NADP+-Specific Isocitrate Dehydrogenase from Oleaginous Yeast Yarrowia lipolytica CLIB122: Biochemical Characterization and Coenzyme Sites Evaluation

Abstract

NADP(+)-dependent isocitrate dehydrogenase from Yarrowia lipolytica CLIB122 (YlIDP) was overexpressed and purified. The molecular mass of YlIDP was estimated to be about 81.3 kDa, suggesting its homodimeric structure in solution. YlIDP was divalent cation dependent and Mg(2+) was found to be the most favorable cofactor. The purified recombinant YlIDP displayed maximal activity at 55 °C and its optimal pH for catalysis was found to be around 8.5. Heat inactivation studies revealed that the recombinant YlIDP was stable below 45 °C, but its activity dropped quickly above this temperature. YlIDP was absolutely dependent on NADP(+) and no NAD-dependent activity could be detected. The K m values displayed for NADP(+) and isocitrate were 59 and 31 μM (Mg(2+)), 120 μM and 58 μM (Mn(2+)), respectively. Mutant enzymes were constructed to tentatively alter the coenzyme specificity of YlIDP. The K m values for NADP(+) of R322D mutant was 2,410 μM, being about 41-fold higher than that of wild type enzyme. NAD(+)-dependent activity was detected for R322D mutant and the K m and k cat values for NAD(+) were 47,000 μM and 0.38 s(-1), respectively. Although the R322D mutant showed low activity with NAD(+), it revealed the feasibility of engineering an eukaryotic IDP to a NAD(+)-dependent one.

Substrates and oxygen dependent citric acid production by Yarrowia lipolytica: insights through transcriptome and fluxome analyses

“…The higher PPP fluxes could be a consequence of higher NADPH requirement on glucose needed for growth and biosynthesis (Table 2). Moreover, the higher PPP fluxes would also reduce the fluxes toward the NADP dependent isocitrate dehydrogenase, reported to be present in Y. lipolytica , the major citrate degrading enzyme [38]. On the other hand, the relative higher fluxes from glycerol 3-phosphate to dihydroxyacetone phosphate via the membrane bound glycerol-3-phosphate dehydrogenase with a concurrent reduction of flavin adenine dinucleotide (FAD) might explain the relatively higher TCA cycle fluxes on glycerol.…”

Metabolic Remodeling during Long-Lasting Cultivation of the Endomyces magnusii Yeast on Oxidative and Fermentative Substrates

“…The NADPH level in the cells is another factor impacting on the activity of the GR/GP system [55]. The content of the latter in the cytosol depends on the activity of the pentose phosphate pathway and, in particular, its first enzyme, NADP-G6PDH [56], and on the activity of some alternative NADPH-producing enzymes, such as NADH-related IDH [57]. Oxidation of isocitrate into 2-oxoglutarate catalyzed by NADP-IDG is closely connected with the enzymatic transformations of GSSG and GSH (2GSH = GSSG + 2H + ) catalyzed by GR.…”

Single Arginine Mutation in Two Yeast Isocitrate Dehydrogenases: Biochemical Characterization and Functional Implication

“…Each of these proteins plays distinct roles in the central metabolism of S. cerevisiae : ScIDH1 is involved in the cellular glutamate synthesis, ScIDH2 provides NADPH for thiol-based antioxidant systems, and ScIDH3 generates NADPH for the β-oxidation of unsaturated fatty acids [10] . In Yarrowia lipolytica , a 'non-conventional' species of oleaginous yeast, has only one NADP + -IDH (YlIDH), which is located in the mitochondria and is closely related to the oleaginicity of Y.lipolytica [11] .…”