BIOCHEMICAL AND PHYLOGENETIC CHARACTERIZATION OF ISOCITRATE DEHYDROGENASE FROM A HYPERTHERMOPHILIC ARCHAEON, ARCHAEOGLOBUS-FULGIDUS

A thermostable homodimeric isocitrate dehydrogenase from the hyperther mophilic sulfate-reducing archaeon Archaeoglobus fulgidus was purified and characterized. The mol. mass of the isocitrate dehydrogenase subu nit was 42 kDa as determined by SDS-PAGE, Following separation by SDS- PAGE, A. fulgidus isocitrate dehydrogenase could be renatured and dete cted in situ by activity staining, The enzyme showed dual coenzyme spe cificity with a high preference for NADP(+). Optimal temperature for a ctivity 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 iden tified the icd gene as an open reading frame encoding a protein of 412 amino acids with a molecular mass corresponding to that determined fo r the purified enzyme. The deduced amino acid sequence closely resembl ed that of the isocitrate dehydrogenase from the archaeon Caldococcus noboribetus (59% identity) and bacterial isocitrate dehydrogenases, wi th 57% identity with isocitrate dehydrogenase from Escherichia coli. A ll 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 isoc itrate dehydrogenase confirmes the presence of Bacteria-type isocitrat e dehydrogenases among Archaea. Multiple alignment of all the availabl e amino acid sequences of di-and multimeric isocitrate dehydrogenases from the three domains of life shows that they can be divided into thr ee distinct phylogenetic groups.