Thr373, Asp375, and Lys260 are in the coenzyme site of porcine NADP-dependent isocitrate dehydrogenase

Thr(373), Lys(374), Asp(375), and Lys(260) were chosen as site-directed mutagenesis targets within porcine NADP-dependent isocitrate dehydrogenase based on structurally corrected sequence alignment among prokaryotic and eukaryotic NADP-isocitrate dehydrogenases. Wild-type and all mutant enzymes were expressed in Escherichia coli and purified to homogeneity. These mutations do not alter the secondary structure or dimerization state of the mutants. The D375N and K260Q mutants exhibit, respectively, a 15- and 28-fold increase in K(m) for NADP, along with marked decreases in V(max) as compared to wild-type enzyme. In contrast, replacing Lys(374), which was previously proposed to contribute to apparent coenzyme affinity, does not change the enzyme's kinetic parameters. T373S exhibits similar kinetic parameters to those of wild-type while T373A and T373V mutations reduce the V(max) values of the resulting enzymes to 1 and 20%, respectively of that of wild-type. We conclude that a hydroxyl group at position 373 is required for effective enzyme function and that Asp(375) and Lys(260) are critical amino acids contributing to coenzyme affinity as well as catalysis by porcine NADP-isocitrate dehydrogenase.     

Evaluation by site-directed mutagenesis of aspartic acid residues in the metal site of pig heart NADP-dependent isocitrate dehydrogenase

Pig heart NADP-dependent isocitrate dehydrogenase requires a divalent metal cation for catalysis. On the basis of affinity cleavage studies [Soundar and Colman (1993) J. Biol. Chem. 268, 5267] and analysis of the crystal structure of E. coli NADP-isocitrate dehydrogenase [Hurley et al. (1991) Biochemistry 30, 8671], the residues Asp(253), Asp(273), Asp(275), and Asp(279) were selected as potential ligands of the divalent metal cation in the pig heart enzyme. Using a megaprimer PCR method, the Asp at each of these positions was mutated to Asn. The wild-type and mutant enzymes were expressed in Escherichia coli and purified. D253N has a specific activity, K(m) values for Mn(2+), isocitrate, and NADP, and also a pH-V(max) profile similar to those of the wild-type enzyme. Thus, Asp(253) is not involved in enzyme function. D273N has an increased K(m) for Mn(2+) and isocitrate with a specific activity 5% that of wild type. The D273N mutation also prevents the oxidative metal cleavage seen with Fe(2+) alone in the wild-type enzyme. As compared to wild type, D275N has greatly increased K(m) values for Mn(2+) and isocitrate, with a specific activity <0.1% that of wild type, and a large increase in pK(a) for the enzyme-substrate complex. D279N has only small increases in K(m) for Mn(2+) and isocitrate, but a specific activity <0.1% that of wild type and a major change in the shape of its pH-V(max) profile. These results suggest that Asp(273) and Asp(275) contribute to metal binding, whereas Asp(279), as well as Asp(275), is critical for catalysis. Asp(279) may function as the catalytic base. Using the Modeler program of Insight II, a structure for porcine NADP-isocitrate dehydrogenase was built based on the X-ray coordinates of the E. coli enzyme, allowing visualization of the metal-isocitrate site.     

Alteration of coenzyme specificity in halophilic NAD(P) glucose dehydrogenase by site-directed mutagenesis

Structural analysis of glucose dehydrogenase from Haloferax mediterranei revealed that the adenosine 2′-phosphate of NADP⁺ was stabilized by the side chains of Arg207 and Arg208. To investigate the structural determinants for coenzyme specificity, several mutants involving residues Gly206, Arg207 and Arg208 were engineered and kinetically characterized. The single mutants G206D and R207I were less efficient with NADP⁺ than the wild type, and the double and triple mutants G206D/R207I and G206D/R207I/R208N showed no activity with NADP⁺.In the single mutant G206D, the relation k_cat/K_NAD⁺ was 1.6 times higher than in the wild type, resulting in an enzyme that preferred NAD⁺ over NADP⁺. The single mutation was sufficient to modify coenzyme specificity, whereas other dehydrogenases usually required more than one or two mutations to change coenzyme specificity. However, the highest reaction rates were reached with the double mutant G206D/R207I and with coenzyme NAD⁺, where the k_cat was 1.6 times higher than the k_cat of the wild-type enzyme with NADP⁺. However, catalytic efficiency with NAD⁺ was lower, as the K_m value for coenzyme was 77 times higher than the wild type with NADP⁺.     

