (straight chain) and biophysical properties of the membrane. The soil bacterium Bacillus subtilis encodes two copies of FabH (BsFabHA and BsFabHB), and here we solve their crystal structures. The substrate-free 1.85 Å and 2.40 Å structures of BsFabHA and BsFabHB show both enzymes have similar residues that line the active site but differ in the architecture surrounding the catalytic residues and oxyanion hole. Branching in the BsFabHB active site may better accommodate the structure of an iso-branched acyl-Coenzyme A molecule and thus confer superior utilization to BsFabHA for this primer type. The 2.02 Å structure of BsFabHA•Coenzyme A shows how the active site architecture changes after binding the first substrate. The other notable difference is an amino acid insertion in BsFabHB that extends a cap that covers the dimer interface. The cap topology is diverse across FabH structures and appears to be a distinguishing feature. FabH enzymes have variable sensitivity to natural product inhibitors and the availability of crystal structures help clarify how nature designs antimicrobials that differentially target FabH homologs.


Assuntos
Proteína de Transporte de Acila , Bacillus subtilis , Especificidade por Substrato , Proteína de Transporte de Acila/química , Ácidos Graxos , Coenzima A
17.
Biochemistry ; 63(6): 806-814, 2024 Mar 19.
Artigo em Inglês | MEDLINE | ID: mdl-38422553

RESUMO

In the field of medical chemistry and other organic chemistry, introducing a methyl group into a designed position has been difficult to achieve. However, owing to the vigorous developments in the field of enzymology, methyltransferases are considered potential tools for addressing this problem. Within the methyltransferase family, Fur6 catalyzes the methylation of C3 of 1,2,4,5,7-pentahydroxynaphthalene (PHN) using S-adenosyl-l-methionine (SAM) as the methyl donor. Here, we report the catalytic mechanism and substrate specificity of Fur6 based on computational studies. Our molecular dynamics (MD) simulation studies reveal the reactive form of PHN and its interactions with the enzyme. Our hybrid quantum mechanics/molecular mechanics (QM/MM) calculations suggest the reaction pathway of the methyl transfer step in which the energy barrier is 8.6 kcal mol-1. Our free-energy calculations with a polarizable continuum model (PCM) indicate that the final deprotonation step of the methylated intermediate occurs after it is ejected into the water solvent from the active center pocket of Fur6. Additionally, our studies on the protonation states, the highest occupied molecular orbital (HOMOs), and the energy barriers of the methylation reaction for the analogs of PHN demonstrate the mechanism of the specificity to PHN. Our study provides valuable insights into Fur6 chemistry, contributing to a deeper understanding of molecular mechanisms and offering an opportunity to engineer the enzyme to achieve high yields of the desired product(s).


Assuntos
Metiltransferases , Simulação de Dinâmica Molecular , Metiltransferases/metabolismo , Especificidade por Substrato , Catálise , Metilação , Teoria Quântica
18.
Nature ; 627(8003): 445-452, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38383785

RESUMO

Reversible modification of target proteins by ubiquitin and ubiquitin-like proteins (UBLs) is widely used by eukaryotic cells to control protein fate and cell behaviour1. UFM1 is a UBL that predominantly modifies a single lysine residue on a single ribosomal protein, uL24 (also called RPL26), on ribosomes at the cytoplasmic surface of the endoplasmic reticulum (ER)2,3. UFM1 conjugation (UFMylation) facilitates the rescue of 60S ribosomal subunits (60S) that are released after ribosome-associated quality-control-mediated splitting of ribosomes that stall during co-translational translocation of secretory proteins into the ER3,4. Neither the molecular mechanism by which the UFMylation machinery achieves such precise target selection nor how this ribosomal modification promotes 60S rescue is known. Here we show that ribosome UFMylation in vivo occurs on free 60S and we present sequential cryo-electron microscopy snapshots of the heterotrimeric UFM1 E3 ligase (E3(UFM1)) engaging its substrate uL24. E3(UFM1) binds the L1 stalk, empty transfer RNA-binding sites and the peptidyl transferase centre through carboxy-terminal domains of UFL1, which results in uL24 modification more than 150 Å away. After catalysing UFM1 transfer, E3(UFM1) remains stably bound to its product, UFMylated 60S, forming a C-shaped clamp that extends all the way around the 60S from the transfer RNA-binding sites to the polypeptide tunnel exit. Our structural and biochemical analyses suggest a role for E3(UFM1) in post-termination release and recycling of the large ribosomal subunit from the ER membrane.


