Quantifying Interactions Within the NADP(H) Enzyme Network in Drosophila melanogaster

In this report, we use synthetic, activity-variant alleles in Drosophila melanogaster to quantify interactions across the enzyme network that reduces nicotinamide adenine dinucleotide phosphate (NADP) to NADPH. We examine the effects of large-scale variation in isocitrate dehydrogenase (IDH) or glucose-6-phosphate dehydrogenase (G6PD) activity in a single genetic background and of smaller-scale variation in IDH, G6PD, and malic enzyme across 10 different genetic backgrounds. We find significant interactions among all three enzymes in adults; changes in the activity of any one source of a reduced cofactor generally result in changes in the other two, although the magnitude and directionality of change differs depending on the gene and the genetic background. Observed interactions are presumably through cellular mechanisms that maintain a homeostatic balance of NADPH/NADP, and the magnitude of change in response to modification of one source of reduced cofactor likely reflects the relative contribution of that enzyme to the cofactor pool. Our results suggest that malic enzyme makes the largest single contribution to the NADPH pool, consistent with the results from earlier experiments in larval D. melanogaster using naturally occurring alleles. The interactions between all three enzymes indicate functional interdependence and underscore the importance of examining enzymes as components of a network.IN traits determined by a network of gene products, the phenotype is a function of the alleles present and of the relative contributions of individual network member genes. Since selection is on phenotype, the total composite genotype, not just individual loci, determines the fitness of an organism. In establishing the connection between genotype and phenotype for such networks, the first challenge is to quantify the relative contribution of each member of the network to the endpoint phenotype. By addressing function on a network-wide basis, interactions and interconnections that may not be apparent in individual gene examinations can be determined (Proulx et al. 2005).In most organisms, reduction of the cofactor nicotinamide adenine dinucleotide phosphate, or NADP, to NADPH is primarily the function of four enzymes: cytosolic malic enzyme (MEN), cytosolic isocitrate dehydrogenase (IDH), and the two oxidative enzymes of the pentose shunt, glucose-6-phosphate dehydrogenase (G6PD) and 6-phosphogluconate (6PGD; recently reviewed in Ying 2008). In larval Drosophila melanogaster, MEN produces ∼30% of the available NADPH, IDH ∼20%, and G6PD and 6PGD the remaining ∼40% (Geer et al. 1979a,b). It is believed that these four enzymes interact to maintain the NADP/NADPH balance and supply of reducing power for lipogenesis and antioxidation (Geer et al. 1976, 1978, 1981; Wilton et al. 1982; Bentley et al. 1983; Geer and Laurie-Ahlberg 1984; Merritt et al. 2005; Pollak et al. 2007; Singh et al. 2007; Ying 2008). Dietary induction studies and observations of natural genetic variation have found connections between MEN activity and the activities of the pentose shunt enzymes to be generally straightforward and compensatory; reductions in one lead to increases in the other. The interactions involving IDH activity, however, have been found to be more complicated and at times counterintuitive; reductions in reducing power sometimes lead to decreases in IDH activity.In an earlier study (Merritt et al. 2005), we quantified the impact of genetic variation in Men activity on IDH and G6PD activities and triglyceride (a strong correlate with total lipid; Clark and Keith 1989) concentration. 6PGD was not independently assayed because earlier works suggest that G6PD and 6PGD activities are highly correlated, likely because of their coupled function in the pentose shunt (Wilton et al. 1982). We examined both naturally occurring Men alleles and synthetic alleles created by P-element excision and found significant associations between MEN activity and induction of the activities of both IDH and G6PD. The apparent interactions between MEN and IDH and G6PD across these 10 different third chromosome lines were quantified as mean elasticity coefficients: = −0.76 ± 0.236 and = −0.88 ± 0.208. Because MEN activity was reduced by 20%, both IDH and G6PD activity varied in a compensatory direction, increasing almost 1:1 with the decrease in MEN.The significant change in enzyme activity of two members of the NADPH network in response to our genetic reduction of the activity of a third strongly suggests that a physiological mechanism coregulates the three enzymes. Such functional interdependence would mean that individual members of the network do not act in isolation and should be examined collectively, not as isolated units. In this study, we characterize the effects of the independently varying activity levels of IDH, G6PD, and MEN on the activity of each other and triglyceride concentration in adult flies. We found significant responses to changes in all three enzymes, although the responses to genetic changes in IDH and G6PD were generally small; variation in MEN caused the greatest changes in the other enzymes.     

