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1.
J Agric Food Chem ; 72(35): 19403-19412, 2024 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-39180506

RESUMEN

Alginate lyases (ALys) whose degrading products, alginate oligosaccharides, exhibit various outstanding biochemical activities have aroused increasing interest of researchers in the marine bioresource field. However, their predominant sourcing from marine bacteria, with limited yields and unclear genetic backgrounds, presents a challenge for industrial production. In this study, ALys (Aly01) from Vibrio natriegens SK 42.001 was expressed in Bacillus subtilis (B. subtilis), a nonpathogenic microorganism recognized as generally safe (GRAS). This accomplishment was realized through a comprehensive strategy involving vector and host selection, promoter and signal peptide screening, and engineering of the ribosome binding site (RBS) and the N-terminal coding sequence (NCS). The optimal combination was identified as the pP43NMK and B. subtilis WB600. Among the 19 reported strong promoters, PnprE exhibited the best performance, showing intracellular enzyme activities of 4.47 U/mL. Despite expectations, dual promoter construction did not yield a significant increase. Further, SPydhT demonstrated the highest extracellular activity (1.33 U/mL), which was further improved by RBS/NCS engineering, reaching 4.58 U/mL. Finally, after fed-batch fermentation, the extracellular activity reached 18.01 U/mL, which was the highest of ALys with a high molecular weight expressed in B. subtilis. These findings are expected to offer valuable insights into the heterologous expression of ALys in B. subtilis.


Asunto(s)
Bacillus subtilis , Proteínas Bacterianas , Polisacárido Liasas , Regiones Promotoras Genéticas , Señales de Clasificación de Proteína , Bacillus subtilis/genética , Bacillus subtilis/enzimología , Bacillus subtilis/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/química , Señales de Clasificación de Proteína/genética , Vibrio/genética , Vibrio/enzimología , Vectores Genéticos/metabolismo , Vectores Genéticos/genética , Vectores Genéticos/química , Iniciación de la Cadena Peptídica Traduccional
2.
Biochem Biophys Res Commun ; 729: 150357, 2024 Oct 15.
Artículo en Inglés | MEDLINE | ID: mdl-39002194

RESUMEN

ß-N-acetylglucosaminidase (GlcNAcase) is an essential biocatalyst in chitin assimilation by marine Vibrio species, which rely on chitin as their main carbon source. Structure-based phylogenetic analysis of the GlcNAcase superfamily revealed that a GlcNAcase from Vibrio campbellii, formerly named V. harveyi, (VhGlcNAcase) belongs to a major clade, Clade A-I, of the phylogenetic tree. Pre-steady-state and steady-state kinetic analysis of the reaction catalysed by VhGlcNAcase with the fluorogenic substrate 4-methylumbelliferyl N-acetyl-ß-D-glucosaminide suggested the following mechanism: (1) the Michaelis-Menten complex is formed in a rapid enzyme-substrate equilibrium with a Kd of 99.1 ± 1 µM. (2) The glycosidic bond is cleaved by the action of the catalytic residue Glu438, followed by the rapid release of the aglycone product with a rate constant (k2) of 53.3 ± 1 s-1. (3) After the formation of an oxazolinium ion intermediate with the assistance of Asp437, the anomeric carbon of the transition state is attacked by a catalytic water, followed by release of the glycone product with a rate constant (k3) of 14.6 s-1, which is rate-limiting. The result clearly indicated a three-step "ping-pong" mechanism for VhGlcNAcase.


Asunto(s)
Acetilglucosaminidasa , Filogenia , Vibrio , Acetilglucosaminidasa/genética , Acetilglucosaminidasa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Cinética , Modelos Moleculares , Especificidad por Sustrato , Vibrio/enzimología , Vibrio/genética
3.
Int J Biol Macromol ; 277(Pt 3): 134221, 2024 Oct.
Artículo en Inglés | MEDLINE | ID: mdl-39069041

RESUMEN

Alginate is a commercially important polysaccharide widely distributed in brown algae. Carbohydrate-binding modules (CBMs), a class of commonly used polysaccharide-binding proteins, have greatly facilitated the investigations of polysaccharides. Few alginate-binding CBMs have been hitherto reported and structurally characterized. Herein, an unknown domain from a potential PL6 family alginate lyase in the marine bacterium Vibrio breoganii was discovered and recombinantly expressed. The obtained protein, designated VbCBM106, displayed the favorable specificity to alginate. The unique sequence and well-defined function of VbCBM106 reveal a new CBM family (CBM106). Moreover, the structure of VbCBM106 was determined at a 1.5 Å resolution by the X-ray crystallography, which shows a typical ß-sandwich fold comprised of two antiparallel ß-sheets. Site-directed mutagenesis assays confirmed that positively charged polar residues are crucial for the ligand binding of VbCBM106. The discovery of VbCBM106 enriches the toolbox of alginate-binding proteins, and the elucidation of critical residues would guide the future practical applications of VbCBM106.