Assuntos
Retículo Endoplasmático , Processamento de Proteína Pós-Traducional , Subunidades Ribossômicas Maiores de Eucariotos , Ubiquitina-Proteína Ligases , Sítios de Ligação , Biocatálise , Microscopia Crioeletrônica , Retículo Endoplasmático/metabolismo , Retículo Endoplasmático/ultraestrutura , Membranas Intracelulares/química , Membranas Intracelulares/metabolismo , Membranas Intracelulares/ultraestrutura , Peptidil Transferases/química , Peptidil Transferases/metabolismo , Peptidil Transferases/ultraestrutura , Ligação Proteica , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Proteínas Ribossômicas/ultraestrutura , Subunidades Ribossômicas Maiores de Eucariotos/química , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Subunidades Ribossômicas Maiores de Eucariotos/ultraestrutura , RNA de Transferência/metabolismo , Especificidade por Substrato , Ubiquitina-Proteína Ligases/química , Ubiquitina-Proteína Ligases/metabolismo , Ubiquitina-Proteína Ligases/ultraestrutura
19.
J Agric Food Chem ; 72(8): 4116-4126, 2024 Feb 28.
Artigo em Inglês | MEDLINE | ID: mdl-38372665

RESUMO

Alginate lyase Aly448, a potential new member of the polysaccharide lyase (PL) 7 family, which was cloned and identified from the macroalgae-associated bacterial metagenomic library, showed bifunctionality. The molecular docking results revealed that Aly448 has two completely different binding sites for alginate (polyMG), poly-α-l-guluronic acid (polyG), and poly-ß-d-mannuronic acid (polyM) substrates, respectively, which might be the molecular basis for the enzyme's bifunctionality. Truncational results confirmed that predicted key residues affected the bifunctionality of Aly448, but did not wholly explain. Besides, Aly448 presented excellent biochemical characteristics, such as higher thermal stability and pH tolerance. Degradation of polyMG, polyM, and polyG substrates by Aly448 produced tetrasaccharide (DP4), disaccharide (DP2), and galactose (DP1), which exhibited excellent antioxidant activity. These findings provide novel insights into the substrate recognition mechanism of bifunctional alginate lyases and pave a new path for the exploitation of natural antioxidant agents.


Assuntos
Antioxidantes , Proteínas de Bactérias , Proteínas de Bactérias/metabolismo , Simulação de Acoplamento Molecular , Polissacarídeo-Liases/química , Alginatos/química , Especificidade por Substrato , Concentração de Íons de Hidrogênio
20.
Arch Biochem Biophys ; 753: 109926, 2024 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-38346547

RESUMO

Of the more than 100 families of glycosyltransferases, family 1 glycosyltransferases catalyze glycosylation using uridine diphosphate (UDP)-sugar as a sugar donor and are thus referred to as UDP-sugar:glycosyl transferases. The blue color of the Nemophila menziesii flower is derived from metalloanthocyanin, which consists of anthocyanin, flavone, and metal ions. Flavone 7-O-ß-glucoside-4'-O-ß-glucoside in the plant is sequentially biosynthesized from flavons by UDP-glucose:flavone 4'-O-glucosyltransferase (NmF4'GT) and UDP-glucose:flavone 4'-O-glucoside 7-O-glucosyltransferase (NmF4'G7GT). To identify the molecular mechanisms of glucosylation of flavone, the crystal structures of NmF4'G7GT in its apo form and in complex with UDP-glucose or luteolin were determined, and molecular structure prediction using AlphaFold2 was conducted for NmF4'GT. The crystal structures revealed that the size of the ligand-binding pocket and interaction environment for the glucose moiety at the pocket entrance plays a critical role in the substrate preference in NmF4'G7GT. The substrate specificity of NmF4'GT was examined by comparing its model structure with that of NmF4'G7GT. The structure of NmF4'GT may have a smaller acceptor pocket, leading to a substrate preference for non-glucosylated flavones (or flavone aglycones).


Assuntos
Flavonas , Glucosiltransferases , Glucosiltransferases/química , Glucosiltransferases/metabolismo , Ligantes , Uridina Difosfato Glucose/química , Glucose , Glicosiltransferases , Glucosídeos , Especificidade por Substrato
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