The NADPH Metabolic Network Regulates Human αB-crystallin Cardiomyopathy and Reductive Stress in Drosophila melanogaster

Dominant mutations in the alpha-B crystallin (CryAB) gene are responsible for a number of inherited human disorders, including cardiomyopathy, skeletal muscle myopathy, and cataracts. The cellular mechanisms of disease pathology for these disorders are not well understood. Among recent advances is that the disease state can be linked to a disturbance in the oxidation/reduction environment of the cell. In a mouse model, cardiomyopathy caused by the dominant CryAB^R120G missense mutation was suppressed by mutation of the gene that encodes glucose 6-phosphate dehydrogenase (G6PD), one of the cell''s primary sources of reducing equivalents in the form of NADPH. Here, we report the development of a Drosophila model for cellular dysfunction caused by this CryAB mutation. With this model, we confirmed the link between G6PD and mutant CryAB pathology by finding that reduction of G6PD expression suppressed the phenotype while overexpression enhanced it. Moreover, we find that expression of mutant CryAB in the Drosophila heart impaired cardiac function and increased heart tube dimensions, similar to the effects produced in mice and humans, and that reduction of G6PD ameliorated these effects. Finally, to determine whether CryAB pathology responds generally to NADPH levels we tested mutants or RNAi-mediated knockdowns of phosphogluconate dehydrogenase (PGD), isocitrate dehydrogenase (IDH), and malic enzyme (MEN), the other major enzymatic sources of NADPH, and we found that all are capable of suppressing CryAB^R120G pathology, confirming the link between NADP/H metabolism and CryAB.     

Glucose-6-phosphate dehydrogenase (G6PD) electrophoretic variants and the PvuII polymorphism in Southern African populations

Summary Southern African Bantu-speaking negroid and San populations were examined with regard to the glucose-6-phosphate dehydrogenase (G6PD) PvuII restriction fragment length polymorphism (RFLP) showing alleles of 4kb and 1.6 kb, called Type 1 and Type 2, respectively. The standardized disequilibrium coefficient for the electrophoretic G6PD types and PvuII alleles in the Southern African population was 0.28. The molecular lesion causing the Gd^A mutation is the same in the San and Southern African negroid populations. Gd^A chromosomes are found in association with both the Type 1 and Type 2 alleles, whereas none of the 62 Gd^B chromosomes from the Southern African populations had the Type 2 allele. Five of the 44 Gd^B chromosomes studied in the American Black population had the Type 2 allele, indicating that the Gd^B allele in the two populations may have different origins. The presence of all 3 A^– deficiency mutations in the G6PD A gene, in a region where the ancestral population was thought to have predominantly G6PD B, may be explained by their origin in Africa after the divergence of the races.     

Methylglyoxal-induced modification of arginine residues decreases the activity of NADPH-generating enzymes

Inadequate control of plasma and cellular glucose and ketone levels in diabetes is associated with increased generation of reactive aldehydes, including methylglyoxal (MGO). These aldehydes react with protein side chains to form advanced glycation end-products (AGEs). Arg residues are particularly susceptible to MGO glycation and are essential for binding NADP⁺ in several enzymes that generate NADPH, a coenzyme for many critical metabolic and antioxidant enzymes. In most animal cells, NADPH is produced predominantly by glucose-6-phosphate dehydrogenase (G6PD) in the oxidative phase of the pentose phosphate pathway and, to a lesser extent, by isocitrate dehydrogenase (IDH) and malic enzyme (ME). In this study, the activities of isolated G6PD, IDH, and ME were inhibited by MGO (0–2.5 mM, 2–3 h, 37 °C), in a dose- and time-dependent manner, with G6PD and IDH more sensitive to modification than ME. Significant inhibition of these two enzymes occurred with MGO levels ≥500 μM. Incubation with radiolabeled MGO (0–500 µM, 0–3 h, 37 °C) demonstrated dose- and time-dependent adduction to G6PD and IDH. HPLC analysis provided evidence for AGE formation and particularly the hydroimidazolones MG-H1 and MG-H2 from Arg residues, with corresponding loss of parent Arg residues. Peptide mass mapping studies confirmed hydroimidazolone formation on multiple peptides in G6PD and IDH, including those critical for NADP⁺ binding, and substrate binding, in the case of IDH. These results suggest that modification of NADPH-producing enzymes by reactive aldehydes may result in alterations to the cellular redox environment, potentially predisposing cells to further damage by oxidants and reactive aldehydes.     

