scholarly journals Characterization of Solanum melongena Thioesterases Related to Tomato Methylketone Synthase 2

Genes ◽  
2019 ◽  
Vol 10 (7) ◽  
pp. 549 ◽  
Author(s):  
Khuat ◽  
Bui ◽  
Tran ◽  
Truong ◽  
Nguyen ◽  
...  
Keyword(s):  
Methyl Salicylate ◽  
Functional Characterization ◽  
Solanum Melongena ◽  
Industrial Applications ◽  
Biofuel Production ◽  
Mechanical Wounding ◽  
Acyl Carrier Proteins ◽  
E Coli ◽  
High Level

2-Methylketones are involved in plant defense and fragrance and have industrial applications as flavor additives and for biofuel production. We isolated three genes from the crop plant Solanum melongena (eggplant) and investigated these as candidates for methylketone production. The wild tomato methylketone synthase 2 (ShMKS2), which hydrolyzes β-ketoacyl-acyl carrier proteins (ACP) to release β-ketoacids in the penultimate step of methylketone synthesis, was used as a query to identify three homologs from S. melongena: SmMKS2-1, SmMKS2-2, and SmMKS2-3. Expression and functional characterization of SmMKS2s in E. coli showed that SmMKS2-1 and SmMKS2-2 exhibited the thioesterase activity against different β-ketoacyl-ACP substrates to generate the corresponding saturated and unsaturated β-ketoacids, which can undergo decarboxylation to form their respective 2-methylketone products, whereas SmMKS2-3 showed no activity. SmMKS2-1 was expressed at high level in leaves, stems, roots, flowers, and fruits, whereas expression of SmMKS2-2 and SmMKS2-3 was mainly in flowers and fruits, respectively. Expression of SmMKS2-1 was induced in leaves by mechanical wounding, and by methyl jasmonate or methyl salicylate, but SmMKS2-2 and SmMKS2-3 genes were not induced. SmMKS2-1 is a candidate for methylketone-based defense in eggplant, and both SmMKS2-1 and SmMKS2-2 are novel MKS2 enzymes for biosynthesis of methylketones as feedstocks to biofuel production.

scholarly journals Identification and functional characterization of a β-glucosidase from Bacillus tequelensis BD69 expressed in bacterial and yeast heterologous systems

PeerJ ◽  
2020 ◽  
Vol 8 ◽  
pp. e8792 ◽  
Author(s):  
Ahmad Raza ◽  
Ratnasri Pothula ◽  
Heba Abdelgaffar ◽  
Saira Bashir ◽  
Juan Luis Jurat-Fuentes
Keyword(s):  
Molecular Docking ◽  
Animal Feed ◽  
Functional Characterization ◽  
Industrial Applications ◽  
Biofuel Production ◽  
Cloning And Expression ◽  
E Coli ◽  
Glucosidase Activity ◽  
Hydrolysis Of

Background The identification and characterization of novel β-glucosidase genes has attracted considerable attention because of their valuable use in a variety of industrial applications, ranging from biofuel production to improved digestibility of animal feed. We previously isolated a fiber-degrading strain of Bacillus tequelensis from buffalo dung samples, and the goal of the current work was to identify β-glucosidase genes in this strain. We describe the cloning and expression of a new β-glucosidase gene (Bteqβgluc) from Bacillus tequelensis strain BD69 in bacterial and yeast hosts. The recombinant Bteqβgluc were used to characterize specificity and activity parameters, and candidate active residues involved in hydrolysis of different substrates were identified through molecular docking. Methods The full length Bteqβgluc gene was cloned and expressed in Escherichia coli and Pichia pastoris cultures. Recombinant Bteqβgluc proteins were purified by immobilized metal affinity or anion exchange chromatography and used in β-glucosidase activity assays measuring hydrolysis of ρ-nitrophenyl-β-D-glucopyranoside (pNPG). Activity parameters were determined by testing relative β-glucosidase activity after incubation under different temperature and pH conditions. Candidate active residues in Bteqβgluc were identified using molecular operating environment (MOE) software. Results The cloned Bteqβgluc gene belongs to glycoside hydrolase (GH) family 4 and encoded a 54.35 kDa protein. Specific activity of the recombinant β-glucosidase was higher when expressed in P. pastoris (1,462.25 U/mg) than in E. coli (1,445.09 U/mg) hosts using same amount of enzyme. Optimum activity was detected at pH 5 and 50 °C. The activation energy (Ea) was 44.18 and 45.29 kJ/mol for Bteqβgluc produced by P. pastoris and E. coli, respectively. Results from other kinetic parameter determinations, including pKa for the ionizable groups in the active site, Gibbs free energy of activation (ΔG‡), entropy of activation (ΔS‡), Michaelis constant (Km) and maximum reaction velocity (Vmax) for pNPG hydrolysis support unique kinetics and functional characteristics that may be of interest for industrial applications. Molecular docking analysis identified Glu, Asn, Phe, Tyr, Thr and Gln residues as important in protein-ligand catalytic interactions.

