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Interaction of bacterial glutathione transferase with hemin.
No full textGlutathione transferases (GSTs, EC 2.5.1.18) are pivotal enzymes for detoxification processes in eukaryotes and prokaryotes (1, 2). GSTs catalyse glutathione conjugation to a broad spectrum of compounds. In addition, functions not related to detoxification have been discovered for GSTs including steroid biosynthesis, signal transduction and intracellular transport (1).
Hemin has been shown to be hazardous to the cell membrane because it inserts into the lipid bilayer and binds to membrane proteins. These interactions may cause membrane changes, which may lead to cellular lysis.
The ability of several eukaryotic GSTs from pluricellular and unicellular organisms to interact with hemin has been widely investigated (3, 4), and this compound was shown to bind the enzyme with high affinity (3, 4).
To verify if this interaction is a prerogative of eukaryotic GSTs or it has a general significance, we studied the binding of hemin to Proteus mirabilis GST (PmGST), a representative bacterial GST belonging to the Beta class. The interaction was studied by means of inhibition assays and fluorescence spectroscopy. Results obtained indicate that PmGST binds hemin efficiently, with a considerable quenching of the intrinsic fluorescence of PmGST. Furthermore, hemin was found to be a strong inhibitor of GST conjugation activity
Glutatione transferasi nel micete opportunista Blastoschizomyces capitatus.
No full textBlastoschizomyces capitatus è un patogeno fungino emergente responsabile di infezioni sistemiche, spesso letali, in pazienti immunocompromessi. B. capitatus è un microrganismo ambientale e nell’uomo è presente come commensale nella flora del tratto intestinale e respiratorio e sulla cute.
Una caratteristica peculiare dei miceti patogeni è il dimorfismo. Molti dei miceti di importanza medica esibiscono due diverse morfologie: lievitiforme unicellulare e miceliale pluricellulare. Nei campioni patologici, B. capitatus è stato isolato in entrambe le forme: piccole cellule lievitiformi e micelio con ife settate ramificate.
Le glutatione transferasi (GSTs), una famiglia di enzimi multifunzionali ampiamente distribuita in natura, sono coinvolte nella detossificazione cellulare sia negli eucarioti che nei procarioti. Al contrario di altre specie eucariotiche, le GSTs nei miceti sono state poco studiate e poco si conosce delle loro proprietà strutturali e funzionali.
B. capitatus, isolato da un campione di sangue nella forma lievitiforme, è stato coltivato in vitro nelle due morfologie e per ognuna si è proceduto all’estrazione della frazione citosolica per la purificazione di eventuali forme di GSTs.
Per ogni morfologia è stata purificata una GST mediante due passaggi cromatografici.
Il confronto tra i risultati ottenuti per le due isoforme non ha mostrato significative differenze nella espressione e nell’attività enzimatica indicando che questa GST non è influenzata dalle condizioni ambientali di crescita
Glutathione transferases in bacteria.
No full textBacterial glutathione transferases (GSTs) are part of a superfamily of enzymes that play a key role in cellular detoxification. GSTs are widely distributed in prokaryotes and are grouped into several classes. Bacterial GSTs are implicated in a variety of distinct processes such as the biodegradation of xenobiotics, protection against chemical and oxidative stresses and antimicrobial drug resistance. In addition to their role in detoxification, bacterial GSTs are also involved in a variety of distinct metabolic processes such as the biotransformation of dichloromethane, the degradation of lignin and atrazine, and the reductive dechlorination of pentachlorophenol. This review article summarizes the current status of knowledge regarding the functional and structural properties of bacterial GSTs
Contribution of two conserved trypthophan residues to the catalytic and structural properties of Proteus mirabilis glutathione S-transferase B1-1
No full textPmGSTB1-1 (Proteus mirabilis glutathione S-transferase B1-1) has two tryptophan residues at positions 97 and 164 in each monomer. Structural data for this bacterial enzyme indicated that Trp97 is positioned in the helix α4, whereas Trp164 is located at the bottom of the helix α6 in the xenobiotic-binding site. To elucidate the role of the two tryptophan residues they were replaced by site-directed mutagenesis. Trp97 and Trp164 were mutated to either phenylalanine or alanine. A double mutant was also constructed. The effects of the replacement on the activity, structural properties and antibiotic-binding capacity of the enzymes were examined. On the basis of the results obtained, Trp97 does not seem to be involved in the enzyme active site and structural stabilization. In contrast, different results were achieved for Trp164 mutants. Conservative substitution of the Trp164 with phenylalanine enhanced enzyme activity 10-fold, whereas replacement with alanine enhanced enzyme activity 17-fold. Moreover, the catalytic efficiency for both GSH and 1-chloro-2,4-dinitrobenzene substrates improved. In particular, the catalytic efficiency for 1-chloro-2,4-dinitrobenzene improved for both W164F (Trp164 → Phe) and W164A by factors of 7- and 22-fold respectively. These results are supported by molecular graphic analysis. In fact, W164A presented a more extensive substrate-binding pocket that could allow the substrates to be better accommodated. Furthermore, both Trp164 mutants were significantly more thermolabile than wild-type, suggesting that the substitution of this residue affects the overall stability of the enzyme. Taken together, these results indicate that Trp164 is an important residue of PmGSTB1-1 in the catalytic process as well as for protein stability
A Narrative Review of the Role of S-Glutathionylation in Bacteria
