Purification and Characterization of NADP-Linked Isocitrate Dehydrogenase from the Copper-tolerant Wood-rotting BasidiomyceteFomitopsis palustris

Abstract

NADP-linked isocitrate dehydrogenase (EC 1.1.1.42), a key enzyme of the tricarboxylic acid cycle, was purified 672-fold as a nearly homogeneous protein from the copper-tolerant wood-rotting basidiomycete Fomitopsis palustris. The purified enzyme, with a molecular mass of 115 kDa, consisted of two 55-kDa subunits, and had the Km of 12.7, 2.9, and 23.9 microM for isocitrate, NADP, and Mg2+, respectively, at the optimal pH of 9.0. The enzyme had maximum activity in the presence of Mg2+, which also helped to prevent enzyme inactivation during the purification procedures and storage. The enzyme activity was competitively inhibited by 2-oxoglutarate (K(i), 127.0 microM). Although adenine nucleotides and other compounds, including some of the metabolic intermediates of glyoxylate and tricarboxylic acid cycles, had no or only slight inhibition, a mixture of oxaloacetate and glyoxylate potently inhibited the enzyme activity and the inhibition pattern was a mixed type.

NADPH-generating systems in bacteria and archaea

“…Together with isocitrate lyase, IDH is a branching point between the TCA cycle and the glyoxylate shunt (Figure 3 ), a pathway needed for growth on non-fermentative carbon sources such as acetate and ethanol. IDH activity is important in controlling the metabolic flux between both pathways and is affected by various regulatory factors, such as metal ions (Murakami et al, 1997 ; Yoon et al, 2003 ), PEP concentration (Ogawa et al, 2007 ), and phosphorylation/dephosphorylation (Walsh and Koshland, 1985 ; Cozzone, 1998 ). In addition, IDH is a component of the reductive tricarboxylic acid (RTCA) cycle.…”

“…Although most IDHs have a strong requirement for either Mg 2+ or Mn 2+ , substitution of other divalent cations, or even univalent ions such as Na + , is sometimes possible. However, in most cases, this leads to little or no activity (Murakami et al, 1997 ; Steen et al, 1997 ; Yoon et al, 2003 ; Banerjee et al, 2005 ).…”

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

“…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.…”

Thermococcus kodakarensis : the key to affordable biohydrogen production

“…Together with isocitrate lyase, IDH is a branching point between the TCA cycle and the glyoxylate shunt (Figure 2.3), a pathway needed for growth on non-fermentative carbon sources such as acetate and ethanol. IDH activity is important in controlling the metabolic flux between both pathways and is affected by various regulatory factors, such as metal ions [225,226], PEP concentration [227], and phosphorylation/dephosphorylation [228,229]. In addition, IDH is a component of the reductive tricarboxylic acid (RTCA) cycle.…”

“…In addition, IDH is a component of the reductive tricarboxylic acid (RTCA) cycle. The RTCA cycle is a CO 2 fixation pathway, present in some bacteria and archaea, in which four molecules of CO 2 are fixed to produce one molecule of oxaloacetate [230][231][232] [225,226,244,245].…”