scholarly journals Functional identification of ygiP as a positive regulator of the ttdA-ttdB-ygjE operon

Microbiology ◽  
2006 ◽  
Vol 152 (7) ◽  
pp. 2129-2135 ◽  
Author(s):  
Taku Oshima ◽  
Francis Biville
Keyword(s):  
Functional Characterization ◽  
Anaerobic Growth ◽  
Functional Identification ◽  
New Members ◽  
E Coli ◽  
Inner Membrane Protein ◽  
Tartrate Dehydratase

Functional characterization of unknown genes is currently a major task in biology. The search for gene function involves a combination of various in silico, in vitro and in vivo approaches. Available knowledge from the study of more than 21 LysR-type regulators in Escherichia coli has facilitated the classification of new members of the family. From sequence similarities and its location on the E. coli chromosome, it is suggested that ygiP encodes a lysR regulator controlling the expression of a neighbouring operon; this operon encodes the two subunits of tartrate dehydratase (TtdA, TtdB) and YgiE, an integral inner-membrane protein possibly involved in tartrate uptake. Expression of tartrate dehydratase, which converts tartrate to oxaloacetate, is required for anaerobic growth on glycerol as carbon source in the presence of tartrate. Here, it has been demonstrated that disruption of ygiP, ttdA or ygjE abolishes tartrate-dependent anaerobic growth on glycerol. It has also been shown that tartrate-dependent induction of the ttdA-ttdB-ygjE operon requires a functional YgiP.

scholarly journals Conjuring up a ghost: structural and functional characterization of FhuF, a ferric siderophore reductase from E. coli

2021 ◽  
Author(s):  
I. B. Trindade ◽  
G. Hernandez ◽  
E. Lebègue ◽  
F. Barrière ◽  
T. Cordeiro ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Paramagnetic Nmr ◽  
E Coli ◽  
The Past ◽  
Internal Cavities ◽  
Structural Differences ◽  
Bond Strengths ◽  
Redox Bohr Effect ◽  
Micromolar Range

AbstractIron is a fundamental element for virtually all forms of life. Despite its abundance, its bioavailability is limited, and thus, microbes developed siderophores, small molecules, which are synthesized inside the cell and then released outside for iron scavenging. Once inside the cell, iron removal does not occur spontaneously, instead this process is mediated by siderophore-interacting proteins (SIP) and/or by ferric-siderophore reductases (FSR). In the past two decades, representatives of the SIP subfamily have been structurally and biochemically characterized; however, the same was not achieved for the FSR subfamily. Here, we initiate the structural and functional characterization of FhuF, the first and only FSR ever isolated. FhuF is a globular monomeric protein mainly composed by α-helices sheltering internal cavities in a fold resembling the “palm” domain found in siderophore biosynthetic enzymes. Paramagnetic NMR spectroscopy revealed that the core of the cluster has electronic properties in line with those of previously characterized 2Fe–2S ferredoxins and differences appear to be confined to the coordination of Fe(III) in the reduced protein. In particular, the two cysteines coordinating this iron appear to have substantially different bond strengths. In similarity with the proteins from the SIP subfamily, FhuF binds both the iron-loaded and the apo forms of ferrichrome in the micromolar range and cyclic voltammetry reveals the presence of redox-Bohr effect, which broadens the range of ferric-siderophore substrates that can be thermodynamically accessible for reduction. This study suggests that despite the structural differences between FSR and SIP proteins, mechanistic similarities exist between the two classes of proteins. Graphic abstract

Sa1806 Molecular and Functional Characterization of Fimh in Crohn's Disease Associated E. Coli