Asunto(s)
Alginatos , Alginatos/química , Alginatos/metabolismo , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/metabolismo , Secuencia de Aminoácidos , Modelos Moleculares , Vibrio/enzimología , Vibrio/genética , Unión Proteica , Cristalografía por Rayos X , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Especificidad por Sustrato
4.
Carbohydr Res ; 543: 109221, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-39067181

RESUMEN

Hyaluronidases are a class of enzymes that can degrade hyaluronic acid and have a wide range of applications in the medical field. In this study, the marine bacterium Vibrio sp. ZG1, which can degrade HA, was isolated, leading to the discovery of two novel hyaluronan lyases, Vhylzx1 and Vhylzx2, through genome sequencing and bioinformatic analysis. These lyases belong to the polysaccharide lyase-8 family. Vhylzx1 and Vhylzx2 specifically degrade HA, with highest activity at 35 °C, pH 5.7 and 50 °C, pH 7.1. Vhylzx1 and Vhylzx2 are endo-type enzymes that can fully degrade HA into unsaturated disaccharides. Sequence homology assessment and site-directed mutagenesis revealed that the catalytic residues of Vhylzx1 are Asn231, His281, and Tyr290, and that the catalytic residues of Vhylzx2 are Asn227, His277, and Tyr286. Moreover, this study used consensus sequences to enhance the specific activity of Vhylzx2 mutants. Notably, the mutants V564I, N742D, L619F, and D658G increases the specific activity by 2.4, 2.2, 1.3, and 1.2-fold. These characteristics are useful for further basic research and applications, and have a promising application in the preparation of biologically active hyaluronic acid oligosaccharides.


Asunto(s)
Clonación Molecular , Ácido Hialurónico , Polisacárido Liasas , Vibrio , Vibrio/enzimología , Vibrio/genética , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/química , Ácido Hialurónico/química , Ácido Hialurónico/biosíntesis , Ácido Hialurónico/metabolismo , Secuencia de Aminoácidos , Especificidad por Sustrato
5.
Int J Mol Sci ; 25(11)2024 May 26.
Artículo en Inglés | MEDLINE | ID: mdl-38891987

RESUMEN

Alginate lyases cleave the 1,4-glycosidic bond of alginate by eliminating sugar molecules from its bond. While earlier reported alginate lyases were primarily single catalytic domains, research on multi-module alginate lyases has been lfiguimited. This study identified VsAly7A, a multi-module alginate lyase present in Vibrio sp. QY108, comprising a "Pro-Asp-Thr(PDT)" fragment and two PL-7 catalytic domains (CD I and CD II). The "PDT" fragment enhances the soluble expression level and increases the thermostability and binding affinity to the substrate. Moreover, CD I exhibited greater catalytic efficiency than CD II. The incorporation of PDT-CD I resulted in an increase in the optimal temperature of VsAly7A, whereas CD II displayed a preference for polyG degradation. The multi-domain structure of VsAly7A provides a new idea for the rational design of alginate lyase, whilst the "PDT" fragment may serve as a fusion tag in the soluble expression of recombinant proteins.


Asunto(s)
Alginatos , Estabilidad de Enzimas , Polisacárido Liasas , Vibrio , Polisacárido Liasas/metabolismo , Polisacárido Liasas/genética , Polisacárido Liasas/química , Vibrio/enzimología , Vibrio/genética , Alginatos/metabolismo , Alginatos/química , Unión Proteica , Dominio Catalítico , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/química , Solubilidad , Secuencia de Aminoácidos , Temperatura , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética
6.
Mar Drugs ; 22(5)2024 May 18.
Artículo en Inglés | MEDLINE | ID: mdl-38786621