Location of the coenzyme binding site in the porcine mitochondrial NADP-dependent isocitrate dehydrogenase

The structure of crystalline porcine mitochondrial NADP-dependent isocitrate dehydrogenase (IDH) has been determined in complex with Mn2+-isocitrate. Based on structural alignment between this porcine enzyme and seven determined crystal structures of complexes of NADP with bacterial IDHs, Arg83, Thr311, and Asn328 were chosen as targets for site-directed mutagenesis of porcine IDH. The circular dichroism spectra of purified wild-type and mutant enzymes are similar. The mutant enzymes exhibit little change in Km for isocitrate or Mn2+, showing that these residues are not involved in substrate binding. In contrast, the Arg83 mutants, Asn328 mutants, and T311A exhibit 3-20-fold increase in the Km(NADP). We propose that Arg83 enhances NADP affinity by hydrogen bonding with the 3'-OH of the nicotinamide ribose, whereas Asn328 hydrogen bonds with N1 of adenine. The pH dependence of Vmax for Arg83 and Asn328 mutants is similar to that of wild-type enzyme, but for all the Thr311 mutants, pK(es) is increased from 5.2 in the wild type to approximately 6.0. We have previously attributed the pH dependence of Vmax to the deprotonation of the metal-bound hydroxyl of isocitrate in the enzyme-substrate complex, prior to the transfer of a hydride from isocitrate to NADP's nicotinamide moiety. Thr311 interacts with the nicotinamide ribose and is the closest of the target amino acids to the nicotinamide ring. Distortion of the nicotinamide by Thr311 mutation will likely be transmitted to Mn2+-isocitrate resulting in an altered pK(es). Because porcine and human mitochondrial NADP-IDH have 95% sequence identity, these results should be applicable to the human enzyme.     

利用定点突变改变树干毕赤酵母(Pichia stipitis)木糖醇脱氢酶辅酶特性的研究

在酿酒酵母中同时表达木糖还原酶基因(xyl1)和木糖醇脱氢酶基因(xyl2)可使酿酒酵母利用木糖发酵生成乙醇.但由于两种酶所依赖的辅酶不同导致酿酒酵母细胞内氧化还原失衡,致使中间产物大量积累,降低了乙醇产率.本研究从树干毕赤酵母中克隆了木糖醇脱氢酶基因,通过与银叶粉虱山梨醇脱氢酶[其活性依赖NADP+(H)]序列进行对...     

Molecular cloning of the cDNA of mouse mitochondrial NADP-dependent isocitrate dehydrogenase and the expression of the gene during lymphocyte activation

The current report documents the molecular cloning of the mouse mitochondrial NADP-dependent isocitrate dehydronegase (mNADP-IDH) cDNA. The cDNA was 1,863 bp in length and contained one open reading frame encoding a 523-residue polypeptide with a predicted molecular weight of 58 kDa. The cDNA and the deduced amino acid (AA) sequence of the mouse mNADP-IDH had a high degree of homology with those of porcine, bovine, alfalfa, and yeast. The recombinant mNADP-IDH expressed in Escherichia coli had active enzymatic function, as well as an expected molecular weight. The heart had the highest constitutive expression of the steady-state mNADP-IDH mRNA, followed by the kidney, while the expression of the gene in other tissues was low. The enzymatic activity of different tissues was in agreement with their mNADP-IDH mRNA levels. The resting lymphocytes had low constitutive expression of the gene, but the steady-state mRNA could be induced 48 h after mitogen stimulation. At the protein level, the resting lymphocytes had low enzymatic activity of mNADP-IDH, but the activity was augmented fivefold after mitogen stimulation. The cytosolic NADP-IDH, on the contrary, remained low or undetectable before and after the mitogen stimulation. Based on our current findings as well as the known roles of the mNADP-IDH in anabolism and in the isocitrate shuttle, it is conceivable that the mNADP-IDH is necessary for optimizing proliferation in lymphocytes. © 1996 Wiley-Liss, Inc.     

