The dietary triterpenoid 18α-Glycyrrhetinic acid protects from MMC-induced genotoxicity through the ERK/Nrf2 pathway.

18α-Glycyrrhetinic acid (18α-GA) is a bioactive triterpenoid that has been shown to activate the nuclear factor (erythroid-derived-2)-like 2 (Nrf2), the main transcription factor that orchestrates the cellular antioxidant response, in both cellular and organismal context. Although various beneficial properties of 18α-GA have been revealed, including its anti-oxidation and anti-aging activity, its possible protective effect against DNA damage has never been addressed. In this study, we investigated the potential beneficial properties of 18α-GA against DNA damage induced by mitomycin C (MMC) treatment. Using human primary fibroblasts exposed to MMC following pre-treatment with 18α-GA, we reveal an Nrf2-mediated protective effect against MMC-induced cell death that depends on extracellular signal-regulated kinase (ERK) signaling. In total, our results reveal an additional beneficial effect of the Nrf2 activator 18α-GA, suggesting that this important phytochemical compound is a potential candidate in preventive and/or therapeutic schemes against conditions (such as aging) or diseases that are characterized by both oxidative stress and DNA damage

Purification and characterization of a new hyperthermostable, allosamidin-insensitive and denaturation-resistant chitinase from the hyperthermophilic archaeon Thermococcus chitonophagus

A new chitinase (1,4-β-D-N-acetyl-glucosaminidase, EC 3.2.1.14) was detected and purified to homogeneity in its native form from the chitinolytic enzyme system of the extremely thermophilic archaeon Thermococcus chitonophagus. This is the first non-recombinant chitinase purified and characterized from archaea and also constitutes the first case of a membrane-associated chitinase isolated from archaea. The enzyme is a monomer with an apparent molecular weight of 70 kDa [therefore named chitinase 70 (Chi70)] and pI of 5.9; it is hydrophobic and appears to be associated with the outer side of the cell membrane. Chi70 is optimally active at 70°C and pH 7.0 and exhibits remarkable thermostability, maintaining 50% activity even after 1 h at 120°C, and therefore the enzyme is the most thermostable chitinase so far isolated. The enzyme was not inhibited by allosamidin, the natural inhibitor of chitinolytic activity, and was also resistant to denaturation by urea and SDS. On the other hand, guanidine hydrochloride significantly reduced enzymatic activity, indicating that, apart from the hydrophobic interactions, ion pairs located on the surface of the protein could be playing an important role in maintaining the protein's fold and enzyme activity. Chi70 showed broad substrate specificity for several chitinous substrates and derivatives. The lowest K m and highest Kcat values were found for pNP(NAG) 2 as substrate and were determined to be 0.14 mM and 23 min -1, respectively. The hydrolysis pattern was similar for oligomers and polymers, with N,N′-diacetylchitobiose [(NAG)2] being the final, major hydrolysis product. Chi70 was classified as an endochitinase due to its ability to release chitobiose from colloidal chitin. Additionally, the enzyme presented considerable cellulolytic activity. Analysis of the NH 2-terminal amino acid sequence showed no detectable homology with other known sequences, suggesting that Chi70 is a new protein. © Springer-Verlag 2002

Insights into the role of the (α + β) insertion in the TIM-barrel catalytic domain, regarding the stability and the enzymatic activity of Chitinase A from Serratia marcescens

Chitinase A (ChiA) from Serratia marcescens is a mesophilic enzyme with high catalytic activity and high stability. The crystal structure of ChiA has revealed a TIM-barrel fold of the catalytic domain, an (α + β) insertion between the B7 β-strand and A7 α-helix of the TIM-barrel, an FnIII domain at the N-terminus of the molecule and a hinge region that connects the latter to the catalytic domain. In this study, the role of the (α + β) domain on the stability, catalytic activity and specificity of the enzyme was investigated by deleting this domain and studying the enzymatic and structural properties of the resulting truncated enzyme. The obtained data clearly show that by removing the (α + β) domain, the thermal stability of the enzyme is substantially reduced, with an apparent Tm of 42.0 ± 1.0 °C, compared to the apparent Tm of 58.1 ± 1.0 °C of ChiA at pH 9.0. The specific activity of ChiAΔ(α + β) was substantially decreased, the pH optimum was shifted from 6.5 to 5.0 and the substrate and product specificities were altered. © 2008 Elsevier B.V. All rights reserved

