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1.
Microbiol Spectr ; 9(3): e0133321, 2021 12 22.
Artigo em Inglês | MEDLINE | ID: mdl-34817221

RESUMO

A novel putative trehalose synthase gene (treM) was identified from an extreme temperature thermal spring. The gene was expressed in Escherichia coli followed by purification of the protein (TreM). TreM exhibited the pH optima of 7.0 for trehalose and trehalulose production, although it was functional and stable in the pH range of 5.0 to 8.0. Temperature activity profiling revealed that TreM can catalyze trehalose biosynthesis in a wide range of temperatures, from 5°C to 80°C. The optimum activity for trehalose and trehalulose biosynthesis was observed at 45°C and 50°C, respectively. A catalytic reaction performed at the low temperature of 5°C yielded trehalose with significantly reduced by-product (glucose) production in the reaction. TreM displayed remarkable thermal stability at optimum temperatures, with only about 20% loss in the activity after heat (50°C) exposure for 24 h. The maximum bioconversion yield of 74% trehalose (at 5°C) and 90% trehalulose (at 50°C) was obtained from 100 mM maltose and 70 mM sucrose, respectively. TreM was demonstrated to catalyze trehalulose biosynthesis utilizing the low-cost feedstock jaggery, cane molasses, muscovado, and table sugar. IMPORTANCE Trehalose is a rare sugar of high importance in biological research, with its property to stabilize cell membrane and proteins and protect the organism from drought. It is instrumental in the cryopreservation of human cells, e.g., sperm and blood stem cells. It is also very useful in the food industry, especially in the preparation of frozen food products. Trehalose synthase is a glycosyl hydrolase 13 (GH13) family enzyme that has been reported from about 22 bacterial species so far. Of these enzymes, to date, only two have been demonstrated to catalyze the biosynthesis of both trehalose and trehalulose. We have investigated the metagenomic data of an extreme temperature thermal spring to discover a novel gene that encodes a trehalose synthase (TreM) with higher stability and dual transglycosylation activities of trehalose and trehalulose biosynthesis. This enzyme is capable of catalyzing the transformation of maltose to trehalose and sucrose to trehalulose in a wide pH and temperature range. The present investigation endorses the thermal aquatic habitat as a promising genetic resource for the biocatalysts with high potential in producing high-value rare sugars.


Assuntos
Dissacarídeos/biossíntese , Glucosiltransferases/genética , Glucosiltransferases/metabolismo , Nocardioides/metabolismo , Thermus/metabolismo , Trealose/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Fontes Termais/microbiologia , Humanos , Metagenoma/genética , Nocardioides/enzimologia , Nocardioides/genética , Thermomonospora/enzimologia , Thermomonospora/genética , Thermomonospora/metabolismo , Thermus/enzimologia , Thermus/genética
2.
Int J Biol Macromol ; 168: 13-21, 2021 Jan 31.
Artigo em Inglês | MEDLINE | ID: mdl-33285196

RESUMO

One of the most desirable properties for industrial enzymes is high thermotolerance, which can reduce the amount of biocatalyst used and lower the production cost. Aiming to improve the thermotolerance of trehalose synthase (TreS, EC 5.4.99.16) from Thermomonospora curvata, four mutants (G78D, V289L, G322A, I323L) and four cyclized TreS variants fused using different Tag/Catcher pairs (SpyTag-TreS-SpyCatcher, SpyTag-TreS-KTag, SnoopTag-TreS-SnoopCatcher, SnoopTagJR-TreS-DogTag) were constructed. The results showed that cyclization led to a much larger increase of thermostability than that achieved via site-directed mutagenesis. The t1/2 of all four cyclized TreS variants at 55 °C increased 2- to 3- fold, while the analysis of kinetic and thermodynamic stability indicated that the T50 of the different cyclized TreS variants increased by between 7.5 °C and 15.5 °C. Molecular dynamics simulations showed that the Rg values of cyclized TreS decreased significantly, indicating that the protein maintained a tight tertiary structure at high temperatures, avoiding exposure of the hydrophobic core to the solvent. Cyclization using a Tag/Catcher pair is a simple and effective method for improving the thermotolerance of enzymes.


Assuntos
Glucosiltransferases/química , Glucosiltransferases/metabolismo , Actinomycetales , Ciclização , Temperatura Alta , Cinética , Mutagênese Sítio-Dirigida , Oligopeptídeos , Thermomonospora/enzimologia , Trealose
3.
Nature ; 576(7786): 321-325, 2019 12.
Artigo em Inglês | MEDLINE | ID: mdl-31597161

RESUMO

Host infection by pathogenic mycobacteria, such as Mycobacterium tuberculosis, is facilitated by virulence factors that are secreted by type VII secretion systems1. A molecular understanding of the type VII secretion mechanism has been hampered owing to a lack of three-dimensional structures of the fully assembled secretion apparatus. Here we report the cryo-electron microscopy structure of a membrane-embedded core complex of the ESX-3/type VII secretion system from Mycobacterium smegmatis. The core of the ESX-3 secretion machine consists of four protein components-EccB3, EccC3, EccD3 and EccE3, in a 1:1:2:1 stoichiometry-which form two identical protomers. The EccC3 coupling protein comprises a flexible array of four ATPase domains, which are linked to the membrane through a stalk domain. The domain of unknown function (DUF) adjacent to the stalk is identified as an ATPase domain that is essential for secretion. EccB3 is predominantly periplasmatic, but a small segment crosses the membrane and contacts the stalk domain. This suggests that conformational changes in the stalk domain-triggered by substrate binding at the distal end of EccC3 and subsequent ATP hydrolysis in the DUF-could be coupled to substrate secretion to the periplasm. Our results reveal that the architecture of type VII secretion systems differs markedly from that of other known secretion machines2, and provide a structural understanding of these systems that will be useful for the design of antimicrobial strategies that target bacterial virulence.