SIRT5 promotes IDH2 desuccinylation and G6PD deglutarylation to enhance cellular antioxidant defense

Excess in mitochondrial reactive oxygen species (ROS) is considered as a major cause of cellular oxidative stress. NADPH, the main intracellular reductant, has a key role in keeping glutathione in its reduced form GSH, which scavenges ROS and thus protects the cell from oxidative damage. Here, we report that SIRT5 desuccinylates and deglutarylates isocitrate dehydrogenase 2 (IDH2) and glucose‐6‐phosphate dehydrogenase (G6PD), respectively, and thus activates both NADPH‐producing enzymes. Moreover, we show that knockdown or knockout of SIRT5 leads to high levels of cellular ROS. SIRT5 inactivation leads to the inhibition of IDH2 and G6PD, thereby decreasing NADPH production, lowering GSH, impairing the ability to scavenge ROS, and increasing cellular susceptibility to oxidative stress. Our study uncovers a SIRT5‐dependent mechanism that regulates cellular NADPH homeostasis and redox potential by promoting IDH2 desuccinylation and G6PD deglutarylation.     

Molecular analysis of G6PD variants in northern Italy: a study on the population from the Ferrara district

In the Ferrara district, an area south of the Po delta, four different variants of glucose-6-phosphate dehydrogenase (G6PD;E.C.1.1.49) have been described as a result of biochemical characterization of the enzyme protein: one was G6PD Mediterranean (G6PD Med) and three were local variants named Ferrara I, II, and III. The Ferrara I variant was recently analysed at the DNA level and shown to correspond to G6PD A^376G/202A, while the mutations causing the variants II and III, still remain unknown. We analysed the G6PD coding region of 18 apparently unrelated G6PD deficient subjects, whose families have lived in the Ferrara district for at least three generations: 12 subjects had G6PD Med^563T/1311T, 3, G6PD Santamaria^376G/542T and 2, G6PD A-^376G/202A. In one subject we found a new mutation, a GA transition at nucleotide 242 causing an ArgHis amino acid replacement at position 81. We named this new variant G6PD Lagosanto^{242 A}. Phenotypically the enzyme has nearly normal kinetic properties and appears different from the variants Ferrara II and III.     

Glucose-6-phosphate dehydrogenase deficiency in northern Mexico and description of a novel mutation

Glucose-6-phosphate dehydrogenase deficiency (G6PD) is the most common enzyme pathology in humans; it is X-linked inherited and causes neonatal hyperbilirubinaemia, chronic nonspherocytic haemolytic anaemia and drug-induced acute haemolytic anaemia. G6PD deficiency has scarcely been studied in the northern region of Mexico, which is important because of the genetic heterogeneity described in Mexican population. Therefore, samples from the northern Mexico were biochemically screened for G6PD-deficiency, and PCR-RFLPs, and DNA sequencing used to identify mutations in positive samples. The frequency of G6PD deficiency in the population was 0.95% (n = 1993); the mutations in 86% of these samples were G6PD A^?202A/376G , G6PD A^?376G/968C and G6PD Santamaria^376G/542T . Contrary to previous reports, we demonstrated that G6PD deficiency distribution is relatively homogenous throughout the country (P = 0.48336), and the unique exception with high frequency of G6PD deficiency does not involve a coastal population (Chihuahua: 2.4%). Analysis of eight polymorphic sites showed only 10 haplotypes. In one individual we identified a new G6PD mutation named Mexico DF^193A>G (rs199474830), which probably results in a damaging functional effect, according to PolyPhen analysis. Proteomic impact of the mutation is also described.     

Genetic regulation of glucose 6-phosphate dehydrogenase activity in the inbred mouse

The erythrocyte glucose 6-phosphate dehydrogenase activity characteristic of each of 16 inbred mouse strains falls into one of three distinct classes. Strains C57L/J and C57BR/cdJ represent the low activity class: strains A/J and A/HeJ represent the high activity class; other strains have intermediate activities. There is no evidence that structural variation is responsible for the variation in G6PD activity, since partially purified enzyme from each class has the same thermal stability, pH-activity profile, Michaelis constants for G6P and NADP, electrophoretic mobility, and activity using 2-deoxy d-glucose 6-phosphate as substrate. The activities of 6-phosphogluconate dehydrogenase and glucose phosphate isomerase do not differ in erythrocytes of the three G6PD activity classes. Young red cells have higher G6PD activities than old red cells and there is evidence that the intracellular stability of the enzyme is reduced in red cells of strain C57L/J. G6PD activities in kidney and skeletal and cardiac muscle from animals with low red cell G6PD are slightly lower than the activities in kidney and muscle from animals with high red cell G6PD activity. The quantitative differences in red cell G6PD activity are not regulated by X-linked genes, but by alleles at two or more autosomal loci. A simple genetic model is proposed in which alleles at two unlinked, autosomal loci, called Gdr-1 and Gdr-2 regulate G6PD activity in the mouse erythrocyte.     

