Expression and characterization of a novel isocitrate dehydrogenase from Streptomyces diastaticus No. 7 strain M1033

Abstract

Isocitrate dehydrogenase (IDH) is one of the key enzymes in tricarboxylic acid cycle, widely distributed in Archaea, Bacteria and Eukarya. Here, we report for the first time the cloning, expression and characterization of a monomeric NADP(+)-dependent IDH from Streptomyces diastaticus No. 7 strain M1033 (SdIDH). Molecular mass of SdIDH was about 80 kDa and showed high amino acid sequence identity with known monomeric IDHs. Maximal activity of SdIDH was observed at pH 8.0 (Mn(2+)) and 9.0 (Mg(2+)), and the optimal temperature was 40 °C (Mn(2+)) and 37 °C (Mg(2+)). Heat-inactivation studies showed that SdIDH remained about 50 % activity after 20 min of incubation at 47 °C. SdIDH displayed a 19,000 and 32,000-fold (k (cat)/K (m)) preference for NADP(+) over NAD(+) with Mn(2+) and Mg(2+), respectively. Our work implicate that SdIDH is a divalent metal ion-dependent monomeric IDH with remarkably high coenzyme preference for NADP(+). This work may provide fundamental information for further investigation on the catalytic mechanism of monomeric IDH and give a clue to disclose the real cause of IDH monomerization.

Molecular cloning, purification, and biochemical characterization of recombinant isocitrate dehydrogenase from Streptomyces coelicolor M-145

“…As described in other actinomycetes 6,16) , S. coelicolor IDH was NADP + and Mn 2+ /Mg 2+ dependent and had a maximum activity of 25.3 μmoles/mg/min. Unexpectedly, in polyacrylamide gel under native conditions ( Fig.…”

“…Despite the fact that ScIDH had high identity (between 80 and 90%) and similar molecular masses (between 70 and 79 KDa) with S. lividans TK54 6) S. diastaticus, 16) and S. avermitilis, 12) their enzymes have been described as monomers, contrary to that observed in S. coelicolor.…”

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

“…pylori NADP + -IDH (176 μM) [15]. The K m value of Mf IDH for DL-isocitrate (8–9 μM) is within the range observed for many characterized IDHs [18]. …”

“…nautica NADP + -IDH (25 μM), and S . diastaticus NADP + -IDH (8.5 μM) [18], but is in the range of those of H . volcanii NADP + -IDH (101 μM) [40] and H .…”

“…Most prokaryotic IDHs that have been investigated are NADP + -dependent and homodimeric [11–17]. A few NADP + -dependent monomeric IDHs [9,18–20] and homotetrameric IDH from T . maritima [17] have also been characterized.…”