RESUMEN

Alginate oligosaccharides (AOS), products of alginate degradation by endotype alginate lyases, possess favorable biological activities and have broad applications. Although many have been reported, alginate lyases with homogeneous AOS products and secretory production by an engineered host are scarce. Herein, the alginate lyase AlyC7 from Vibrio sp. C42 was characterized as a trisaccharide-producing lyase exhibiting high activity and broad substrate specificity. With PelB as the signal peptide and 500 mM glycine as the additive, the extracellular production of AlyC7 in Escherichia coli reached 1122.8 U/mL after 27 h cultivation in Luria-Bertani medium. The yield of trisaccharides from sodium alginate degradation by the produced AlyC7 reached 758.6 mg/g, with a purity of 85.1%. The prepared AOS at 20 µg/mL increased the root length of lettuce, tomato, wheat, and maize by 27.5%, 25.7%, 9.7%, and 11.1%, respectively. This study establishes a robust foundation for the industrial and agricultural applications of AlyC7.


Asunto(s)
Escherichia coli , Polisacárido Liasas , Trisacáridos , Vibrio , Polisacárido Liasas/metabolismo , Trisacáridos/biosíntesis , Vibrio/enzimología , Especificidad por Sustrato , Alginatos , Zea mays , Oligosacáridos
7.
Carbohydr Res ; 540: 109144, 2024 Jun.
Artículo en Inglés | MEDLINE | ID: mdl-38733729

RESUMEN

Chitooligosaccharides, the hydrolysis products of chitin, have superior biological activities and application value to those of chitin itself; however, the ordered and highly crystalline structure of chitin renders its degradation by chitinase difficult. Herein, the effects of plasma-activated water (PAW) pre-treatment on the physicochemical properties, crystal structure, and enzymatic hydrolysis of chitin were investigated. The hydrolysis of PAW-pre-treated chitin (PAW activation time of 5 min) using chitinase from Vibrio harveyi (VhChit2) yielded 71 % more reducing sugar, compared with that from untreated chitin, with the degree of chitin hydrolysis increasing from 13 % without pre-treatment to 23 % post-treatment. Moreover, the amount of VhChit2 adsorbed by chitin increased from 41.7 to 58.2 mg/g. Fourier transform infrared spectrometry revealed that PAW could break the ß-1,4-glycosidic bonds of chitin (but had no effects on the hydrogen and amido bonds), thereby decreasing the molecular weight and crystallinity of the polysaccharide, which caused its structural damage and enhanced its enzymatic hydrolysis by chitinase. Consequently, PAW pre-treatment can be considered a simple, effective, and environmentally-friendly method for the biotransformation of chitin as its easier hydrolysis yields high-value products.


Asunto(s)
Quitina , Quitinasas , Peso Molecular , Vibrio , Agua , Quitinasas/química , Quitinasas/metabolismo , Quitina/química , Quitina/metabolismo , Quitina/análogos & derivados , Agua/química , Hidrólisis , Vibrio/enzimología
8.
J Agric Food Chem ; 72(22): 12618-12629, 2024 Jun 05.
Artículo en Inglés | MEDLINE | ID: mdl-38778776

RESUMEN

Microbial nitrogen fixation presents a viable alternative to chemical fertilizers, yet the limited colonization and specificity of naturally occurring nitrogen-fixing microorganisms present significant challenges to their widespread application. In this study, we identified a nitrogen fixation gene cluster (VNnif) in Vibrio natriegens (VN) and tested its nitrogenase activity through the acetylene reduction assay. We investigated the potential utilization of nitrogenase by incorporating the nitrogenase gene cluster from VN into plant growth-promoting rhizosphere bacteria Pseudomonas protegens CHA0 and enhancing its activity to 48.16 nmol C2H2/mg/h through promoter replacement and cluster rearrangement. The engineered strain CHA0-PVNnif was found to positively impact the growth of Arabidopsis thaliana col-0 and Triticum aestivum L. (wheat). This study expanded the role of plant growth-promoting rhizobacteria (PGPR) and provided a research foundation for enhancing nitrogenase activity.