Red cell glucose 6 phosphate dehydrogenase genotypes of the population of two West African villages

Summary Blood samples from 1109 individuals, residents of two villages in The Gambia, West Africa, have been examined for red cell G6PD. Using both starch gel electrophoresis and a spectrophotometric assay, preliminary phenotypes were assigned to the 519 males and 590 females. The G6PD genotypes were established by reference to the family trees of the two village populations. In addition to the G6PD alleles B⁺, A⁺ and A^-, a fourth allele, representing a new variant of human G6PD was discovered. A significant difference in the frequency of G6PD deficiency was discovered between the two villages, despite their being of the same tribal origin and only five miles apart.     

Tissue Distribution of Glucose-6-phosphate Dehydrogenase Isoenzymes and the Effect of Alloxan Diabetes and Insulin

ACTIVITY of glucose-6-phosphate dehydrogenase (G₆PD) in liver¹ and adipose tissue² has been shown to be decreased in diabetic rats and restored by insulin treatment. In this report we supply data on the distribution of electrophoretically distinguishable forms of G₆PD in four rat tissues and the effect of diabetes and insulin treatment.     

Immunological Difference between Glucose-6-P Dehydrogenase and Hexose-6-P Dehydrogenase from Human Liver

SHAW and Barto¹ have demonstrated the presence of an autosomally inherited glucose-6-P dehydrogenase (G6PD) in the deer mouse. Subsequently, Ohno et al.² found a similar enzyme in trout and showed that this enzyme and the autosomally inherited mouse enzyme differed from the sex-linked G6PD in possessing marked catalytic activity with galactose-6-P. This autosomally inherited G6PD was therefore named hexose-6-P dehydrogenase (H6PD)^2,3. It was shown to oxidize glucose-6-P, galactose-6-P, mannose-6-P and 2-deoxy glucose-6-P with a K_m of the order of 10^?5 M. It also oxidizes glucose with a K_m of 0.7 M³. It appears to be identical to the so-called “glucose dehydrogenase”. The enzyme utilizes both NAD and NADP and is microsome-bound. G6PD is localized in the soluble fraction of the cells of various tissues. Although it has been shown that two dehydrogenases from liver have different substrate specificity, molecular weight and elec-trophoretic mobility^3,4, it has been suggested that the two enzymes are merely isozymes and they might be interconvertible^5–7. We have now partially purified the two enzymes from human liver and show that they have different immunological properties.     

Primate red cell enzymes: glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase

Glucose-6-phosphate dehydrogenase (E. C.: 1.1.1.49) phenotypes and 6-phosphogluconate dehydrogenase (E. C.: 1.1.1.44) phenotypes were determined by starch-gel electrophoresis of red cell hemolysates of Galago crassicaudatus subspp., Propithecus verreauxi, Lemur spp., Hapalemur griseus, and Macaca mulatta. A single glucose-6-phosphate dehydrogenase (G6PD) phenotype was found in each species. A single 6-phosphogluconate dehydrogenase (6PGD) phenotype was found in Lemur spp., Hapalemur griseus, and Galago crassicaudatus argentatus. In a group of six Propithecus verreauxi, three 6PGD phenotypes, PGD A, PGD AB, and PGD B, were found. Three phenotypes, PGD A, PGD AB, and PGD B, were found in 38 G. c. crassicaudatus. The three phenotypes in each species are apparently the products of two codominant autosomal alleles, PGD^A and PGD^B. The frequency of PGD^A in G. c. crassicaudatus is 0.263. A population of 260 free-ranging macaques displays a polymorphism at the 6PGD locus. Three phenotypes, PGD A, PGD AB, and PGD B, were found. These also appear to be controlled by two codominant autosomal alleles, PGD^A and PGD^B the frequency of PGD^A = 0.913. Additional analysis of three well-defined troops within the macaque population indicated that there are no significant differences between the troops or within the population at the 6PGD locus.     