Eubacterial isocitrate dehydrogenase with dual specificity for NAD and NADP from Rhodomicrobium vannielii

Abstract Cell-free extracts of the photosynthetic eubacterium Rhodomicrobium vannielii contained both NADP and NAD-linked isocitrate dehydrogenase activities. Apparent K _m values of 12 μM for NADP, 0.75 mM for NAD, 9.3 μM for isocitrate (NADP utilising) and 8.2 μM for isocitrate (NAD utilising) were determined in such extracts. Four lines of evidence indicated that one enzyme was responsible for the two activities; (i) non-additivity of reaction rates in the presence of both NADP and NAD (ii) the presence of one band which stained for activity with both cofactors on non-denaturing polyacrylamide gels (iii) identical heat inactivation kinetics for both activities (iv) co-elution of both activities after ion-exchange and hydrophobic interaction chromatography. This is the first report of a eubacterial isocitrate dehydrogenase with dual cofactor specificity.     

NADP-dependent isocitrate dehydrogenase from the halophilic archaeon Haloferax volcanii: cloning,sequence determination and overexpression in Escherichia coli

A gene encoding NADP-dependent Ds-threo-isocitrate dehydrogenase was isolated from Haloferax volcanii genomic DNA by using a combination of polymerase chain reaction and screening of a lambda EMBL3 library. Analysis of the nucleotide sequence revealed an open reading frame of 1260 bp encoding a protein of 419 amino acids with 45837 Da molecular mass. This sequence is highly similar to previously sequenced isocitrate dehydrogenases. In the alignment of the amino acid sequences with those from several archaeal and mesophilic NADP-dependent isocitrate dehydrogenases, the residues involved in dinucleotide binding and isocitrate binding are well conserved. We have developed methods for the expression in Escherichia coli and purification of the enzyme from H. volcanii. This expression was carried out in E. coli as inclusion bodies using the cytoplasmic expression vector pET3a. The enzyme was refolded by solubilisation in 8 M urea followed by dilution into a buffer containing EDTA, MgCl(2) and 3 M NaCl. Maximal activity was obtained after several hours incubation at room temperature.     

Evaluation by mutagenesis of the roles of His309, His315, and His319 in the coenzyme site of pig heart NADP-dependent isocitrate dehydrogenase

Sequence alignment predicts that His(309) of pig heart NADP-dependent isocitrate dehydrogenase is equivalent to His(339) of the Escherichia coli enzyme, which interacts with the coenzyme in the crystal structure [Hurley et al. (1991) Biochemistry 30, 8671-8688], and porcine His(315) and His(319) are close to that site. The mutant porcine enzymes H309Q, H309F, H315Q, and H319Q were prepared by site-directed mutagenesis, expressed in E. coli, and purified. The H319Q mutant has K(m) values for NADP, isocitrate, and Mn(2+) similar to those of wild-type enzyme, and V(max) = 20.1, as compared to 37.8 micromol of NADPH min(-1) (mg of protein)(-1) for wild type. Thus, His(319) is not involved in coenzyme binding and has a minimal effect on catalysis. In contrast, H315Q exhibits a K(m) for NADP 40 times that of wild type and V(max) = 16.2 units/mg of protein, with K(m) values for isocitrate and Mn(2+) similar to those of wild type. These results implicate His(315) in the region of the NADP site. Replacement of His(309) by Q or F yields enzyme with no detectable activity. The His(309) mutants bind NADPH poorly, under conditions in which wild type and H319Q bind 1.0 mol of NADPH/mol of subunit, indicating that His(309) is important for the binding of coenzyme. The His(309) mutants bind isocitrate stoichiometrically, as do wild-type and the other mutant enzymes. However, as distinguished from the wild-type enzyme, the His(309) mutants are not oxidatively cleaved by metal isocitrate, implying that the metal ion is not bound normally. Since circular dichroism spectra are similar for wild type, H315Q, and H319Q, these amino acid substitutions do not cause major conformational changes. In contrast, replacement of His(309) results in detectable change in the enzyme's CD spectrum and therefore in its secondary structure. We propose that His(309) plays a significant role in the binding of coenzyme, contributes to the proper coordination of divalent metal ion in the presence of isocitrate, and maintains the normal conformation of the enzyme.     