Extreme environments as a resource for microorganisms and novel biocatalysts

The steady increase in the number of newly isolated extremophilic microorganisms and the discovery of their enzymes by academic and industrial institutions underlines the enormous potential of extremophiles for application in future biotechnological processes. Enzymes from extremophilic microorganisms offer versatile tools for sustainable developments in a variety of industrial application as they show important environmental benefits due to their biodegradability, specific stability under extreme conditions, improved use of raw materials and decreased amount of waste products. Although major advances have been made in the last decade, our knowledge of the physiology, metabolism, enzymology and genetics of this fascinating group of extremophilic microorganisms and their related enzymes is still limited. In-depth information on the molecular properties of the enzymes and their genes, however, has to be obtained to analyze the structure and function of proteins that are catalytically active around the boiling and freezing points of water and extremes of pH. New techniques, such as genomics, metanogenomics, DNA evolution and gene shuffling, will lead to the production of enzymes that are highly specific for countless industrial applications. Due to the unusual properties of enzymes from extremophiles, they are expected to optimize already existing processes or even develop new sustainable technologies. © Springer-Verlag 2005

Structure of a complete four-domain chitinase from Moritella marina, a marine psychrophilic bacterium

X-ray crystallography reveals chitinase from the psychrophilic bacterium Moritella marina to be an elongated molecule which in addition to the catalytic β/α-barrel domain contains two Ig-like domains and a chitin-binding domain, all linked in a chain. A ligand-binding study using NAG oligomers showed the enzyme to be active in the crystal lattice and resulted in complexes of the protein with oxazolinium ion (the reaction intermediate) and with NAG2, a reaction product. The characteristic motif DXDXE, containing three acidic amino-acid residues, which is a signature of type 18 chitinases, is conserved in the enzyme. Further analysis of the unliganded enzyme with the two protein-ligand complexes and a comparison with other known chitinases elucidated the roles of other conserved residues near the active site. Several features have been identified that are probably important for the reaction mechanism, substrate binding and the efficiency of the enzyme at low temperatures. The chitin-binding domain and the tryptophan patch on the catalytic domain provide general affinity for chitin, in addition to the affinity of the binding site; the two Ig-like domains give the protein a long reach over the chitin surface, and the flexible region between the chitin-binding domain and the adjacent Ig-like domain suggests an ability of the enzyme to probe the surface of the substrate, while the open shallow substrate-binding groove allows easy access to the active site. © 2013 International Union of Crystallography Printed in Singapore - all rights reserved

A straightforward access to TMG-chitooligomycins and their evaluation as β-N-acetylhexosaminidase inhibitors

International audienc

Crystallographic analysis of a thermoactive nitrilase

The nitrilase superfamily is a large and diverse superfamily of enzymes that catalyse the cleavage of various types of carbon-nitrogen bonds using a Cys-Glu-Lys catalytic triad. Thermoactive nitrilase from Pyrococcus abyssi (PaNit) hydrolyses small aliphatic nitriles like fumaro- and malononitryl. Yet, the biological role of this enzyme is unknown. We have analysed several crystal structures of PaNit: without ligands, with an acetate ion bound in the active site and with a bromide ion in the active site. In addition, docking calculations have been performed for fumaro- and malononitriles. The structures provide a proof for specific binding of the carboxylate ion and a general affinity for negatively changed ligands. The role of residues in the active site is considered and an enzymatic reaction mechanism is proposed in which Cys146 acts as the nucleophile, Glu42 as the general base, Lys113/Glu42 as the general acid, WatA as the hydrolytic water and Nζ_Lys113 and N_Phe147 form the oxyanion hole. © 2010 Elsevier Inc

BRCA1-BRCT mutations alter the subcellular localization of BRCA1 in vitro

Background/Aim: Numerous missense mutations have been determined in the BRCT domain of the BRCA1 gene, affecting localization and interaction of BRCA1 with other proteins. Materials and Methods: We examined whether the M1775K and V1809F mutations in the BRCT domain affect BRCA1 cellular localization. Cells were transfected with pEGFP-C3-BRCA1 and detected by fluorescence microscopy. Results: Following induction of DNA damage, cytoplasmic mislocalization was observed for both M1775K and V1809F mutants compared to EGFP-BRCA1wt and the less common variant M1652I. These results indicate that M1775K and V1809F mutations may change the function of the protein by affecting BRCA1 localization. Conclusion: There is a correlation between subcellular localization of BRCA1 and diminished DNA repair observed in breast cancer cells, which may be explained by structural variations and altered binding properties of phosphopeptides. © 2021 International Institute of Anticancer Research. All rights reserved