2013 ◽  
Vol 144 (5) ◽  
pp. S-310
Author(s):  
Brendan Chandler ◽  
Belgin Dogan ◽  
Ellen J. Scherl ◽  
Kenneth W. Simpson
Keyword(s):  
Crohn’S Disease ◽  
Crohn's Disease ◽  
Functional Characterization ◽  
E Coli

P. vortex-mediated strategies for polysaccharides decomposition

TECHNOLOGY ◽  
2015 ◽  
Vol 03 (02n03) ◽  
pp. 80-83
Author(s):  
Mark Polikovsky ◽  
Eshel Ben-Jacob ◽  
Alin Finkelshtein
Keyword(s):  
Degradation Products ◽  
Cellulose Degradation ◽  
Cellulose Hydrolysis ◽  
Industrial Applications ◽  
Biofuel Production ◽  
E Coli ◽  
Novel Approach ◽  
Textile Manufacture ◽  
Hydrolysis Of Cellulose ◽  
The Relationship

Cellulose hydrolysis has many industrial applications such as biofuel production, food, paper and textile manufacture. Here, we present a novel approach to cellulose hydrolysis using a consortium of motile bacteria, Paenibacillus vortex, that can swarm on solid medium carrying a non-motile recombinant E. coli cargo strain expressing the β-glucosidase and cellulase genes that facilitate the hydrolysis of cellulose. These two species cooperate; the relationship is mutually beneficial: the E. coli is dispersed over long distances, while the P. vortex bacteria gain from the supply of cellulose degradation products. This enables the use of such consortia in this area of biotechnology.

scholarly journals Molecular and Functional Characterization of Protein 4.1B, a Novel Member of the Protein 4.1 Family with High Level, Focal Expression in Brain

2000 ◽  
Vol 275 (5) ◽  
pp. 3247-3255 ◽  
Author(s):  
Marilyn Parra ◽  
Philippe Gascard ◽  
Loren D. Walensky ◽  
J. Aura Gimm ◽  
Seth Blackshaw ◽  
...  
Keyword(s):  
Functional Characterization ◽  
Protein 4.1 ◽  
High Level ◽  
Focal Expression ◽  
Protein 4.1B

scholarly journals Expression, secretion and functional characterization of three laccases in E. coli

2022 ◽  
Vol 7 (1) ◽  
pp. 474-480
Author(s):  
Yating Mo ◽  
Hou Ip Lao ◽  
Sau Wa Au ◽  
Ieng Chon Li ◽  
Jeremy Hu ◽  
...  
Keyword(s):  
Functional Characterization ◽  
E Coli

Cloning and functional characterization of a superfamily of microbial inwardly rectifying potassium channels

2006 ◽  
Vol 26 (1) ◽  
pp. 1-7 ◽  
Author(s):  
Si Sun ◽  
Jo Han Gan ◽  
Jennifer J. Paynter ◽  
Stephen J. Tucker
Keyword(s):  
Functional Properties ◽  
Functional Characterization ◽  
Unicellular Eukaryote ◽  
Channel Blockers ◽  
E Coli ◽  
Inwardly Rectifying Potassium Channels ◽  
Genes Encoding ◽  
Functionally Diverse ◽  
Inwardly Rectifying

Our understanding of the mammalian inwardly rectifying family of K+ channels (Kir family) has recently been advanced by X-ray crystal structures of two homologous prokaryotic orthologs (KirBac1.1 and KirBac3.1). However, the functional properties of these KirBac channels are still poorly understood. To address this problem, we cloned and characterized genes encoding KirBac orthologs from a wide variety of different prokaryotes and a simple unicellular eukaryote. The functional properties of these KirBacs were then examined by growth complementation in a K+ uptake-deficient strain of Escherichia coli (TK2420). Whereas some KirBac genes exhibited robust growth complementation, others either did not complement or showed temperature-dependent complementation including KirBac1.1 and KirBac3.1. In some cases, KirBac expression was also toxic to the growth of E. coli. The KirBac family exhibited a range of sensitivity to the K+ channel blockers Ba2+ and Cs+ as well as differences in their ability to grow on very low-K+ media, thus demonstrating major differences in their permeation properties. These results reveal the existence of a functionally diverse superfamily of microbial KirBac genes and present an excellent resource for the structural and functional analysis of this class of K+ channels. Furthermore, the complementation assay used in this study provides a simple and robust method for the functional characterization of a range of prokaryotic K+ channels that are difficult to study by traditional methods.

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