Regulation of high glucose-induced apoptosis by mitochondrial NADP+-dependent isocitrate dehydrogenase

A high concentration of glucose has been implicated as a causal factor in initiation and progression of diabetic kidney complications, and there is evidence to suggest that hyperglycemia increases the production of free radicals and oxidant stress. Recently, we demonstrated that the control of mitochondrial redox balance and the cellular defense against oxidative damage is one of the primary functions of mitochondrial NADP(+)-dependent isocitrate dehydrogenase (IDPm) to supply NADPH for antioxidant systems. In this report, we demonstrate that modulation of IDPm activity in HEK293 cells, an embryonic kidney cell line, regulates high glucose-induced apoptosis. When we examined the protective role of IDPm against high glucose-induced apoptosis with HEK293 cells transfected with the cDNA for mouse IDPm in sense and antisense orientations, a clear inverse relationship was observed between the amount of IDPm expressed in target cells and their susceptibility to apoptosis. The results suggest that IDPm plays an important protective role in apoptosis of HEK293 cells induced by a high concentration of glucose and may contribute to various pathologies associated with the long-term complications of diabetes.     

Regulation of NAD- and NADP-linked isocitrate dehydrogenase by hydrocortisone in the brain and liver of male rats of various ages

The activity and hormonal regulation of NAD- and NADP-linked isocitrate dehydrogenase (EC 1.1.1.41 and 1.1.1.42, respectively) in the brain and liver of rats of various ages were investigated. The activity of NAD-linked isocitrate dehydrogenase of the brain was greater than cytoplasmic or mitochondrial NADP-linked isocitrate dehydrogenase. In contrast, the cytoplasmic NADP-isocitrate dehydrogenase of the liver predominates over both NAD- and mitochondrial NADP-isocitrate dehydrogenases at the three ages studied. The activity of NAD-isocitrate dehydrogenase increased in the brain (139%) and liver (17%) of rats upt o 33 weeks of age and decreased (57 and 39%, respectively) in old rats (85-week-old). The activity of cytoplasmic NADP-isocitrate dehydrogenase was maximum in immature (6-week-old) rat brain and decreased as the age of the rats increased; whereas, in liver, the activity of this enzyme was found to be maximum in adult rats (33-week-old). Brain mitochondrial NADP-isocitrate dehydrogenase activity increased (64%) in adult rats, but in liver it decreased (45 and 33% in 33- and 85-week-old rats, respectively). In both tissues, adrenalectomy and hydrocortisone treatment showed differential age-dependent response. Hydrocortisone-mediated induction of the level of enzymes was inhibited by actinomycin D.     

