Biochemical and molecular characterization of the NAD⁺-dependent isocitrate dehydrogenase from the chemolithotrophAcidithiobacillus thiooxidans

Abstract

An isocitrate dehydrogenase (ICDH) with an unique coenzyme specificity from Acidithiobacillus thiooxidans was purified and characterized, and its gene was cloned. The native enzyme was homodimeric with a subunit of M(r) 45000 and showed a 78-fold preference for NAD(+) over NADP(+). The cloned ICDH gene (icd) was expressed in an icd-deficient strain of Escherichia coli EB106; the activity was found in the cell extract. The gene encodes a 429-amino acid polypeptide and is located between open reading frames encoding a putative aconitase gene (upstream of icd) and a putative succinyl-CoA synthase beta-subunit gene (downstream of icd). A. thiooxidans ICDH showed high sequence similarity to bacterial NADP(+)-dependent ICDH rather than eukaryotic NAD(+)-dependent ICDH, but the NAD(+)-preference of the enzyme was suggested due to residues conserved in the coenzyme binding site of the NAD(+)-dependent decarboxylating dehydrogenase.

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

“…mobilis (pH 8.5) [26] and A . thiooxidans NAD + -IDHs (pH 8.5) [21], but is lower than that of the H . thermophilus NAD + -IDH (pH 10.5) [24].…”

“…maritima [17] have also been characterized. Recently, numerous prokaryotic and archaeal homodimeric NAD + -IDHs and, a few monomeric NAD + -IDHs, have been reported [21–27]. However, NAD + -dependency is relatively rare in prokaryotic IDHs, and true double coenzyme specificity has never been reported.…”

Isocitrate Dehydrogenase from Streptococcus mutans: Biochemical Properties and Evaluation of a Putative Phosphorylation Site at Ser102

“…SmIDH was found to have an optimal pH range of 7.5–8.5, with the optimal pH at 7.8 (Figure 2A), lower than that of the NAD + -IDHs from A . thiooxidans (pH 8.5) [2] and Hydrogenobacter thermophilus (pH 10.5) [38]. When compared with the broad optimum pH range of NADP + -IDHs from other sources, such as Streptomyces lividans (pH 8.5–10.0) [39], Fomitopsis palustri s (pH 8.0–10.0) [40] and Aspergillus niger (pH 6.0–8.0) [41], it was narrower for SmIDH, suggesting that SmIDH was sensitive to pH changes.…”

“…As a better phenotype acquired through evolution, most IDHs exhibit the NADP + dependence, causing NAD + dependence comparatively uncommon among the bacterial IDHs. A small number of NAD + -IDHs have been characterized in some bacteria and archaea [2]–[7]. A general feature shared by these organisms is that they have an incomplete tricarboxylic acid (TCA) cycle due to the absence of one or more TCA cycle enzymes [3].…”

“…A general feature shared by these organisms is that they have an incomplete tricarboxylic acid (TCA) cycle due to the absence of one or more TCA cycle enzymes [3]. Thus, these NAD + -IDHs have been proposed to be reminiscent of the enzyme that participates in CO 2 fixation as well as glutamate biosynthesis [2], [8]. Eukaryotic NAD + -IDHs localize exclusively in the mitochondria and play a central catabolic role in energy production.…”