NADP+ -dependent isocitrate dehydrogenase from a psychrophilic bacterium, Psychromonas marina

Extremophiles : Life Under Extreme Conditions
Ryo HirotaY Takada

Abstract

The gene encoding NADP+-dependent isocitrate dehydrogenase (IDH; EC 1.1.1.42) of a psychrophilic bacterium, Psychromonas marina, was cloned and sequenced. The open reading frame of the gene encoding IDH of P. marina (PmIDH) was 2229 bp in length and corresponded to a polypeptide composed of 742 amino acids. The molecular mass of IDH was calculated as 80,426 Da. The deduced amino acid sequence of PmIDH exhibited high degrees of homology with the monomeric IDH from other bacteria such as Colwellia maris (62% identity) and Azotobacter vinelandii (AvIDH) (64%). His-tagged PmIDH overexpressed in Escherichia coli cells was purified and characterized. The optimum temperature of PmIDH activity was about 35 °C; however, the enzyme lost 74% of the activity after incubation for 10 min at 30 °C, indicating that this enzyme is thermolabile. Chimeric enzymes produced through domain swapping between PmIDH and mesophilic AvIDH were constructed and their optimum temperatures and thermostability were determined. The results suggest that regions 2 and 3, especially region 3, of the two IDHs are involved in their catalytic activities and optimum temperature and thermostability for activity.

References

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Methods Mentioned

BETA
PCR
column
PCRs
electrophoresis

Related Concepts

Idp1 protein, S pombe
Genes, Bacterial
Isocitrate Dehydrogenase-I
Western Blot
Homologous Sequences, Amino Acid
Gammaproteobacteria
Escherichia coli
Genes
Isocitrate Dehydrogenase (NAD+)
NADP

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