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To survive at low temperatures, psychrophiles seem to produce cold-adapted enzymes with a high flexibility around active sites for high catalytic efficiency. To gain insights into the cold-adaptation of psychrophilic enzymes in atomic detail, we determined the crystal structure of 5-enolpyruvylshikimate-3-phosphate synthase ( Cps EPSPS) from Colwellia psychrerythraea , a psychrophilic bacterium. EPSPS is the primary target for the broad-spectrum herbicide, glyphosate, and a promising target for the development of antimicrobial and antiparasitic agents since it is absent in animals. The crystal structure of unliganded, open Cps EPSPS was determined at 2.2 Å resolution in space group P 2 1 with two protomers per asymmetric unit. Superposition of separate domain I and II of Cps EPSPS structure with those of Escherichia coli EPSPS ( Eco EPSPS) structure showed relatively small differences of RMSD values of 0.423 Å and 0.693 Å for domains I and II, respectively, implying the residues in ligand binding and catalysis of cold-adapted Cps EPSPS showed no significant flexibility. This result is conflicting to other cases of cold-adapted proteins. We also observed that hydrogen-bond forming residues in the surface of Eco EPSPS was mutated to non- or lesser hydrogen-bond forming one in Cps EPSPS, which makes the protein surface softer and eventually makes the protein more active at low temperature. In addition, domain rotation angle between open and closed states of Cps EPSPS was smaller than those of any EPSPSs whose structures are known. The restriction of the domain closure, which reduces the entropy cost of ligand binding and catalysis, may be a novel molecular adaptations of cold-adapted enzymes. [ABSTRACT FROM AUTHOR]

Background: Colwellia psychrerythraea 34H is a psychrophilic bacterium able to produce docosahexaenoic acid (DHA). Polyketide synthase pathway is assumed to be responsible for DHA production in marine bacteria. Results: Five pfa genes from strain 34H were confirmed to be responsible for DHA formation by heterogeneous expression in Escherichia coli. The complexity of fatty acid profile of this strain was revealed by GC and GC-MS. Treatment of cells with cerulenin resulted in significantly reduced level of C16 monounsaturated fatty acid (C16:1Δ9t, C16:1Δ7). In contrast, the amount of saturated fatty acids (C10:0, C12:0, C14:0), hydroxyl fatty acids (3-OH C10:0 and 3-OH C12:0), as well as C20:4ω3, C20:5ω3 and C22:6ω3 were increased. RNA sequencing (RNA-Seq) revealed the altered gene expression pattern when C. psychrerythraea cells were treated with cerulenin. Genes involved in polyketide synthase pathway and fatty acid biosynthesis pathway were not obviously affected by cerulenin treatment. In contrast, several genes involved in fatty acid degradation or β-oxidation pathway were dramatically reduced at the transcriptional level. Conclusions: Genes responsible for DHA formation in C. psychrerythraea was first cloned and characterized. We revealed the complexity of fatty acid profile in this DHA-producing strain. Cerulenin could substantially change the fatty acid composition by affecting the fatty acid degradation at transcriptional level. Acyl-CoA dehydrogenase gene family involved in the first step of β-oxidation pathway may be important to the selectivity of degraded fatty acids. In addition, inhibition of FabB protein by cerulenin may lead to the accumulation of malonyl-CoA, which is the substrate for DHA formation. [ABSTRACT FROM AUTHOR]

