ABSTRACT
In pathogens, the thioredoxin system forms part of the defense against oxidative stress and ensures the formation of the proper disulfide bonds to ensure protein function. In Corynebacterium pseudotuberculosis, the role and mechanism of TrxA1 has not been elucidated, but, the significant homology among different Trxs and the conservation of the residues that form their active sites underline the importance of the Trx systems. Proteins involved in redox metabolism and low molecular weight thiols, which might interact with them, become attractive targets to modulate the activity of pathogens. The activity of the protein was investigated using a turbidimetric assay system. The influence of different pH and low molecular weight thiols were tested. Additionally, this assay was used to investigate the inhibitory potential of ligands from different molecular families, such as, polyanions (suramin and heparin) and flavonoids (hesperetin and hesperidin). All four compounds showed inhibition of the protein activity by approximately 80%. The interactions between these compounds and Cp-TrxA1 were investigated using CD spectroscopy, NMR, molecular docking and dynamics. Our results demonstrate that suramin and hesperetin can serve as lead molecules for the development of specific inhibitors for the C. pseudotuberculosis TrxA1.
Subject(s)
Corynebacterium pseudotuberculosis/metabolism , Flavonoids/chemistry , Flavonoids/pharmacology , Polymers/chemistry , Polymers/pharmacology , Thioredoxins/antagonists & inhibitors , Thioredoxins/chemistry , Catalytic Domain , Corynebacterium pseudotuberculosis/genetics , Ligands , Magnetic Resonance Spectroscopy , Molecular Conformation , Molecular Docking Simulation , Molecular Dynamics Simulation , Oxidation-Reduction , Polyelectrolytes , Protein Binding , Recombinant Proteins , Structure-Activity Relationship , Thioredoxins/genetics , Thioredoxins/isolation & purificationABSTRACT
Glutaredoxin A1 from Corynebacterium pseudotuberculosis was shown to be a mycoredoxin protein. In this study, we established a process to overexpress and purify glutaredoxin A1. The aim of this study was the investigation of the Glutaredoxin A1 from C. pseudotuberculosis behavior under different redox environments and the identification of lead molecules, which can be used for specific inhibitor development for this protein family. A quantitative assay was performed measuring the rate of insulin reduction spectrophotometrically at 640nm through turbidity formation from the precipitation of the free insulin. Glutaredoxin A1, at 5µM concentration, accelerated the reduction process of 0.2mM insulin and 1mM DTT. The pH optimum of the reaction was 7.4. In the presence of DTT and ESH the glutaredoxin A1 presents similar activity, and its activity is reduced by 50% in the presence of GSH. Additional function for ESH in the redox metabolism of C. pseudotuberculosis is suggested. A combined STD and Chemical Shift - NMR approach was employed to study the effects of potential inhibitors on the structure of glutaredoxin A1 from Corynebacterium pseudotuberculosis. The inhibitory potential of four ligands (heparin, suramin, hesperetin - Hst, and hesperidin - Hsp) against glutaredoxin A1 is discussed.
Subject(s)
Bacterial Proteins/chemistry , Biophysical Phenomena , Corynebacterium pseudotuberculosis/metabolism , Glutaredoxins/chemistry , Amino Acid Sequence , Circular Dichroism , Glutaredoxins/isolation & purification , Humans , Insulin/metabolism , Ligands , Magnetic Resonance Spectroscopy , Models, Molecular , Oxidation-Reduction , Oxidoreductases/metabolism , Sequence Analysis, Protein , Structural Homology, ProteinABSTRACT
The conformational stability of the Cold shock protein A (CspA) from C. pseudotuberculosis (Cp), a nucleic acid binding protein in function of pH and salt concentration was examined by using differential scanning calorimetry and CD spectroscopy in combination with computational analysis to identify the specify amino acids undergoing change. Our approach identified a sodiumbinding site in CpCspA and at pH 8.0 a significant reduction in the ß-sheet content was observed which resulted in a decrease of the protein thermal stability. The computational analyses identified His30 and His65 as the amino acids with the largest charge shifts at different pHs. His30/His65 are part of the extensive hydrogen bonding network and along with the ion-binding site are essential for the conformational stability of CspA.
Subject(s)
Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Corynebacterium pseudotuberculosis/chemistry , Corynebacterium pseudotuberculosis/metabolism , Hydrogen/chemistry , Hydrogen/metabolism , Hydrogen-Ion Concentration , Models, Molecular , Protein Binding , Protein Stability , Protein Structure, Secondary , Recombinant Proteins/chemistry , Recombinant Proteins/metabolism , Sodium/chemistry , Sodium/metabolism , Static ElectricityABSTRACT
Cianobactérias são microrganismos procariontes que, durante o crescimento celular, são capazes de produzir exopolissacarídeos (EPS). Devido à diversidade bioquímica destes, podem ser excelentes para vários fins biotecnológicos, tendo aplicações em indústrias alimentícias, têxteis, de tintas, cosméticos, de papel, e farmacêuticas, como floculantes, espessantes ou estabilizadores, substituindo os polissacarídeos de macroalgas e plantas. Além disso, as cianobactérias apresentam taxas maiores de crescimento e são mais fáceis de manipular do que plantas e macroalgas. Este estudo teve por objetivo otimizar a produção de EPS no meio BG11, com relação a diferentes concentrações de nitrogênio e glicose do meio de cultivo na produção de EPS e biomassa pela cianobactéria Nostoc sp.
