Isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Pseudomonas psychrophila

Matsuo, Shougo; Shirai, Haruna; Takada, Yasuhiro

doi:10.1007/s00203-010-0595-3

Cited by 18 publications

(31 citation statements)

References 32 publications

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“…Various psychrophilic enzymes [178, 184, 248–250, 275–282] have been engineered in order to check the involvement of specific adaptive mutations within or close to the active site. These studies have revealed a complex pattern of effects on kinetic parameters, activation energy, and stability that cannot be interpreted in simple terms.…”

Section: Activity-flexibility-stability Relationships: Current Issuesmentioning

confidence: 99%

Psychrophilic Enzymes: From Folding to Function and Biotechnology

2013

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Psychrophiles thriving permanently at near-zero temperatures synthesize cold-active enzymes to sustain their cell cycle. Genome sequences, proteomic, and transcriptomic studies suggest various adaptive features to maintain adequate translation and proper protein folding under cold conditions. Most psychrophilic enzymes optimize a high activity at low temperature at the expense of substrate affinity, therefore reducing the free energy barrier of the transition state. Furthermore, a weak temperature dependence of activity ensures moderate reduction of the catalytic activity in the cold. In these naturally evolved enzymes, the optimization to low temperature activity is reached via destabilization of the structures bearing the active site or by destabilization of the whole molecule. This involves a reduction in the number and strength of all types of weak interactions or the disappearance of stability factors, resulting in improved dynamics of active site residues in the cold. These enzymes are already used in many biotechnological applications requiring high activity at mild temperatures or fast heat-inactivation rate. Several open questions in the field are also highlighted.

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Section: Activity-flexibility-stability Relationships: Current Issuesmentioning

confidence: 99%

Psychrophilic Enzymes: From Folding to Function and Biotechnology

2013

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“…Many bacteria, including Escherichia coli, possess only one of either type IDH [1,2,5,6,17]. However, psychrophilic bacteria, Colwellia maris strain ABE-1 (JCM 10085) [20,26] and Colwellia psychrerythraea [3,4], and a psychrotolerant bacterium, Pseudomonas psychrophila strain E-3 (JCM 10889) [19,27], have been found to hold both of the two type IDHs [13][14][15][16]. Although these two types of IDH catalyze the same reaction, their amino acid sequences and immunological cross-reactivities are different from each other [6][7][8]17].…”

Section: Introductionmentioning

confidence: 99%

Contribution of Three Different Regions of Isocitrate Dehydrogenases from Psychrophilic and Psychrotolerant Bacteria to Their Thermal Properties

Mouri

Takada

2018

Curr Microbiol

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Monomeric isocitrate dehydrogenases of a psychrophilic bacterium, Colwellia maris, and a psychrotolerant bacterium, Pseudomonas psychrophila, (CmIDH and PpIDH) are cold-adapted and mesophilic, respectively. On the other hand, previous studies revealed that the monomeric IDH of Azotobacter vinelandii (AvIDH) is also mesophilic and the regions 2 and 3 among three regions of this enzyme are involved in the thermal properties. Therefore, to examine whether the region(s) responsible for the mesophilic properties are common between PpIDH and AvIDH, the genes of chimeric IDHs exchanging three regions of PpIDH and CmIDH in various combinations were constructed and overexpressed as His-tagged recombinant proteins in the Escherichia coli cells, and the chimeric and wild-type PpIDH and CmIDH were purified with Ni-chelating affinity column chromatography. The swapping chimeras of the regions 2 or 3 in PpIDH and CmIDH showed lower and higher optimum temperatures for activities and their thermostabilities than the wild-type ones, respectively. On the other hand, the exchange of the respective region 1 hardly influenced these properties of the two IDHs. Therefore, the regions 2 and 3 of the two IDHs were confirmed to be involved in their thermal properties. These results were coincident with those of the previous study on chimeric IDHs between AvIDH and CmIDH, indicating that the common regions of AvIDH and PpIDH are responsible for their mesophilic properties and the amino acid residues involved in their thermal properties are present in the regions 2 and 3.

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“…tuberculosis , P . psychrophila or Vibrio sp ., have two structurally different isozymes [1,5–7]. For example, both C .…”

Section: Introductionmentioning

confidence: 99%

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus

Romkina

Kiriukhin

2017

PLoS ONE

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The isocitrate dehydrogenase (MfIDH) with unique double coenzyme specificity from Methylobacillus flagellatus was purified and characterized, and its gene was cloned and overexpressed in E. coli as a fused protein. This enzyme is homodimeric,—with a subunit molecular mass of 45 kDa and a specific activity of 182 U mg -1 with NAD+ and 63 U mg -1 with NADP+. The MfIDH activity was dependent on divalent cations and Mn2+ enhanced the activity the most effectively. MfIDH exhibited a cofactor-dependent pH-activity profile. The optimum pH values were 8.5 (NAD+) and 6.0 (NADP+).The Km values for NAD+ and NADP+ were 113 μM and 184 μM respectively, while the Km values for DL-isocitrate were 9.0 μM (NAD+), 8.0 μM (NADP+). The MfIDH specificity (kcat/Km) was only 5-times higher for NAD+ than for NADP+. The purified MfIDH displayed maximal activity at 60°C. Heat-inactivation studies showed that the MfIDH was remarkably thermostable, retaining full activity at 50°C and losting ca. 50% of its activity after one hour of incubation at 75°C. The enzyme was insensitive to the presence of intermediate metabolites, with the exception of 2 mM ATP, which caused 50% inhibition of NADP+-linked activity. The indispensability of the N6 amino group of NAD(P)+ in its binding to MfIDH was demonstrated. MfIDH showed high sequence similarity with bacterial NAD(P)+-dependent type I isocitrate dehydrogenases (IDHs) rather than with eukaryotic NAD+-dependent IDHs. The unique double coenzyme specificity of MfIDH potentially resulted from the Lys340, Ile341 and Ala347 residues in the coenzyme-binding site of the enzyme. The discovery of a type I IDH with double coenzyme specificity elucidates the evolution of this subfamily IDHs and may provide fundamental information for engineering enzymes with desired properties.

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Isocitrate dehydrogenase isozymes from a psychrotrophic bacterium, Pseudomonas psychrophila

Cited by 18 publications

References 32 publications

Psychrophilic Enzymes: From Folding to Function and Biotechnology

Psychrophilic Enzymes: From Folding to Function and Biotechnology

Contribution of Three Different Regions of Isocitrate Dehydrogenases from Psychrophilic and Psychrotolerant Bacteria to Their Thermal Properties

Biochemical and molecular characterization of the isocitrate dehydrogenase with dual coenzyme specificity from the obligate methylotroph Methylobacillus Flagellatus

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