Asunto(s)
Proteínas Bacterianas , Fijación del Nitrógeno , Nitrogenasa , Vibrio , Arabidopsis/genética , Arabidopsis/microbiología , Arabidopsis/enzimología , Arabidopsis/crecimiento & desarrollo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Familia de Multigenes , Nitrogenasa/metabolismo , Nitrogenasa/genética , Rizosfera , Triticum/microbiología , Triticum/genética , Triticum/crecimiento & desarrollo , Triticum/metabolismo , Vibrio/genética , Vibrio/crecimiento & desarrollo , Vibrio/enzimología
9.
Proc Natl Acad Sci U S A ; 121(19): e2317954121, 2024 May 07.
Artículo en Inglés | MEDLINE | ID: mdl-38683976

RESUMEN

Several microbial genomes lack textbook-defined essential genes. If an essential gene is absent from a genome, then an evolutionarily independent gene of unknown function complements its function. Here, we identified frequent nonhomologous replacement of an essential component of DNA replication initiation, a replicative helicase loader gene, in Vibrionaceae. Our analysis of Vibrionaceae genomes revealed two genes with unknown function, named vdhL1 and vdhL2, that were substantially enriched in genomes without the known helicase-loader genes. These genes showed no sequence similarities to genes with known function but encoded proteins structurally similar with a viral helicase loader. Analyses of genomic syntenies and coevolution with helicase genes suggested that vdhL1/2 encodes a helicase loader. The in vitro assay showed that Vibrio harveyi VdhL1 and Vibrio ezurae VdhL2 promote the helicase activity of DnaB. Furthermore, molecular phylogenetics suggested that vdhL1/2 were derived from phages and replaced an intrinsic helicase loader gene of Vibrionaceae over 20 times. This high replacement frequency implies the host's advantage in acquiring a viral helicase loader gene.


Asunto(s)
ADN Helicasas , Replicación del ADN , Filogenia , Vibrionaceae , Vibrionaceae/genética , Vibrionaceae/enzimología , ADN Helicasas/metabolismo , ADN Helicasas/genética , Proteínas Bacterianas/metabolismo , Proteínas Bacterianas/genética , Proteínas Virales/genética , Proteínas Virales/metabolismo , Bacteriófagos/genética , Bacteriófagos/enzimología , Evolución Molecular , Genoma Bacteriano , AdnB Helicasas/metabolismo , AdnB Helicasas/genética , Vibrio/genética , Vibrio/enzimología
10.
Chembiochem ; 25(10): e202400107, 2024 May 17.
Artículo en Inglés | MEDLINE | ID: mdl-38536122

RESUMEN

This study characterizes the acceptor specificity of levansucrases (LSs) from Gluconobacter oxydans (LS1), Vibrio natriegens (LS2), Novosphingobium aromaticivorans (LS3), and Paraburkholderia graminis (LS4) using sucrose as fructosyl donor and selected phenolic compounds and carbohydrates as acceptors. Overall, V. natriegens LS2 proved to be the best biocatalyst for the transfructosylation of phenolic compounds. More than one fructosyl unit could be attached to fructosylated phenolic compounds. The transfructosylation of epicatechin by P. graminis LS4 resulted in the most diversified products, with up to five fructosyl units transferred. In addition to the LS source, the acceptor specificity of LS towards phenolic compounds and their transfructosylation products were found to greatly depend on their chemical structure: the number of phenolic rings, the reactivity of hydroxyl groups and the presence of aliphatic chains or methoxy groups. Similarly, for carbohydrates, the transfructosylation yield was dependent on both the LS source and the acceptor type. The highest yield of fructosylated-trisaccharides was Erlose from the transfructosylation of maltose catalyzed by LS2, with production reaching 200 g/L. LS2 was more selective towards the transfructosylation of phenolic compounds and carbohydrates, while reactions catalyzed by LS1, LS3 and LS4 also produced fructooligosaccharides. This study shows the high potential for the application of LSs in the glycosylation of phenolic compounds and carbohydrates.


Asunto(s)
Biocatálisis , Hexosiltransferasas , Fenoles , Hexosiltransferasas/metabolismo , Hexosiltransferasas/química , Fenoles/metabolismo , Fenoles/química , Glicosilación , Especificidad por Sustrato , Vibrio/enzimología , Gluconobacter oxydans/enzimología , Gluconobacter oxydans/metabolismo , Carbohidratos/química
11.
J Biol Chem ; 299(5): 104639, 2023 05.
Artículo en Inglés | MEDLINE | ID: mdl-36965614