Glutathione reductases catalyze the reduction of oxidized glutathione (glutathione disulfide, GSSG) using NADPH as the substrate to produce reduced glutathione (GSH), which is an important antioxidant molecule that helps maintain the proper reducing environment of the cell. A recombinant form of glutathione reductase from Colwellia psychrerythraea, a marine psychrophilic bacterium, has been biochemically characterized to determine its molecular and enzymatic properties. C. psychrerythraea glutathione reductase was shown to be a homodimer with a molecular weight of 48.7 kDa using SDS-PAGE, MALDI-TOF mass spectrometry and gel filtration. The C. psychrerythraea glutathione reductase sequence shows significant homology to that of Escherichia coli glutathione reductase (66 % identity), and it possesses the FAD and NADPH binding motifs, as well as absorption spectrum features which are characteristic of flavoenzymes such as glutathione reductase. The psychrophilic C. psychrerythraea glutathione reductase exhibits higher k and k/ K at lower temperatures (4 °C) compared to mesophilic Baker's yeast glutathione reductase. However, C. psychrerythraea glutathione reductase was able to complement an E. coli glutathione reductase deletion strain in oxidative stress growth assays, demonstrating the functionality of C. psychrerythraea glutathione reductase over a broad temperature range, which suggests its potential utility as an antioxidant enzyme in heterologous systems. [ABSTRACT FROM AUTHOR]

In the two cold-adapted monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea ( CmIDH and CpIDH, respectively), the combined substitutions of amino acid residues between the Leu693, Leu724 and Phe735 residues of CmIDH and the corresponding Phe693, Gln724 and Leu735 residues of CpIDH were introduced by site-directed mutagenesis. A double mutant of CmIDH substituted its Leu724 and Phe735 residues by the corresponding ones of CpIDH, CmL724Q/F735L, and the triple mutant of CpIDH, CpF693L/Q724L/L735F, showed the most decrease and increase of activity, respectively, of each wild-type and its all mutated enzymes. In the case of CmIDH, the substitutions of these three amino acid residues resulted in the decrease of catalytic activity and thermostability for activity, but the combined substitutions of amino acid residues did not necessarily exert additive effects on these properties. On the other hand, similar substitutions in CpIDH had quite opposite effects to CmIDH, and the effects of the combined substitutions were additive. All multiple mutants of CmIDH and CpIDH showed lower and higher catalytic efficiency ( k/ K) values than the respective wild-type enzymes. Single and multiple mutations of the substituted amino acid residues in the CmIDH and CpIDH led to the increase and decrease of sensitivity to tryptic digestion, indicating that the stability of protein structure was decreased and increased by the mutations, respectively. [ABSTRACT FROM AUTHOR]

Monomeric isocitrate dehydrogenases from psychrophilic bacteria, Colwellia maris and Colwellia psychrerythraea (CmIDH-II and CpIDH-M, respectively) are cold-adapted enzymes and show a high degree of amino acid sequential identity to each other (77%). However, maximum activity of CpIDH-M at optimum temperature is much less than that of CmIDH-II. In the C-terminal region 3 of these enzymes, which was suggested from previous study to be responsible for their distinct catalytic ability, several sequential differences of amino acid residue are present. Among them, ten amino acid residues were exchanged between them by site-directed mutagenesis and several properties of the mutated enzymes were examined in this study. The mutated enzymes of CmIDH-II substituted its Gln671, Leu724 and Phe735 residues with the corresponding residues of CpIDH-M (termed Q671K, L724Q and F735L, respectively) showed lower specific activity and thermostability for activity than the wild-type enzyme. Furthermore, the decreased specific activity was also observed in L693F. In contrast, the corresponding mutants of CpIDH-M, F693L, Q724L and L735F, showed the increased specific activity and thermostability for activity. The catalytic efficiency (k cat/K m) values of these mutated CmIDH-II and CpIDH-M were lower and higher than those of their wild-type IDHs, respectively. These results suggest that the Gln671, Leu693, Leu724 and Phe735 residues of CmIDH-II are important for exerting its high catalytic ability. [Copyright &y& Elsevier]

The putative lipase CpsLip from the psychrophilic bacterium Colwellia psychrerythraea 34H encodes a 34 538 Da, 308-amino-acid protein. In this study, CpsLip (UniProtKB code Q486T5) was expressed as an N-terminal hexahistidine fusion protein in Escherichia coli and purified by affinity and size-exclusion chromatography. The expression and purification of CpsLip enabled characterization of the lipase enzymatic properties of the protein. The optimal activity temperature and pH of the recombinant protein were 298 K and pH 7, respectively. CpsLip maintained over 80% activity in the low-temperature range (278-288 K), thereby suggesting that CpsLip is a cold-active lipase. Substrate-specificity analysis demonstrated that CpsLip exhibits maximum activity towards the C12 acyl group. In addition, sequence-alignment results revealed that CpsLip has a highly conserved catalytic triad in the active site consisting of residues Ser111, Asp135 and His283. Moreover, purified CpsLip was successfully crystallized using the hanging-drop vapour-diffusion method and a complete diffraction data set was collected to 4.0 Å resolution using synchrotron radiation on the BL-5A beamline of the Photon Factory. [ABSTRACT FROM AUTHOR]

