Heterologous expression in Escherichia coli of Neurospora crassa neutral trehalase as an active enzyme

Neutral trehalase from Neurospora crassa was expressed in Escherichia coli as a polypeptide of 84 kDa in agreement with the theoretical size calculated from the corresponding cDNA. The recombinant neutral trehalase, purified by affinity chromatography exhibited a specific activity of 80–150 mU/mg protein. Optima of pH and temperature were 7.0 and 30 °C, respectively. The enzyme was absolutely specific for trehalose, and was quite sensitive to incubation at 40 °C. The recombinant enzyme was totally dependent on calcium, and was inhibited by ATP, copper, silver, aluminium and cobalt. K_M was 42 mM, and V_max was 30.6 nmol of glucose/min. The recombinant protein was phosphorylated by cAMP-dependent protein kinase, but not significantly activated. Immunoblotting with polyclonal antiserum prepared against the recombinant protein showed that neutral trehalase protein levels increased during exponential phase of N. crassa growth and dropped at the stationary phase. This is the first report of a neutral trehalase produced in E. coli with similar biochemical properties described for fungi native neutral trehalases, including calcium-dependence.     

Identification of a protein mediating respiratory supercomplex stability

The complexes of the electron transport chain associate into large macromolecular assemblies, which are believed to facilitate efficient electron flow. We have identified a conserved mitochondrial protein, named respiratory supercomplex factor 1 (Rcf1-Yml030w), that is required for the normal assembly of respiratory supercomplexes. We demonstrate that Rcf1 stably and independently associates with both Complex III and Complex IV of the electron transport chain. Deletion of the RCF1 gene caused impaired respiration, probably as a result of destabilization of respiratory supercomplexes. Consistent with the hypothetical function of these respiratory assemblies, loss of RCF1 caused elevated mitochondrial oxidative stress and damage. Finally, we show that knockdown of HIG2A, a mammalian homolog of RCF1, causes impaired supercomplex formation. We suggest that Rcf1 is a member of an evolutionarily conserved protein family that acts to promote respiratory supercomplex assembly and activity.     

Purification and biochemical characterization of an endoxylanase from Aspergillus versicolor

An endoxylanase (β-1,4-xylan xylanohydrolase, EC 3.2.1.8) was purified from the culture filtrate of a strain of Aspergillus versicolor grown on oat wheat. The enzyme was purified to homogeneity by chromatography on DEAE-cellulose and Sephadex G-75. The purified enzyme was a monomer of molecular mass estimated to be 19 kDa by SDS-PAGE and gel filtration. The enzyme was glycoprotein with 71% carbohydrate content and exhibited a pI of 5.4. The purified xylanase was specific for xylan hydrolysis. The enzyme had a K _m of 6.5 mg ml⁻¹ and a V _max of 1440 U (mg protein)⁻¹.     

Molecular Modeling of Mycobacterium Tuberculosis DNA Gyrase and its Molecular Docking Study with Gatifloxacin Inhibitors

Abstract

Mycobacterium tuberculosis (Mt) is a leading cause of infectious disease in the world today. This outlook is aggravated by a growing number of Mt infections in individuals who are immunocompromised as a result of HIV infections. Thus, new and more potent anti-tuberculosis agents are necessary. Therefore, DNA gyrase was selected as a target enzyme to combat Mt. In this work, the first three-dimensional molecular model of the hypothetical structures for the Mycobacterium tuberculosis DNA gyrase (mtDNAg) was elucidated by a homology modeling method. In addition, the orientations and binding affinities of some gatifloxacin analogs with those new structures were investigated. Our findings could be helpful for the design of new more potent gatifloxacin analogs.     

Downregulation of metallothionein 2A reduces migration,invasion and proliferation activities in human squamous cell carcinoma cells

Molecular Biology Reports - The invasive behaviour of squamous cell carcinoma (SCC), a common malignant tumour of the mouth, is a process mediated by cell proliferation, extracellular matrix...     

Inhibition of α-Glycosidase by Lippia dulcis Trevir. (Verbenaceae) Preparations,Quantification of Verbascoside,and Study of Its Molecular Docking

This study aimed to quantify verbascoside (VEB), perform molecular docking studies of VEB with the α-glucosidase (GL) of Bacillus stearothermophilus, and evaluate the inhibition of the enzyme by L. dulcis preparations. The substrate concentration and presence of reduced glutathione were evaluated for their effect on the in vitro inhibition of the GL enzyme. Assays were also performed in the presence and absence of simulated gastric fluid. The antidiabetic fractions 2 and 3 were the most inhibited GL, but their activity were significantly decreased in the presence of gastric fluid. Chromatographic analyses confirmed the predominant presence of VEB in the samples. The samples had VEB concentrations between 49.9 and 243.5 mg/g. Simulation of the molecular docking of VEB were consistent with its GL-inhibitory activity. It can conclude that the crude ethanol extract and fractions show inhibitory activity against the GL enzyme.