Some Mexican glucose-6-phosphate dehydrogenase variants revisited

Summary Glucose-6-phosphate dehydrogenase (G6PD) deficiency appears to be fairly common in Mexico. We have now examined the DNA of three previously reported electrophoretically fast Mexican G6PD variants, — G6PD Distrito Federal, G6PD Tepic, and G6PD Castilla. All three of these variants, believed on the basis of biochemical characterization and population origin to be unique, have the GA transition at nucleotide 202 and the AG transition at nucleotide 376, mutations that we now recognize to be characteristic of G6PD A —. Two other Mexican males with G6PD deficiency were found to have the same mutation. All five have the (NlaIII/ FokI/PvuII/PstI) haplotype characteristic of G6PD A in Africa. Since the PvuII+ genotype seems to be rare in Europe, we conclude that all of these G6PD A-genes had their ancient origin in Africa, although in many of the Mexican patients with G6PD A –^202A/376G the gene may have been imported more recently from Spain, where this variant, formerly known as G6PD Betica, is also prevalent.     

Glioma-derived mutations in isocitrate dehydrogenase 2 beneficial to traditional chemotherapy

Heterozygous mutations in either the R132 residue of isocitrate dehydrogenase I (IDH1) or the R172 residue of IDH2 in human gliomas were recently highlighted. In the present study, we report that mutations of IDH1 and IDH2 are not detected in the rat C6 glioma cell line model, which suggests that these mutations are not required for the development of glioblastoma induced by N,N′-nitroso-methylurea. The effects of IDH2 and IDH2^R172G on C6 cells proliferation and sensitivity to chemotherapy and the possible mechanism are analyzed at the cellular level. IDH1 and IDH2 mutations lead to simultaneous loss and gain of activities in the production of α-ketoglutarate (α-KG) and 2-hydroxyglutarate (2HG), respectively, and result in lowering NADPH levels even further. The low NADPH levels can sensitize tumors to chemotherapy, and account for the prolonged survival of patients harboring the mutations. Our data extrapolate potential importance of the in vitro rat C6 glioma cell model, show that the IDH2^R172G mutation in gliomas may give a benefit to traditional chemotherapy of this cancer and serve as an important complement to existing research on this topic.     

Effect of astaxanthin and aluminum chloride on erythrocyte G6PD and 6PGD enzyme activities in vivo and on erythrocyte G6PD in vitro in rats

In this study, we investigated the effect of astaxanthin (Ast) and aluminum (Al) on the erythrocyte glucose‐6‐phosphate dehydrogenase (G6PD) and 6‐phosphogluconate dehydrogenase (6PGD) enzymes activities in vivo and on G6PD enzyme in vitro in rats. For in vitro studies, G6PD enzyme was purified from rat erythrocyte by using 2′,5′‐ADP‐Sepharose 4B affinity gel. The effects of Ast and Al³⁺ ion were investigated on the purified enzyme. It was determined that Ast increased the enzyme activity, whereas Al³⁺ inhibited the enzyme activity noncompetitively (IC₅₀ values; 0.679 mM, K_i values 1.32 mM). For in vivo studies, the rats were divided into the groups: control (Cont.), Al, Ast, and Al + Ast. The last three groups were compared with the control group. In Al group, a significant degree of inhibition was observed in the activity of G6PD and 6PGD enzymes when compared with the control group (P < 0.05), whereas there was an increase in the activities of G6PD and 6PGD enzymes in Ast and Al + Ast groups (P < 0.05).     

Dinucleotide Sequences at the 5' Ends of Vaccinia Virus mRNA's Synthesized In Vitro

The diversity of dinucleotide sequences at the 5′ ends of vaccinia virus mRNA's was determined by a two-dimensional electrophoresis procedure. RNA labeled with S-adenosyl[methyl-³H]methionine was synthesized in vitro by enzymes present in vaccinia virus cores. The RNA, ending in m⁷G(5′)pppN^mpN−, was β-eliminated and treated with alkaline phosphatase. After digestion with RNases T₂, T₁, and A, all eight possible dinucleotides containing G^m and A^m were identified. They are, in decreasing order of abundance: G^mpUp (22%), A^mpCp (18%), G^mpAp (16%), G^mpCp (15%), A^mpAp (11%), A^mpUp (10%), A^mpGp (7%), and G^mpGp (2%).     