Structural studies of Saccharomyces cerevesiae mitochondrial NADP-dependent isocitrate dehydrogenase in different enzymatic states reveal substantial conformational changes during the catalytic reaction

Isocitrate dehydrogenases (IDHs) catalyze oxidative decarboxylation of isocitrate (ICT) into alpha-ketoglutarate (AKG). We report here the crystal structures of Saccharomyces cerevesiae mitochondrial NADP-IDH Idp1p in binary complexes with coenzyme NADP, or substrate ICT, or product AKG, and in a quaternary complex with NADPH, AKG, and Ca(2+), which represent different enzymatic states during the catalytic reaction. Analyses of these structures identify key residues involved in the binding of these ligands. Comparisons among these structures and with the previously reported structures of other NADP-IDHs reveal that eukaryotic NADP-IDHs undergo substantial conformational changes during the catalytic reaction. Binding or release of the ligands can cause significant conformational changes of the structural elements composing the active site, leading to rotation of the large domain relative to the small and clasp domains along two hinge regions (residues 118-124 and residues 284-287) while maintaining the integrity of its secondary structural elements, and thus, formation of at least three distinct overall conformations. Specifically, the enzyme adopts an open conformation when bound to NADP, a quasi-closed conformation when bound to ICT or AKG, and a fully closed conformation when bound to NADP, ICT, and Ca(2+) in the pseudo-Michaelis complex or with NADPH, AKG, and Ca(2+) in the product state. The conformational changes of eukaryotic NADP-IDHs are quite different from those of Escherichia coli NADP-IDH, for which significant conformational changes are observed only between two forms of the apo enzyme, suggesting that the catalytic mechanism of eukaryotic NADP-IDHs is more complex than that of EcIDH, and involves more fine-tuned conformational changes.     

Antisense inhibition of cytosolic NADP-dependent isocitrate dehydrogenase in transgenic potato plants

Cytosolic NADP-dependent isocitrate dehydrogenase (cyt-NADP-ICDH; EC 1.1.1.42) has been suggested to play a major role in the production of 2-oxoglutarate, an important precursor for amino acid synthesis. Using an antisense RNA approach under the control of the cauliflower mosaic virus 35S promoter, transgenic potato plants were created in which NADP-ICDH activity was reduced to 8% of the wild-type level in leaves. Residual activity was almost completely due to mitochondrial and chloroplastic NADP-ICDH isoforms. Activity staining after non-denaturing polyacrylamide gel electrophoresis revealed the complete absence of a major activity band in leaves of antisense plants. No differences in growth or development, including flower formation and tuber yield, were observed between transgenic and wild-type plants. Photosynthesis and respiration were also unchanged. Levels of amino acids were the same in wild-type and cyt-NADP-ICDH antisense plants, even when accumulation of amino acids was induced by incubation of detached leaves in tap water in the dark (`induced senescence'). Consistent with a reduction in NADP-ICDH activity, however, were slight increases in the levels of isocitrate (up to 2.5-fold) and citrate (up to 2-fold). 2-Oxoglutarate was not reduced. Our data indicate that potato plants can cope with a severe reduction in cyt-NADP-ICDH activity without major shifts in growth and metabolism. Received: 28 July 1997 / Accepted: 3 November 1997     

A comparison of the phosphorylated and unphosphorylated forms of isocitrate dehydrogenase from Escherichia coli ML308

NADP+ can protect active isocitrate dehydrogenase against attack by several proteases. Inactive phosphorylated isocitrate dehydrogenase is much less susceptible to proteolysis than the active enzyme, and it is not protected by NADP+. The results suggest that binding of NADP+ to, or phosphorylation of, active isocitrate dehydrogenase induces similar conformational states. Fluorescence titration experiments show that NADPH can bind to active but not to inactive isocitrate dehydrogenase. It is suggested that the phosphorylation of isocitrate dehydrogenase may occur close to its coenzyme binding site.     

