Biochemical and phylogenetic characterization of isocitrate dehydrogenase from a hyperthermophilic archaeon, Archaeoglobus fulgidus

• Published in: Archives of microbiology 1997-11, Vol.168 (5), p.412-420
• Main Authors: STEEN, I. H, LIEN, T, BIRKELAND, N.-K
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; Archaeoglobus fulgidus - enzymology; Archaeoglobus fulgidus - genetics; Bacteriology; Biological and medical sciences; Cloning, Molecular; Enzyme Stability; Fundamental and applied biological sciences. Psychology; Genes, Archaeal - genetics; Hot Temperature; Isocitrate Dehydrogenase - chemistry; Isocitrate Dehydrogenase - genetics; Isocitrate Dehydrogenase - isolation & purification; Isocitrate Dehydrogenase - metabolism; Isoelectric Point; Kinetics; Metabolism. Enzymes; Microbiology; Molecular Sequence Data; Molecular Weight; Phylogeny; Sequence Analysis, DNA; Sequence Homology, Amino Acid; Space life sciences
• ISSN: 0302-8933; 1432-072X
• DOI: 10.1007/s002030050516

A thermostable homodimeric isocitrate dehydrogenase from the hyperthermophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subunit was 42 kDa as determined by SDS-PAGE. Following separation by SDS-PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and detected in situ by activity staining. The enzyme showed dual coenzyme specificity with a high preference for NADP+. Optimal temperature for activity was 90 degrees C or above, and a half-life of 22 min was found for the enzyme when incubated at 90 degrees C in a 50 mM Tricine-KOH buffer (pH 8.0). Based on the N-terminal amino acid sequence, the gene encoding the isocitrate dehydrogenase was cloned. DNA sequencing identified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined for the purified enzyme. The deduced amino acid sequence closely resembled that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, with 57% identity with isocitrate dehydrogenase from Escherichia coli. All the amino acid residues directly contacting substrate and coenzyme (except Ile-320) in E. coli isocitrate dehydrogenase are conserved in the enzyme from A. fulgidus. The primary structure of A. fulgidus isocitrate dehydrogenase confirmes the presence of Bacteria-type isocitrate dehydrogenases among Archaea. Multiple alignment of all the available amino acid sequences of di- and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into three distinct phylogenetic groups.