Assuntos
Microscopia Crioeletrônica , Mycobacterium smegmatis/química , Sistemas de Secreção Tipo VII/química , Sistemas de Secreção Tipo VII/ultraestrutura , Actinobacteria/química , Actinobacteria/enzimologia , Adenosina Trifosfatases/química , Adenosina Trifosfatases/isolamento & purificação , Adenosina Trifosfatases/ultraestrutura , Trifosfato de Adenosina/metabolismo , Modelos Moleculares , Mycobacterium smegmatis/enzimologia , Mycobacterium smegmatis/ultraestrutura , Domínios Proteicos , Estrutura Quaternária de Proteína , Subunidades Proteicas/química , Subunidades Proteicas/isolamento & purificação , Relação Estrutura-Atividade , Thermomonospora , Sistemas de Secreção Tipo VII/isolamento & purificação
4.
J Biol Chem ; 294(31): 11934-11943, 2019 08 02.
Artigo em Inglês | MEDLINE | ID: mdl-31209106

RESUMO

An aldolase from the bile acid-degrading actinobacterium Thermomonospora curvata catalyzes the C-C bond cleavage of an isopropyl-CoA side chain from the D-ring of the steroid metabolite 17-hydroxy-3-oxo-4-pregnene-20-carboxyl-CoA (17-HOPC-CoA). Like its homolog from Mycobacterium tuberculosis, the T. curvata aldolase is a protein complex of Ltp2 with a DUF35 domain derived from the C-terminal domain of a hydratase (ChsH2DUF35) that catalyzes the preceding step in the pathway. We determined the structure of the Ltp2-ChsH2DUF35 complex at 1.7 Å resolution using zinc-single anomalous diffraction. The enzyme adopts an αßßα organization, with the two Ltp2 protomers forming a central dimer, and the two ChsH2DUF35 protomers being at the periphery. Docking experiments suggested that Ltp2 forms a tight complex with the hydratase but that each enzyme retains an independent CoA-binding site. Ltp2 adopted a fold similar to those in thiolases; however, instead of forming a deep tunnel, the Ltp2 active site formed an elongated cleft large enough to accommodate 17-HOPC-CoA. The active site lacked the two cysteines that served as the nucleophile and general base in thiolases and replaced a pair of oxyanion-hole histidine residues with Tyr-246 and Tyr-344. Phenylalanine replacement of either of these residues decreased aldolase catalytic activity at least 400-fold. On the basis of a 17-HOPC-CoA -docked model, we propose a catalytic mechanism where Tyr-294 acts as the general base abstracting a proton from the D-ring hydroxyl of 17-HOPC-CoA and Tyr-344 as the general acid that protonates the propionyl-CoA anion following C-C bond cleavage.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/metabolismo , Frutose-Bifosfato Aldolase/metabolismo , Hidrolases/metabolismo , Sequência de Aminoácidos , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Sítios de Ligação , Domínio Catalítico , Cristalografia por Raios X , Frutose-Bifosfato Aldolase/química , Frutose-Bifosfato Aldolase/genética , Hidrolases/química , Hidrolases/genética , Cinética , Simulação de Acoplamento Molecular , Estrutura Quaternária de Proteína , Proteínas Recombinantes de Fusão/biossíntese , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/isolamento & purificação , Alinhamento de Sequência , Esteroides/química , Esteroides/metabolismo , Especificidade por Substrato , Thermomonospora
5.
Protein Eng Des Sel ; 32(1): 25-32, 2019 09 10.
Artigo em Inglês | MEDLINE | ID: mdl-31251342

RESUMO

Some bacteria belonging to the actinobacteria and proteobacteria groups can accumulate neutral lipids expressing enzymes of the wax ester synthase/acyl coenzyme A: diacylglycerol acyltransferase (WS/DGAT) family. tDGAT is a WS/DGAT-like enzyme from Thermomonospora curvata able to produce TAGs and WEs when heterologously expressed in Escherichia coli. In this study, a protocol for the directed evolution of bacterial lipid-producing enzymes based on fluorimetry is developed and tested. tDGAT has been successfully evolved towards the improvement of TAG production with an up to 2.5 times increase in TAG accumulation. Mutants with no ability to produce TAGs but able to accumulate waxes were also selected during the screening. The localization of the mutations that enhance TAG production in the outer surface of tDGAT points out possible new mechanisms that contribute to the activity of this family of enzymes. This Nile red-based high throughput screening provides an evolution platform for other WS/DGAT-like enzymes.


Assuntos
Actinobacteria/enzimologia , Proteínas de Bactérias/química , Diacilglicerol O-Aciltransferase/química , Evolução Molecular Direcionada , Actinobacteria/genética , Proteínas de Bactérias/genética , Diacilglicerol O-Aciltransferase/genética , Thermomonospora
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