RESUMEN

Luciferase-based gene reporters generating bioluminescence signals are important tools for biomedical research. Amongst the luciferases, flavin-dependent enzymes use the most economical chemicals. However, their applications in mammalian cells are limited due to their low signals compared to other systems. Here, we constructed Flavin Luciferase from Vibrio campbellii (Vc) for Mammalian Cell Expression (FLUXVc) by engineering luciferase from V. campbellii (the most thermostable bacterial luciferase reported to date) and optimizing its expression and reporter assays in mammalian cells which can improve the bioluminescence light output by >400-fold as compared to the nonengineered version. We found that the FLUXVc reporter gene can be overexpressed in various cell lines and showed outstanding signal-to-background in HepG2 cells, significantly higher than that of firefly luciferase (Fluc). The combined use of FLUXVc/Fluc as target/control vectors gave the most stable signals, better than the standard set of Fluc(target)/Rluc(control). We also demonstrated that FLUXVc can be used for testing inhibitors of the NF-κB signaling pathway. Collectively, our results provide an optimized method for using the more economical flavin-dependent luciferase in mammalian cells.


Asunto(s)
Biotecnología , Genes Reporteros , Luciferasas , Mediciones Luminiscentes , Animales , Genes Reporteros/genética , Luciferasas/genética , Luciferasas/metabolismo , Mediciones Luminiscentes/normas , Mamíferos/metabolismo , Vibrio/enzimología , Proteínas Recombinantes de Fusión/metabolismo , Vectores Genéticos , Biotecnología/métodos
12.
Mar Drugs ; 20(12)2022 Nov 26.
Artículo en Inglés | MEDLINE | ID: mdl-36547893

RESUMEN

Alginate is abundant in the cell walls of brown algae. Alginate lyases can degrade alginate, and thus play an important role in the marine carbon cycle and industrial production. Currently, most reported alginate lyases contain only one functional alginate lyase domain. AlyC8 is a putative alginate lyase with two alginate lyase domains (CD1 and CD2) from the marine alginate-degrading strain Vibrio sp. C42. To characterize AlyC8 and its two catalytic domains, AlyC8 and its two catalytic domain-deleted mutants, AlyC8-CD1 and AlyC8-CD2, were expressed in Escherichia coli. All three proteins have noticeable activity toward sodium alginate and exhibit optimal activities at pH 8.0-9.0 and at 30-40 °C, demonstrating that both CD1 and CD2 are functional. However, CD1 and CD2 showed opposite substrate specificity. The differences in substrate specificity and degradation products of alginate between the mutants and AlyC8 demonstrate that CD1 and CD2 can act synergistically to enable AlyC8 to degrade various alginate substrates into smaller oligomeric products. Moreover, kinetic analysis indicated that AlyC8-CD1 plays a major role in the degradation of alginate by AlyC8. These results demonstrate that AlyC8 is a novel alginate lyase with two functional catalytic domains that are synergistic in alginate degradation, which is helpful for a better understanding of alginate lyases and alginate degradation.


Asunto(s)
Proteínas Bacterianas , Polisacárido Liasas , Vibrio , Alginatos/química , Concentración de Iones de Hidrógeno , Cinética , Polisacárido Liasas/química , Polisacárido Liasas/genética , Polisacárido Liasas/aislamiento & purificación , Especificidad por Sustrato , Vibrio/enzimología , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/aislamiento & purificación , Mutación , Dominio Catalítico
13.
Nat Methods ; 19(2): 205-215, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-35132245

RESUMEN

Transgenic expression of bacterial nitroreductase (NTR) enzymes sensitizes eukaryotic cells to prodrugs such as metronidazole (MTZ), enabling selective cell-ablation paradigms that have expanded studies of cell function and regeneration in vertebrates. However, first-generation NTRs required confoundingly toxic prodrug treatments to achieve effective cell ablation, and some cell types have proven resistant. Here we used rational engineering and cross-species screening to develop an NTR variant, NTR 2.0, which exhibits ~100-fold improvement in MTZ-mediated cell-specific ablation efficacy, eliminating the need for near-toxic prodrug treatment regimens. NTR 2.0 therefore enables sustained cell-loss paradigms and ablation of previously resistant cell types. These properties permit enhanced interrogations of cell function, extended challenges to the regenerative capacities of discrete stem cell niches, and novel modeling of chronic degenerative diseases. Accordingly, we have created a series of bipartite transgenic reporter/effector resources to facilitate dissemination of NTR 2.0 to the research community.