Structural and Functional analysis of Staphylococcus aureus NADP-dependent IDH and its comparison with Bacterial and Human NADPdependent IDH

Staphylococcus aureus a natural inhabitant of nasopharyngeal tract mainly survives as biofilms and possess complete Krebs cycle which plays major role in its pathogenesis. This TCA cycle is regulated by Isocitrate dehydrogenase (IDH) we have earlier cloned, sequenced ({"type":"entrez-nucleotide","attrs":{"text":"HM067707","term_id":"297186241","term_text":"HM067707"}}HM067707), expressed and characterized this enzyme from S. aureus ATCC12600. We have observed only one type of IDH in all the strains of S. aureus which dictates the flow of carbon thereby controlling the virulence and biofilm formation, this phenomenon is variable among bacteria. Therefore in the present study comparative structural and functional analysis of IDH was undertaken. As the crystal structure of S. aureus IDH was not available therefore using the deduced amino sequence of complete gene the 3D structure of IDH was built in Modeller 9v8. The PROCHECK and ProSAweb analysis showed the built structure was close to the crystal structure of Bacillus subtilis. This structure when superimposed with other bacterial IDH structures exhibited extensive structural variations as evidenced from the RMSD values correlating with extensive sequential variations. Only 24% sequence identity was observed with both human NADP dependent IDHs (PDB: 1T09 and 1T0L) and the structural comparative studies indicated extensive structural variations with an RMSD values of 14.284Å and 10.073Å respectively. Docking of isocitrate to both human IDHs and S. aureus IDH structures showed docking scores of -11.6169 and -10.973 respectively clearly indicating higher binding affinity of isocitrate to human IDH.     

Identification by mutagenesis of arginines in the substrate binding site of the porcine NADP-dependent isocitrate dehydrogenase

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase is the most extensively studied among the mammalian isocitrate dehydrogenases. The crystal structure of Escherichia coli isocitrate dehydrogenase and sequence alignment of porcine with E. coli isocitrate dehydrogenase suggests that the porcine Arg(101), Arg(110), Arg(120), and Arg(133) are candidates for roles in substrate binding. The four arginines were separately mutated to glutamine using a polymerase chain reaction method. Wild type and mutant enzymes were each expressed in E. coli, isolated as maltose binding fusion proteins, then cleaved with thrombin, and purified to yield homogeneous porcine isocitrate dehydrogenase. The R120Q mutant has a specific activity, as well as K(m) values for isocitrate, Mn(2+), and NADP(+) similar to wild type enzyme, indicating that Arg(120) is not needed for function. The specific activities of R101Q, R110Q, and R133Q are 1.73, 1.30, and 19.7 micromols/min/mg, respectively, as compared with 39.6 units/mg for wild type enzyme. The R110Q and R133Q enzymes exhibit K(m) values for isocitrate that are increased more than 400- and 165-fold, respectively, as compared with wild type. The K(m) values for Mn(2+), but not for NADP(+), are also elevated indicating that binding of the metal-isocitrate complex is impaired in these mutants. It is proposed that the positive charges of Arg(110) and Arg(133) normally strengthen the binding of the negatively charged isocitrate by electrostatic attraction. The R101Q mutant shows smaller, but significant increases in the K(m) values for isocitrate and Mn(2+); however, the marked decrease in k(cat) suggests a role for Arg(101) in catalysis. The V(max) of wild type enzyme depends on the ionized form of an enzymic group of pK 5.5, and this pK(aes) is similar for the R101Q and R120Q enzymes. In contrast, the pK(aes) for R110Q and R133Q enzymes increases to 6.4 and 7.4, respectively, indicating that the positive charges of Arg(110) and Arg(133) normally lower the pK of the nearby catalytic base to facilitate its ionization. These results may be understood in terms of the structure of the porcine NADP-specific isocitrate dehydrogenase generated by the Insight II Modeler Program, based on the x-ray coordinates of the E. coli enzyme.     

