Abstract:Abstract. Psychrophilic organisms have successfully provides enhanced abilities to undergo conformational colonized polar and alpine regions and are able to grow changes during catalysis. Thermal instability of coldefficiently at sub-zero temperatures. At the enzymatic adapted enzymes is therefore regarded as a consequence level, such organisms have to cope with the reduction of of their conformational flexibility. A survey of the psychrophilic enzymes studied so far reveals only minor chemical reaction rates … Show more
“…Life at low temperatures requires that psychrophilic organisms maintain sufficiently high metabolic fluxes despite thermal inhibition of enzyme reaction rates. In the case of intracellular enzymes, adaptation to cold can be achieved either by increasing k cat, or reducing K m , or both (39). This assumption is confirmed in the case of the psychrophilic PGK which adjusts both k cat and K m parameters, leading to a 2-fold improved catalytic efficiency with respect to the mesophilic PGK at 25°C.…”
Section: Resultsmentioning
confidence: 73%
“…3) from psychrophilic to thermophilic PGK strongly suggest their involvement in temperature adaptation by modulation of the external shell compactness. In addition, it is known that the extremities of a protein are preferential sites for denaturation (20,39). In T. maritima and B. stearothermophilus PGK the N and C termini are linked by two salt bridges (D2-K397 and K3-E387, T. maritima PGK numbering) whereas only one connects the extremities of the mesophilic enzymes (K5-E413 in yeast).…”
The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25°C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heatlabile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.
“…Life at low temperatures requires that psychrophilic organisms maintain sufficiently high metabolic fluxes despite thermal inhibition of enzyme reaction rates. In the case of intracellular enzymes, adaptation to cold can be achieved either by increasing k cat, or reducing K m , or both (39). This assumption is confirmed in the case of the psychrophilic PGK which adjusts both k cat and K m parameters, leading to a 2-fold improved catalytic efficiency with respect to the mesophilic PGK at 25°C.…”
Section: Resultsmentioning
confidence: 73%
“…3) from psychrophilic to thermophilic PGK strongly suggest their involvement in temperature adaptation by modulation of the external shell compactness. In addition, it is known that the extremities of a protein are preferential sites for denaturation (20,39). In T. maritima and B. stearothermophilus PGK the N and C termini are linked by two salt bridges (D2-K397 and K3-E387, T. maritima PGK numbering) whereas only one connects the extremities of the mesophilic enzymes (K5-E413 in yeast).…”
The gene encoding the phosphoglycerate kinase (PGK) from the Antarctic Pseudomonas sp. TACII18 has been cloned and found to be inserted between the genes encoding for glyceraldhyde-3-phosphate dehydrogenase and fructose aldolase. The His-tagged and the native recombinant PGK from the psychrophilic Pseudomonas were expressed in Escherichia coli. The wild-type and the native recombinant enzymes displayed identical properties, such as a decreased thermostability and a 2-fold higher catalytic efficiency at 25°C when compared with the mesophilic PGK from yeast. These properties, which reflect typical features of cold-adapted enzymes, were strongly altered in the His-tagged recombinant PGK. The structural model of the psychrophilic PGK indicated that a key determinant of its low stability is the reduced number of salt bridges, surface charges, and aromatic interactions when compared with mesophilic and thermophilic PGK. Differential scanning calorimetry of the psychrophilic PGK revealed unusual variations in its conformational stability for the free and substrate-bound forms. In the free form, a heatlabile and a thermostable domain unfold independently. It is proposed that the heat-labile domain acts as a destabilizing domain, providing the required flexibility around the active site for catalysis at low temperatures.
“…It has been shown that when the medium temperature decreases from 37 to 0°C, the enzymatic reaction rates can be reduced by 30-to 80-fold (Lonhienne, Gerday, & Feller, 2000). In order to cope with the reduction in chemical reaction rates and further to maintain sufficient metabolic fluxes, enzymes produced by psychrophiles generally feature a higher catalytic efficiency (K cat /K m ) at low temperatures than their warm-active counterparts (Feller & Gerday, 1997). The main strategy adopted by the psychrophilic enzymes to adapt to cold environments is believed to be their high intrinsic structural flexibility (Feller & Gerday, 1997;Lonhienne et al, 2000;Zecchinon et al, 2001).…”
Section: Introductionmentioning
confidence: 99%
“…In order to cope with the reduction in chemical reaction rates and further to maintain sufficient metabolic fluxes, enzymes produced by psychrophiles generally feature a higher catalytic efficiency (K cat /K m ) at low temperatures than their warm-active counterparts (Feller & Gerday, 1997). The main strategy adopted by the psychrophilic enzymes to adapt to cold environments is believed to be their high intrinsic structural flexibility (Feller & Gerday, 1997;Lonhienne et al, 2000;Zecchinon et al, 2001). An increase in the structural flexibility can reduce the activation free energy which has to be overcome to reach the reaction intermediates, thus leading to a more efficient substrate turnover (Georlette et al, 2004).…”
“…Psychrophilic bacteria have been compared with mesophilic bacteria to provide clues for cold-adaptation. 1 The potential importance of molecular chaperones in efficient folding processes has been addressed for subzero temperatures.…”
Psychrophilic bacteria have acquired cold-resistance in order to protect themselves against freezing temperatures, which would otherwise be lethal. DnaK/DnaJ/GrpE systems are molecular chaperones which facilitate proper folding of newly synthesized proteins. Efficient folding processes are of great importance especially in a cold environment, such as the Arctic. In order to understand the protection mechanisms of psychrophilic bacteria against cold temperatures, we have explored a genome of KOPRI22215, tentatively identified as Psychromonas arctica, whose genome sequence has not yet been discovered. With an aim of searching for a coding gene of DnaK from KOPRI22215, we have applied a series of polymerase chain reactions (PCR) with homologous primers designed from other Psychromonas species and LA PCR in vitro cloning. 1917 bp complete coding sequence of dnaK from KOPRI22215 was identified including upstream promoter sites. Recombinant plasmids to overexpress PaDnaK along with EcDnaK (DnaK of E. coli) were then constructed in pAED4 vector and the pET-based system to induce PaDnaK expression by IPTG. Characterization assays of expressed PaDnaK were carried out by measuring survival rates upon 4 day incubation at 4 o C: a refolding assay as molecular chaperone, and ATPase assay for functional activity. Taking account of all the data together, we conclude that PaDnaK was identified, successfully expressed, and found to be more efficient in providing cold-resistance for bacterial cells.
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