Asunto(s)
Metronidazol/farmacología , Nitrorreductasas/metabolismo , Profármacos/química , Animales , Animales Modificados Genéticamente , Células CHO , Cricetulus , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Células HEK293 , Humanos , Metronidazol/farmacocinética , Nitrorreductasas/química , Nitrorreductasas/genética , Profármacos/farmacología , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Retina/citología , Retina/efectos de los fármacos , Vibrio/enzimología , Pez Cebra/genética
14.
Nat Commun ; 13(1): 566, 2022 01 28.
Artículo en Inglés | MEDLINE | ID: mdl-35091565

RESUMEN

The collagenases of Vibrio species, many of which are pathogens, have been regarded as an important virulence factor. However, there is little information on the structure and collagenolytic mechanism of Vibrio collagenase. Here, we report the crystal structure of the collagenase module (CM) of Vibrio collagenase VhaC and the conformation of VhaC in solution. Structural and biochemical analyses and molecular dynamics studies reveal that triple-helical collagen is initially recognized by the activator domain, followed by subsequent cleavage by the peptidase domain along with the closing movement of CM. This is different from the peptidolytic mode or the proposed collagenolysis of Clostridium collagenase. We propose a model for the integrated collagenolytic mechanism of VhaC, integrating the functions of VhaC accessory domains and its collagen degradation pattern. This study provides insight into the mechanism of bacterial collagenolysis and helps in structure-based drug design targeting of the Vibrio collagenase.


Asunto(s)
Proteínas Bacterianas/química , Colágeno/metabolismo , Colagenasas/química , Conformación Proteica , Vibrio/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión/genética , Biocatálisis , Cromatografía Liquida , Colagenasas/genética , Colagenasas/metabolismo , Cristalografía por Rayos X , Espectrometría de Masas , Microscopía de Fuerza Atómica , Simulación de Dinámica Molecular , Péptidos/genética , Péptidos/metabolismo , Unión Proteica , Vibrio/enzimología , Vibrio/genética
15.
Acta Biochim Biophys Sin (Shanghai) ; 53(9): 1124-1133, 2021 Aug 31.
Artículo en Inglés | MEDLINE | ID: mdl-34169308

RESUMEN

Vibrio natriegens is known to be the fastest-growing free-living bacterium with the potential to be a novel protein expression system other than Escherichia coli. Seven sampled genes of interest (GOIs) encoding biocatalyst enzymes, including Ochrobactrum anthropi-derived ω-transaminase (OATA), were strongly expressed in E. coli but weakly in V. natriegens using the pET expression system. In this study, we fused the C-terminal of OATA with green fluorescent protein (GFP) and obtained V. natriegens mutants that could increase both protein yield and enzyme activity of OATA as well as the other three GOIs by ultraviolet mutagenesis, fluorescence-activated cell sorting (FACS), and OATA colorimetric assay. Furthermore, next-generation sequencing and strain reconstruction revealed that the Y457 variants in the conserved site of endogenous RNA polymerase (RNAP) ß' subunit rpoC are responsible for the increase in recombinant protein yield. We speculated that the mutation of rpoC Y457 may reprogram V. natriegens's innate gene transcription, thereby increasing the copy number of pET plasmids and soluble protein yield of certain GOIs. The increase in GOI expression may partly be attributed to the increase in copy number. In conclusion, GOI-GFP fusion combined with FACS is a powerful tool of forward genetics that can be used to obtain a superior expression chassis. If more high-expression-related targets are found for more GOIs, it would make the construction of next-generation protein expression chassis more time-saving.


Asunto(s)
Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/genética , Vibrio/enzimología , Vibrio/genética , Biotecnología/métodos , ARN Polimerasas Dirigidas por ADN/química , ARN Polimerasas Dirigidas por ADN/genética , Citometría de Flujo , Proteínas Fluorescentes Verdes/biosíntesis , Proteínas Fluorescentes Verdes/genética , Secuenciación de Nucleótidos de Alto Rendimiento , Ensayos Analíticos de Alto Rendimiento , Biología Molecular/métodos , Mutagénesis , Ochrobactrum anthropi/enzimología , Ochrobactrum anthropi/genética , Plásmidos , Transaminasas/biosíntesis , Transaminasas/genética
16.
Bioorg Med Chem ; 43: 116271, 2021 08 01.
Artículo en Inglés | MEDLINE | ID: mdl-34171757

