Isocitrate dehydrogenase from a Gram-positive psychrotrophic bacterium, strain G-4

Abstract

By western blot analysis with antibodies against monomeric and dimeric isocitrate dehydrogenases (IDHs), a Gram-positive psychrotrophic bacterium, strain G-4, was confirmed to possess only monomeric IDH. The gene encoding this enzyme (G-4IDH) was cloned and sequenced. The open reading frame of this gene was 2,220 bp in length, corresponding to a polypeptide composed of 739 amino acids. The transcriptional initiation site for the gene was guanine, located at 695 bases upstream of the translational start codon. The deduced amino acid sequence showed high identities with those of the monomeric IDHs from Gram-negative bacteria such as Colwellia maris and Azotobacter vinelandii (62% and 64% identity, respectively) and, particularly, the putative IDHs of Gram-positive bacteria, Arthrobacter alpinus andPaenarthrobacter aurescens (91% and 81% identity, respectively). The His-tagged G-4IDH was overproduced in the Escherichia coli cells and was purified and characterized. The G-4IDH showed the highest activity at 45°C, indicating that it is mesophilic. However, the residual activity after incubation for 10 min at 35°C was 37%, and G-4IDH was more thermolabile than a typical mesophilic IDH of A. vinelandii.