NADP-dependent isocitrate dehydrogenase from the halophilic archaeon<i>Haloferax volcanii</i>: cloning, sequence determination and overexpression in<i>Escherichia coli</i>

Camacho, Mónica; Rodríguez-Arnedo, Adoración; Bonete, Marı́a José

doi:10.1111/j.1574-6968.2002.tb11125.x

Cited by 19 publications

(6 citation statements)

References 23 publications

(33 reference statements)

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“…These findings contrast with the kinetic properties of ICDHs purified from the halophiles Haloferax volcanii and Halobacterium salinarum , which are salt-activated enzymes reaching a V max comparable to the E . coli enzyme [ 53 , 54 ].…”

Section: Resultsmentioning

confidence: 99%

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum

et al. 2018

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Hypersaline environments pose major challenges to their microbial residents. Microorganisms have to cope with increased osmotic pressure and low water activity and therefore require specific adaptation mechanisms. Although mechanisms have already been thoroughly investigated in the green alga Dunaliella salina and some halophilic yeasts, strategies for osmoadaptation in other protistan groups (especially heterotrophs) are neither as well known nor as deeply investigated as for their prokaryotic counterpart. This is not only due to the recent awareness of the high protistan diversity and ecological relevance in hypersaline systems, but also due to methodological shortcomings. We provide the first experimental study on haloadaptation in heterotrophic microeukaryotes, using the halophilic ciliate Schmidingerothrix salinarum as a model organism. We established three approaches to investigate fundamental adaptation strategies known from prokaryotes. First, proton nuclear magnetic resonance (1H-NMR) spectroscopy was used for the detection, identification, and quantification of intracellular compatible solutes. Second, ion-imaging with cation-specific fluorescent dyes was employed to analyze changes in the relative ion concentrations in intact cells. Third, the effect of salt concentrations on the catalytic performance of S. salinarum malate dehydrogenase (MDH) and isocitrate dehydrogenase (ICDH) was determined. 1H-NMR spectroscopy identified glycine betaine (GB) and ectoine (Ect) as the main compatible solutes in S. salinarum. Moreover, a significant positive correlation of intracellular GB and Ect concentrations and external salinity was observed. The addition of exogenous GB, Ect, and choline (Ch) stimulated the cell growth notably, indicating that S. salinarum accumulates the solutes from the external medium. Addition of external 13C2-Ch resulted in conversion to 13C2-GB, indicating biosynthesis of GB from Ch. An increase of external salinity up to 21% did not result in an increase in cytoplasmic sodium concentration in S. salinarum. This, together with the decrease in the catalytic activities of MDH and ICDH at high salt concentration, demonstrates that S. salinarum employs the salt-out strategy for haloadaptation.

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Section: Resultsmentioning

confidence: 99%

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum

et al. 2018

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“…The use of E. coli as host for recombinant haloarchaeal protein production has been challenging, probably due to the requirement of these proteins of molar concentrations of ions to fold properly. Since in most of the cases the studied proteins are obtained as inclusion bodies (Martínez-Espinosa, 2020), several protocols have been described to solubilize and refold different proteins (Connaris et al, 1999;Camacho et al, 2002;Díaz et al, 2006). This was the case for the expression of HvG6PDH in E. coli, where we used a solubilization and refolding protocol, similar to the one described by Pire (Pire et al, 2001), that enabled us to obtain the purified enzyme in a soluble and active form, with kinetic parameters comparable to those reported by Pickl and Schönheit (2015), who characterized the enzyme obtained from the original organism (H. volcanii).…”

Section: Discussionmentioning

confidence: 99%

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii

et al. 2021

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Halophilic enzymes need high salt concentrations for activity and stability and are considered a promising source for biotechnological applications. The model study for haloadaptation has been proteins from the Halobacteria class of Archaea, where common structural characteristics have been found. However, the effect of salt on enzyme function and conformational dynamics has been much less explored. Here we report the structural and kinetic characteristics of glucose-6-phosphate dehydrogenase from Haloferax volcanii (HvG6PDH) belonging to the short-chain dehydrogenases/reductases (SDR) superfamily. The enzyme was expressed in Escherichia coli and successfully solubilized and refolded from inclusion bodies. The enzyme is active in the presence of several salts, though the maximum activity is achieved in the presence of KCl, mainly by an increment in the kcat value, that correlates with a diminution of its flexibility according to molecular dynamics simulations. The high KM for glucose-6-phosphate and its promiscuous activity for glucose restrict the use of HvG6PDH as an auxiliary enzyme for the determination of halophilic glucokinase activity. Phylogenetic analysis indicates that SDR-G6PDH enzymes are exclusively present in Halobacteria, with HvG6PDH being the only enzyme characterized. Homology modeling and molecular dynamics simulations of HvG6PDH identified a conserved NLTX2H motif involved in glucose-6-phosphate interaction at high salt concentrations, whose residues could be crucial for substrate specificity. Structural differences in its conformational dynamics, potentially related to the haloadaptation strategy, were also determined.

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“…mediterranei have been successfully overexpressed in E. coli [38][39][40][41]. The recombinant proteins are usually obtained as inclusion bodies, which are solubilized in the presence of buffers containing urea and refolded in hypersaline solutions to recover their native structure [42,43]. However, homologous overexpression using a halophilic host avoid these disadvantages.…”

Section: Overexpression Purification and Determination Of The Molecular Mass Of Hfx Mediterranei Lrpmentioning

confidence: 99%

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator

Matarredona

Camacho

García-Bonete

et al. 2021

Genes

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Haloferax mediterranei is an extremely halophilic archaeon, able to live in hypersaline environments with versatile nutritional requirements, whose study represents an excellent basis in the field of biotechnology. The transcriptional machinery in Archaea combines the eukaryotic basal apparatus and the bacterial regulation mechanisms. However, little is known about molecular mechanisms of gene expression regulation compared with Bacteria, particularly in Haloarchaea. The genome of Hfx. mediterranei contains a gene, lrp (HFX_RS01210), which encodes a transcriptional factor belonging to Lrp/AsnC family. It is located downstream of the glutamine synthetase gene (HFX_RS01205), an enzyme involved in ammonium assimilation and amino acid metabolism. To study this transcriptional factor more deeply, the lrp gene has been homologously overexpressed and purified under native conditions by two chromatographic steps, namely nickel affinity and gel filtration chromatography, showing that Lrp behaves asa tetrameric protein of approximately 67 kDa. Its promoter region has been characterized under different growth conditions using bgaH as a reporter gene. The amount of Lrp protein was also analyzed by Western blotting in different nitrogen sources and under various stress conditions. To sum up, regarding its involvement in the nitrogen cycle, it has been shown that its expression profile does not change in response to the nitrogen sources tested. Differences in its expression pattern have been observed under different stress conditions, such as in the presence of hydrogen peroxide or heavy metals. According to these results, the Lrp seems to be involved in a general response against stress factors, acting as a first-line transcriptional regulator.

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NADP-dependent isocitrate dehydrogenase from the halophilic archaeonHaloferax volcanii: cloning, sequence determination and overexpression inEscherichia coli

Cited by 19 publications

References 23 publications

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum

Identification of osmoadaptive strategies in the halophile, heterotrophic ciliate Schmidingerothrix salinarum

Structural and Kinetic Insights Into the Molecular Basis of Salt Tolerance of the Short-Chain Glucose-6-Phosphate Dehydrogenase From Haloferax volcanii

Analysis of Haloferax mediterranei Lrp Transcriptional Regulator

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