RESUMEN

Apremilast is an important active pharmaceutical ingredient that relies on a resolution to produce the key chiral amine intermediate. To provide a new catalytic and enzymatic process for Apremilast, we performed the directed evolution of the amine transaminase fromVibriofluvialis. Six rounds of evolution resulted in the VF-8M-E variant with > 400-fold increase specific activity over the wildtype enzyme. A homology model of VF-8M-E was built and a molecular docking study was performed to explain the increase in activity. The purified VF-8M-E was successfully applied to produce the key chiral amine intermediate in enantiopure form and 49% conversion via a kinetic resolution, representing a new enzymatic access towards Apremilast.


Asunto(s)
Aminas/metabolismo , Talidomida/análogos & derivados , Transaminasas/metabolismo , Aminas/química , Biocatálisis , Cinética , Estructura Molecular , Talidomida/química , Talidomida/metabolismo , Transaminasas/química , Vibrio/enzimología
17.
Acta Crystallogr D Struct Biol ; 77(Pt 5): 674-689, 2021 May 01.
Artículo en Inglés | MEDLINE | ID: mdl-33950022

RESUMEN

Vibrio species play a crucial role in maintaining the carbon and nitrogen balance between the oceans and the land through their ability to employ chitin as a sole source of energy. This study describes the structural basis for the action of the GH20 ß-N-acetylglucosaminidase (VhGlcNAcase) in chitin metabolism by Vibrio campbellii (formerly V. harveyi) strain ATCC BAA-1116. Crystal structures of wild-type VhGlcNAcase in the absence and presence of the sugar ligand, and of the unliganded D437A mutant, were determined. VhGlcNAcase contains three distinct domains: an N-terminal carbohydrate-binding domain linked to a small α+ß domain and a C-terminal (ß/α)8 catalytic domain. The active site of VhGlcNAcase has a narrow, shallow pocket that is suitable for accommodating a small chitooligosaccharide. VhGlcNAcase is a monomeric enzyme of 74 kDa, but its crystal structures show two molecules of enzyme per asymmetric unit, in which Gln16 at the dimeric interface of the first molecule partially blocks the entrance to the active site of the neighboring molecule. The GlcNAc unit observed in subsite -1 makes exclusive hydrogen bonds to the conserved residues Arg274, Tyr530, Asp532 and Glu584, while Trp487, Trp546, Trp582 and Trp505 form a hydrophobic wall around the -1 GlcNAc. The catalytic mutants D437A/N and E438A/Q exhibited a drastic loss of GlcNAcase activity, confirming the catalytic role of the acidic pair (Asp437-Glu438).


Asunto(s)
Acetilglucosaminidasa/química , Quitina/metabolismo , Vibrio/enzimología , Unión Proteica , Dominios Proteicos , Especificidad por Sustrato
18.
Biochemistry ; 60(15): 1214-1225, 2021 04 20.
Artículo en Inglés | MEDLINE | ID: mdl-33830741

RESUMEN

Phosphonates represent an important source of bioavailable phosphorus in certain environments. Accordingly, many microorganisms (particularly marine bacteria) possess catabolic pathways to degrade these molecules. One example is the widespread hydrolytic route for the breakdown of 2-aminoethylphosphonate (AEP, the most common biogenic phosphonate). In this pathway, the aminotransferase PhnW initially converts AEP into phosphonoacetaldehyde (PAA), which is then cleaved by the hydrolase PhnX to yield acetaldehyde and phosphate. This work focuses on a pyridoxal 5'-phosphate-dependent enzyme that is encoded in >13% of the bacterial gene clusters containing the phnW-phnX combination. This enzyme (which we termed PbfA) is annotated as a transaminase, but there is no obvious need for an additional transamination reaction in the established AEP degradation pathway. We report here that PbfA from the marine bacterium Vibrio splendidus catalyzes an elimination reaction on the naturally occurring compound (R)-1-hydroxy-2-aminoethylphosphonate (R-HAEP). The reaction releases ammonia and generates PAA, which can be then hydrolyzed by PhnX. In contrast, PbfA is not active toward the S enantiomer of HAEP or other HAEP-related compounds such as ethanolamine and d,l-isoserine, indicating a very high substrate specificity. We also show that R-HAEP (despite being structurally similar to AEP) is not processed efficiently by the PhnW-PhnX couple in the absence of PbfA. In summary, the reaction catalyzed by PbfA serves to funnel R-HAEP into the hydrolytic pathway for AEP degradation, expanding the scope and the usefulness of the pathway itself.