Regulation of ethanol-induced toxicity by mitochondrial NADP-dependent isocitrate dehydrogenase

Chronic alcohol administration has been known to increase peroxynitrite hepatotoxicity by enhancing concomitant production of nitric oxide and superoxide. We previously reported that control of the mitochondrial redox balance and the cellular defense against oxidative damage are primary functions of mitochondrial NADP⁺-dependent isocitrate dehydrogenase (IDPm) through to supply NADPH for antioxidant systems. In the present study, we demonstrate that modulation of IDPm expression in HepG2 cells regulates ethanol-induced toxicity. We observed the significantly enhanced protection to cell killing, lipid peroxidation, protein oxidation, oxidative DNA damage, and decrease in generation of intracellular reactive oxygen species and reactive nitrogen species in IDPm-overexpressed cells compared to control cells upon exposure to ethanol. In contrast, transfection of HepG2 cells with IDPm short interfering RNA markedly decreased the expression of IDPm, modulating cellular redox status and subsequently enhancing the susceptibility of ethanol-induced toxicity. These studies support the hypothesis that IDPm plays an important role in regulating the toxicity induced by ethanol presumably through maintaining the cellular redox status.     

Intracellular distribution of NADP-linked isocitrate dehydrogenase,fumarase and citrate synthase in bovine heart muscle

By fractional extraction of minced bovine heart muscle with iso-osmotic sucrose and phosphate buffer solutions, it is shown that less than 4% of the total citrate synthase in the tissue is in the cytosol. Using citrate synthase as a marker for broken mitochondria, two methods of fractionation of 750 × g supernatants from homogenates of bovine heart muscle show that 10% of the total fumarase and NADP-linked isocitrate dehydrogenase activities are present in the cytoplasm. Homogenates prepared by sonication and osmotic shock and by sand-grinding gave closely similar results as regards enzyme distributions and extent of mitochondrial breakage. The results are compared with those reported for other tissues.     

Valine dehydrogenase from Streptomyces albus: gene cloning, heterologous expression and identification of active site by site-directed mutagenesis

A gene encoding valine dehydrogenase (Vdh) has been cloned from Streptomyces albus, a salinomycin producer, and expressed in Escherichia coli. The S. albus Vdh is composed of 364 amino acids that showed high homology with several other amino acid dehydrogenases as well as Vdhs from Streptomyces spp. and leucine and phenylalanine dehydrogenases (Ldh and Pdh) from Bacillus spp. A protein of 38 kDa, corresponding to the approximate mass of the predicted S. albus Vdh product (38.4 kDa) exhibiting specific Vdh activity, was observed when the S. albus vdh gene was overexpressed in E. coli under the controlled T7 promoter and was subsequently purified to homogeneity. Among branched- and straight-chain amino acids, L-valine and L-alpha-aminobutyrate were the preferred substrates for the enzyme. Lys-79 and Lys-91 of S. albus Vdh were highly conserved in the corresponding region of NAD(P)(+)-dependent amino acid dehydrogenase sequences. To elucidate the functional roles of the lysyl residues, the Lys residues have individually been replaced with Ala by site-directed mutagenesis. Kinetic analyses of the Lys-79 and Lys-91-mutated enzymes revealed that they are involved in the substrate binding site and catalysis, respectively, analogous to the corresponding residues in the homologous Ldh and Pdh.     

Peroxisomal localization and function of NADP-specific isocitrate dehydrogenases in yeast

Yeast peroxisomal NADP⁺-specific isocitrate dehydrogenase (IDP3) contains a canonical type I peroxisomal targeting sequence (a carboxyl-terminal Cys-Lys-Leu tripeptide), and provides the NADPH required for β-oxidation of some fatty acids in that organelle. Cytosolic yeast IDP2 carrying a PTS1 (IDP2^+CKL) was only partially localized to peroxisomes, and the enzyme was able to function in lieu of either peroxisomal IDP3 or cytosolic IDP2. The analogous isocitrate dehydrogenase enzyme (IDPA) from Aspergillus nidulans, irrespective of the presence or absence of a putative PTS1, was found to exhibit patterns of dual compartmental distribution and of dual function in yeast similar to those observed for IDP2^+CKL. To test a potential cellular limit on peroxisomal levels, authentic yeast IDP3, which is normally strictly peroxisomal, was over-expressed. This also resulted in dual distribution and function of the enzyme in both the cytosol and in peroxisomes, supporting the possibility of a restriction on organellar amounts of IDP.