Full text linkProtein glutathionylation is defined as a reversible, ubiquitous post-translational modification, resulting in the formation of mixed disulfides between glutathione and proteins’ cysteine residues. Glutathionylation has been implicated in several cellular mechanisms ranging from protection from oxidative stress to the control of cellular homeostasis and the cell cycle. A significant body of research has examined the multifaceted effects of this post-translational modification under physiological conditions in eukaryotes, with a particular focus on its impact on the development of various diseases in humans. In contrast, the role of glutathionylation in prokaryotic organisms remains to be extensively investigated. However, there has been a recent increase in the number of studies investigating this issue, providing details about the role of glutathione and other related thiols as post-translational modifiers of selected bacterial proteins. It can be concluded that in addition to the classical role of such thiols in protecting against cysteine oxidation and consequent protein inactivation, many more specialized roles of glutathionylation in bacterial pathogenicity, virulence, interspecies competition and survival, and control of gene expression are emerging, and new ones may emerge in the future. In this short review, we aim to summarize the current state-of-the-art in this field of research
Glutathione transferases: substrates, inihibitors and pro-drugs in cancer and neurodegenerative diseases
Full text linkAbstractGlutathione transferase classical GSH conjugation activity plays a critical role in cellular detoxification against xenobiotics and noxious compounds as well as against oxidative stress. However, this feature is also exploited by cancer cells to acquire drug resistance and improve their survival. As a result, various members of the family were found overexpressed in a number of different cancers. Moreover several GST polymorphisms, ranging from null phenotypes to point mutations, were detected in members of the family and found to correlate with the onset of neuro-degenerative diseases. In the last decades, a great deal of research aimed at clarifying the role played by GSTs in drug resistance, at developing inhibitors to counteract this activity but also at exploiting GSTs for prodrugs specific activation in cancer cells. Here we summarize some of the most important achievements reached in this lively area of research.</jats:p
Distribution of glutathione transferases in Gram-positive bacteria and Archaea
No full textGlutathione transferases (GSTs) have been widely studied in Gram-negative bacteria and the structure and function of several representatives have been elucidated. Conversely, limited information is available about the occurrence, classification and functional features of GSTs both in Gram-positive bacteria and in Archaea.
An analysis of 305 fully-sequenced Gram-positive genomes highlights the presence of 49 putative GST genes in the genera of both Firmicutes and Actinobacteria phyla. We also performed an analysis on 81 complete genomes of the Archaea domain. Eleven hits were found in the Halobacteriaceae family of the Euryarchaeota phylum and only one in the Crenarchaeota phylum.
A comparison of the identified sequences with well-characterized GSTs belonging to both Gram- negative and eukaryotic GSTs sheds light on their putative function and the evolutionary relationships within the large GST superfamily.
This analysis suggests that the identified sequences mainly cluster in the new Xi class, while Beta class GSTs, widely distributed in Gram-negative bacteria, are under-represented in Gram-positive bacteria and absent in Archaea
Glutamic acid 65 is an essential residue for catalysis in Proteus mirabilis glutathione transferase B1-1
No full textThe functional role of three conserved amino acid residues in Proteus mirabilis glutathione S-transferase B1-1 (PmGST B1-1) has been investigated by site-directed mutagenesis. Crystallographic analyses indicated that Glu65, Ser103 and Glu104 are in hydrogen-bonding distance of the N-terminal amino group of the γ-glutamyl moiety of the co-substrate, GSH. Glu65 was mutated to either aspartic acid or leucine, and Ser103 and Glu104 were both mutated to alanine. Glu65 mutants (Glu65Asp and Glu65Leu) lost all enzyme activity, and a drastic decrease in catalytic efficiency was observed for Ser103Ala and Glu104 Ala mutants toward both 1-chloro-2,4-dinitrobenzene and GSH. On the other hand, all mutants displayed similar intrinsic fluorescence, CD spectra and thermal stability, indicating that the mutations did not affect the structural integrity of the enzyme. Taken together, these results indicate that Ser103 and Glu104 are significantly involved in the interaction with GSH at the active site of PmGST B1-1, whereas Glu65 is crucial for catalysis
Evolutionarily conserved structural motifs in bacterial GST (glutathione S-transferase) are involved in protein folding and stability.
No full textThe bacterium Proteus mirabilis expresses a cytosolic class beta glutathione S-transferase (PmGST B1-1) that is part of a family of multifunctional detoxication enzymes. Like other cytosolic GSTs, PmGST B1-1 possesses two local structural motifs, an N-capping box and a hydrophobic staple motif, both of which are located between amino acids 151 and 156. The N-capping box consists of a reciprocal hydrogen bonding interaction of Thr152 with Asp155, whereas the hydrophobic staple motif consists of a hydrophobic interaction between Phe151 and Ala156. By contrast with other GSTs, PmGST B1-1 displays distinct hydrogen bond interactions in the N-capping box. In mammalian GSTs these structural elements are critical for protein folding and stability. To investigate the role played by these two motifs in a distantly related organism on the evolutionary scale, site-directed mutagenesis was used to generate several mutants of both motifs in PmGST B1-1. All mutants were efficiently overexpressed and purified, but they were quite unstable, although at different levels, indicating that protein folding was significantly destabilized. The analysis of the T152A and D155G variants indicated that the N-capping box motif plays an important role in the stability and correct folding of the enzyme. The analysis of F151A and A156G mutants revealed that the hydrophobic staple motif influences the structural maintenance of the protein and is implicated in the folding process of PmGST B1-1. Finally, the replacement of Thr152 and Asp155, as well as Phe151 and Ala156 residues influences the catalytic efficiency of the enzyme
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