We investigated the protein folding mechanism of the GroEL system of a psychrophilic bacterium, Colwellia psychrerythraea 34H. The amount of mRNA of the groESL operon of C. psychrerythraea was increased about 6-fold after a temperature upshift from 8 to 18 °C for 30 min, suggesting that this temperature causes heat stress in this bacterium. A σ-type promoter was found upstream of the groESL, suggesting that the C. psychrerythraea groESL is regulated by the σ system, like the groESL in E. coli. The maximum ATPase and CTPase activities of CpGroEL were observed at 45 and 35 °C, respectively, which are much higher than the growth temperatures of C. psychrerythraea. We found that the refolding activity of the CpGroEL system in the presence of ATP is lower than that in the presence of CTP. This suggests that ATP is not the optimum energy source of the CpGroEL system. Analyses for the interaction of CpGroEL- CpGroES revealed that CTP could weaken this interaction, resulting in effective refolding function of the CpGroEL system. From these findings, we consider that the CpGroEL system possesses an energy-saving mechanism for avoiding excess consumption of ATP to ensure growth in a low-temperature environment. [ABSTRACT FROM AUTHOR]

Abstract: The relationships between structure, activity, stability and flexibility of a cold-adapted aminopeptidase produced by a psychrophilic marine bacterium have been investigated in comparison with a mesophilic structural and functional human homolog. Differential scanning calorimetry, fluorescence monitoring of thermal- and guanidine hydrochloride-induced unfolding and fluorescence quenching were used to show that the cold-adapted enzyme is characterized by a high activity at low temperatures, a low structural stability versus thermal and chemical denaturants and a greater structural permeability to a quenching agent relative to the mesophilic homolog. These findings support the hypothesis that cold-adapted enzymes maintain their activity at low temperatures as a result of increased global or local structural flexibility, which results in low stability. Analysis of the thermodynamic parameters of irreversible thermal unfolding suggests that entropy-driven factors are responsible for the fast unfolding rate of the cold-adapted aminopeptidase. A reduced number of proline residues, a lower degree of hydrophobic residue burial and a decreased surface accessibility of charged residues may be responsible for this effect. On the other hand, the reduction in enthalpy-driven interactions is the primary determinant of the weak conformational stability. [Copyright &y& Elsevier]

The gene ( icl) encoding cold-adapted isocitrate lyase (ICL) of a psychrophilic bacterium, Colwellia psychrerythraea, was cloned and sequenced. Open reading frame of the gene was 1,587 bp in length and corresponded to a polypeptide composed of 528 amino acids. The deduced amino acid sequence showed high homology with that of cold-adapted ICL from other psychrophilic bacterium, C. maris (88% identity), but the sequential homology with that of the Escherichia coli ICL was low (28% identity). Primer extension analysis revealed that transcriptional start site for the C. psychrerythraea icl gene was guanine, located at 87 bases upstream of translational initiation codon. The expression of this gene in the cells of an E. coli mutant defective in ICL was induced by not only low temperature but also acetate. However, cis-acting elements for cold-inducible expression known in the several other bacterial genes were absent in the promoter region of the C. psychrerythraea icl gene. The substitution of Ala214 for Ser in the C. psychrerythraea ICL introduced by point mutation resulted in the increased thermostability and lowering of the specific activity at low temperature, indicating that Ala214 is important for psychrophilic properties of this enzyme. [ABSTRACT FROM AUTHOR]