Maternal effect for genes encoding 6-phosphogluconate dehydrogenase and glucose-6-phosphate dehydrogenase in Drosophila melanogaster

We studied the maternal effect for two enzymes of the pentose cycle, 6-phosphogluconate dehydrogenase (6PGD) and glucose-6-phosphate dehydrogenase (G6PD), using a genetic system based on the interaction of Pgd^? and Zw^? alleles, which inactivate 6PGD and G6PD, respectively. The presence and formation of the enzymes was investigated in those individuals that had not received the corresponding genes from the mother. We revealed maternal forms of the enzymes, detectable up to the pupal stage. The activities of “maternal” 6PGD and G6PD per individual increased 20-fold to 30-fold from the egg stage to the 3rd larval instar even in the absence of normal Pgd and Zw genes. Immunologic studies have shown that the increase in 6PGD activity is due to an accumulation of the maternal form of the enzyme molecules. We revealed a hybrid isozyme resulting from an aggregation of the subunits of isozymes controlled by the genes of the mother and embryo itself. These results indicate that the maternal effect in the case of 6PGD is due to a long-lived stable mRNA transmitted with the egg cytoplasm and translated during the development of Drosophila melanogaster.     

Base-pair exchange kinetics of the imino and amino protons of the 3′-phenazinium tethered DNA-RNA duplex,r(5′GAUUGAA3′):d(5′TCAATC3′-Pzn), and their comparison with those of B-DNA duplex

The dynamics of the opening-closing of the constituent base-pairs as well as of the exchange kinetics of the base-paired imino and amino protons with water in a DNA-RNA hybrid, [^5′r(G¹A²U³U⁴G⁵A⁶A⁷)^3′]:^5′p[d(T⁸C⁹A¹⁰A¹¹T¹²C¹³)]^3′-Pzn] duplex (I), are reported here in details for the first time. The exchange kinetics of amino and imino protons in the DNA-RNA hybrid (duplex I) have been compared with identical studies on the following B-DNA duplexes: d(C¹G²T³A⁴C⁵G⁶)₂ (II), d[p(^5′T¹G²T³T⁴T⁵G⁶ G⁷C⁸)^3′]:d[p(^5′C⁹C¹⁰A¹¹A¹²A¹³C¹⁴A¹⁵)^3′] (III), d(C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶)₂ (IV) and d(C¹G²C³G⁴C⁵G⁶C⁷G⁸A⁹A¹⁰T¹¹T¹²C¹³G¹⁴C¹⁵G¹⁶C¹⁷G¹⁸C¹⁹G²⁰)₂ (V). This comparative study shows that the life-times τ_o of various base-pairs in the DNA-RNA hybrid (I) varies in the range of ∼ 1 ms, and they are quite comparable to those of the shorter B-DNA duplexes (II) and (III), but very different from the τ_o of the larger duplexes (IV) and (V): the τ_o for the base pair of T¹¹ and T¹² residues in the 20-mer (duplex V) are 2.9 ± 2.3 ms and 23.2 ± 8.9 ms, respectively, while the corresponding τ_o in the 12-mer (duplex IV) are 2.8 ± 2.2 ms and 17.4 ± 5.4 ms. It has also been shown that the total energy of activation (E_a) assessed from the exchange rates of both imino and amino protons, representing energetic contributions from both base-pair and helix opening-closing as well as from the exchange process of the imino protons from the open state with the bound water, is close to the E_a of the short B-DNA duplex (E_a ≈ 28–47 kcal/mol).     

Enzymatic characterization of a monomeric isocitrate dehydrogenase from Streptomyces lividans TK54

Isocitrate dehydrogenase (IDH) is one of the key enzymes in the citric acid cycle, which involves in providing energy and biosynthetic precursors for metabolism. Here, we report for the first time the enzymatic characterization of a monomeric NADP⁺-dependent IDH from Streptomyces lividans TK54 (SlIDH). The icd gene (GenBank database accession number EU661252) encoding IDH was cloned and overexpressed in Escherichia coli. The molecular mass of SlIDH was about 80 kDa, typical of a monomeric NADP-IDH, and showed high amino acid sequence identity with known monomeric IDHs. The optimal activity of the 6His-tagged SlIDH was found at pH values 8.5 (Mn²⁺) and 9.0 (Mg²⁺), and the optimal temperature was around 46 °C. Heat-inactivation studies showed that about 50% SlIDH activity was preserved at 38 °C after 20 min of incubation. The recombinant SlIDH displayed a 62,000-fold (k_cat/K_m) preference for NADP⁺ over NAD⁺ with Mn²⁺, and a 85,000-fold greater specificity for NADP⁺ than NAD⁺ with Mg²⁺. Therefore, SlIDH is a divalent cation-dependent monomeric IDH with remarkably high coenzyme preference for NADP⁺.