Asunto(s)
Amoníaco-Liasas/metabolismo , Organofosfonatos/metabolismo , Vibrio/enzimología , Biocatálisis , Hidrólisis , Cinética , Organofosfonatos/química , Especificidad por Sustrato
19.
Nucleic Acids Res ; 49(3): 1708-1723, 2021 02 22.
Artículo en Inglés | MEDLINE | ID: mdl-33450012

RESUMEN

Many modification-dependent restriction endonucleases (MDREs) are fusions of a PUA superfamily modification sensor domain and a nuclease catalytic domain. EVE domains belong to the PUA superfamily, and are present in MDREs in combination with HNH nuclease domains. Here, we present a biochemical characterization of the EVE-HNH endonuclease VcaM4I and crystal structures of the protein alone, with EVE domain bound to either 5mC modified dsDNA or to 5mC/5hmC containing ssDNA. The EVE domain is moderately specific for 5mC/5hmC containing DNA according to EMSA experiments. It flips the modified nucleotide, to accommodate it in a hydrophobic pocket of the enzyme, primarily formed by P24, W82 and Y130 residues. In the crystallized conformation, the EVE domain and linker helix between the two domains block DNA binding to the catalytic domain. Removal of the EVE domain and inter-domain linker, but not of the EVE domain alone converts VcaM4I into a non-specific toxic nuclease. The role of the key residues in the EVE and HNH domains of VcaM4I is confirmed by digestion and restriction assays with the enzyme variants that differ from the wild-type by changes to the base binding pocket or to the catalytic residues.


Asunto(s)
Enzimas de Restricción del ADN/química , ADN/química , 5-Metilcitosina/análogos & derivados , 5-Metilcitosina/química , Dominio Catalítico , Cristalografía por Rayos X , ADN de Cadena Simple/química , Modelos Moleculares , Motivos de Nucleótidos , Dominios Proteicos , Dispersión del Ángulo Pequeño , Vibrio/enzimología , Difracción de Rayos X
20.
Eur J Med Chem ; 209: 112883, 2021 Jan 01.
Artículo en Inglés | MEDLINE | ID: mdl-33035924

RESUMEN

Methionine aminopeptidases (MetAPs) have been recognized as drug targets and have been extensively studied for discovery of selective inhibitors. MetAPs are essential enzymes in all living cells. While most prokaryotes contain a single gene, some prokaryotes and all eukaryotes including human have redundancy. Due to the similarity in the active sites of the MetAP enzyme between the pathogens and human limited the success of discovering selective inhibitors. We recently have discovered that MetAPs with small inserts within the catalytic domain to have different susceptibilities against some inhibitors compared to those that do not have. Using this clue we used bioinformatic tools to identify new variants of MetAPs with inserts in pathogenic species. Two new isoforms were identified in Vibrio species with two and three inserts in addition to an isoform without any insert. Multiple sequence alignment suggested that inserts are conserved in several of the Vibrio species. Two of the three inserts are common between two and three insert isoforms. One of the inserts is identified to have "NNKNN" motif that is similar to well-characterized quorum sensing peptide, "NNWNN". Another insert is predicted to have a posttranslational modification site. Three Vibrio proteins were cloned, expressed, purified, enzyme kinetics established and inhibitor screening has been performed. Several of the pyridinylpyrimidine derivatives selectively inhibited MetAPs with inserts compared to those that do not have, including the human enzyme. Crystal structure and molecular modeling studies provide the molecular basis for selective inhibition.


Asunto(s)
Proteínas Bacterianas/antagonistas & inhibidores , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/farmacología , Metionil Aminopeptidasas/antagonistas & inhibidores , Vibrio/enzimología , Secuencia de Aminoácidos , Antibacterianos/química , Antibacterianos/farmacología , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Dominio Catalítico/efectos de los fármacos , Cristalografía por Rayos X , Humanos , Metionil Aminopeptidasas/química , Metionil Aminopeptidasas/metabolismo , Simulación del Acoplamiento Molecular , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/química , Isoformas de Proteínas/metabolismo , Pirimidinas/química , Pirimidinas/farmacología , Vibrio/química , Vibrio/metabolismo
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