Abstract: Direct evidence for metabolism in a variety of frozen environments has pushed temperature limits for bacterial activity to increasingly lower temperatures, so far to −20°C. To date, the metabolic activities of marine psychrophilic bacteria, important components of sea-ice communities, have not been studied in laboratory culture, not in ice and not below −12°C. We measured [3H]-leucine incorporation into macromolecules (further fractionated biochemically) by the marine psychrophilic bacterium Colwellia psychrerythraea strain 34H over a range of anticipated activity-permissive temperatures, from +13 to −20°C, including expected negative controls at −80 and −196°C. For incubation temperatures below −1°C, the cell suspensions [all in artificial seawater (ASW)] were first quick-frozen in liquid nitrogen. We also examined the effect of added extracellular polymeric substances (EPS) on [3H]-leucine incorporation. Results showed that live cells of strain 34H incorporated substantial amounts of [3H]-leucine into TCA-precipitable material (primarily protein) down to −20°C. At temperatures from −1 to −20°C, rates were enhanced by EPS. No activity was detected in the killed controls for strain 34H (or in Escherichia coli controls), which included TCA-killed, heat-killed, and sodium azide- and chloramphenicol-treated samples. Surprisingly, evidence for low but significant rates of intracellular incorporation of [3H]-leucine into protein was observed for both ASW-only and EPS-amended (and live only) samples incubated at −80 and −196°C. Mechanisms that could explain the latter results require further study, but the process of vitrification promoted by rapid freezing and the presence of salts and organic polymers may be relevant. Overall, distinguishing between intracellular and extracellular aspects of bacterial activity appears important to understanding behavior at sub-freezing temperatures. [Copyright &y& Elsevier]

Two structurally different monomeric and dimeric types of isocitrate dehydrogenase (IDH; EC 1.1.1.42) isozymes were confirmed to exist in a psychrophilic bacterium, Colwellia psychrerythraea, by Western blot analysis and the genes encoding them were cloned and sequenced. Open reading frames of the genes ( icd-M and icd-D) encoding the monomeric and dimeric IDHs of this bacterium, IDH-M and IDH-D, were 2,232 and 1,251 bp in length and corresponded to polypeptides composed of 743 and 416 amino acids, respectively. The deduced amino acid sequences of the IDH-M and IDH-D showed high homology with those of monomeric and dimeric IDHs from other bacteria, respectively. Although the two genes were located in tandem, icd-M then icd-D, on the chromosomal DNA, a Northern blot analysis and primer extension experiment revealed that they are transcribed independent of each other. The expression of the monomeric and dimeric IDH isozyme genes in C. maris, a psychrophilic bacterium of the same genus as C. psychrerythraea, is known to be induced by low temperature and acetate, respectively, but no such induction in the expression of the C. psychrerythraea icd-M and icd-D genes was detected. IDH-M and IDH-D overexpressed in Escherichia coli were purified and characterized. In C. psychrerythraea, the IDH-M isozyme is cold-active whereas IDH-D is mesophilic, which is similar to C. maris that contains both cold-adapted and mesophilic isozymes of IDH. Experiments with chimeric enzymes between the cold-adapted monomeric IDHs of C. psychrerythraea and C. maris (IDH-M and ICD-II, respectively) suggested that the C-terminal region of the C. maris IDH-II is involved in its catalytic activity. [ABSTRACT FROM AUTHOR]

DNA gyrase is a type II topoisomerase that is essential for chromosome segregation and cell division owing to its ability to modify the topological forms of bacterial DNA. In this study, the N-terminal breakage-reunion domain of the GyrA subunit of DNA gyrase from Colwellia psychrerythraea 34H was overexpressed in Escherichia coli, purified and crystallized. Diffraction data were collected to 2.60 Å resolution using a synchrotron-radiation source. The crystal belonged to space group P212121, with unit-cell parameters a = 98.98, b = 101.56, c = 141.83 Å. The asymmetric unit contained two molecules, with a corresponding VM of 3.18 Å3 Da−1 and a solvent content of 59.9%. [ABSTRACT FROM AUTHOR]