NAD+-specific D-arabinose dehydrogenase and its contribution to erythroascorbic acid production in Saccharomyces cerevisiae

Erythroascorbic acid (eAsA) is a five-carbon analog of ascorbic acid, and it is synthesized from D-arabinose by D-arabinose dehydrogenase (ARA) and D-arabinono-gamma-lactone oxidase. We found an NAD+-specific ARA activity which is operative under submillimolar level of d-arabinose in the extracts of Saccharomyces cerevisiae. The hypothetical protein encoded by YMR041c showed a significant homology to a l-galactose dehydrogenase which plays in plant ascorbic acid biosynthesis, and we named it as Ara2p. Recombinant Ara2p showed NAD+-specific ARA activity with Km=0.78 mM to d-arabinose, which is 200-fold lower than that for the conventional NADP+-specific ARA, Ara1p. Gene disruptant of ARA2 lost entire NAD+-specific ARA activity and the conspicuous increase in intracellular eAsA by exogenous d-arabinose feeding, while the double knockout mutant of ARA1 and ARA2 still retained measurable amount of eAsA. It demonstrates that Ara2p, not Ara1p, mainly contributes to the production of eAsA from d-arabinose in S. cerevisiae.     

NADP(+)-dependent D-arabinose dehydrogenase shows a limited contribution to erythroascorbic acid biosynthesis and oxidative stress resistance in Saccharomyces cerevisiae

The molecular aspects and physiological significance of NADP(+)-dependent D-arabinose dehydrogenase (ARA), which is thought to function in the biosynthesis of an analog of ascorbic acid, D-erythroascorbic acid in yeasts, were examined. A large subunit of ARA, Ara1p produced in E. coli, was purified as a homodimer, some of which was degraded at the N-terminus. It showed sufficient ARA activity. Degradation of Ara1p occurs naturally in yeast cells, and the small subunit of ARA previously thought as is, in fact, a naturally occuring degradation product of Ara1p. A deficient mutant of ARA1 lost almost all NADP(+)-ARA activity, but intracellular D-erythroascorbic acid was only halved. This mutant showed increased susceptibility to H(2)O(2) and diamide but not to menadione or tert-butylhydroperoxide. Feeding D-arabinose to mutant cells led to increases in intracellular D-erythroascorbic acid, suggesting the presence of another ARA isozyme. The deficient mutant of ARA1 recovered resistance to H(2)O(2) with feeding of D-arabinose. Our results suggest that the direct contributions of Ara1p both to D-erythroascorbic acid biosynthesis and to oxidative stress resistance are quite limited.     

Genetic and biochemical characterization of D-arabinose dehydrogenase from Neurospora crassa.

D-Arabinose dehydrogenase has been purified to homogeneity from wild-type Neurospora crassa 74-A (FGSC 262) and from two colonial mutants, col-15a (FGSC 1391) and col-16a (FGSC 1349), found to contain more of the enzyme. The enzymes were characterized by measurement of several kinetic and physicochemical parameters. The enzymes were the same in all characteristics studied thus far. Immunological studied performed with enzyme preparations from the three strains showed antigenic identity and indicated that those colonial strains contain more normal enzyme, rather than the usual amount of an altered "improved" enzyme. Quantitation of the enzyme in crude extracts, performed by single radial immunodiffusion, showed that the colonial strains have twice the level of enzyme as the wild-type strain. Genetic characterization, performed by analysis of meiotic products, heterokaryosis, and reversions, indicated that the difference in D-arabinose dehydrogenase activity detected among the three strains is probably determined by one gene. The genetic control, structural or regulatory of this enzyme activity is different from that determining the morphological alterations exhibited by mutant strains carrying the col-15 or col-16 gene.     

The Saccharomyces cerevisiae succinate dehydrogenase does not require heme for ubiquinone reduction

The coupling of succinate oxidation to the reduction of ubiquinone by succinate dehydrogenase (SDH) constitutes a pivotal reaction in the aerobic generation of energy. In Saccharomyces cerevisiae, SDH is a tetramer composed of a catalytic dimer comprising a flavoprotein subunit, Sdh1p and an iron-sulfur protein, Sdh2p and a heme b-containing membrane-anchoring dimer comprising the Sdh3p and Sdh4p subunits. In order to investigate the role of heme in SDH catalysis, we constructed an S. cerevisiae strain expressing a mutant enzyme lacking the two heme axial ligands, Sdh3p His-106 and Sdh4p Cys-78. The mutant enzyme was characterized for growth on a non-fermentable carbon source, for enzyme assembly, for succinate-dependent quinone reduction and for its heme b content. Replacement of both Sdh3p His-106 and Sdh4p Cys-78 with alanine residues leads to an undetectable level of cytochrome b(562). Although enzyme assembly is slightly impaired, the apocytochrome SDH retains a significant ability to reduce quinone. The enzyme has a reduced affinity for quinone and its catalytic efficiency is reduced by an order of magnitude. To better understand the effects of the mutations, we employed atomistic molecular dynamic simulations to investigate the enzyme's structure and stability in the absence of heme. Our results strongly suggest that heme is not required for electron transport from succinate to quinone nor is it necessary for assembly of the S. cerevisiae SDH.     

Crystal structure and biochemical properties of the D-arabinose dehydrogenase from Sulfolobus solfataricus

Sulfolobus solfataricus metabolizes the five-carbon sugar d-arabinose to 2-oxoglutarate by an inducible pathway consisting of dehydrogenases and dehydratases. Here we report the crystal structure and biochemical properties of the first enzyme of this pathway: the d-arabinose dehydrogenase. The AraDH structure was solved to a resolution of 1.80 A by single-wavelength anomalous diffraction and phased using the two endogenous zinc ions per subunit. The structure revealed a catalytic and cofactor binding domain, typically present in mesophilic and thermophilic alcohol dehydrogenases. Cofactor modeling showed the presence of a phosphate binding pocket sequence motif (SRS-X2-H), which is likely to be responsible for the enzyme's preference for NADP+. The homo-tetrameric enzyme is specific for d-arabinose, l-fucose, l-galactose and d-ribose, which could be explained by the hydrogen bonding patterns of the C3 and C4 hydroxyl groups observed in substrate docking simulations. The enzyme optimally converts sugars at pH 8.2 and 91 degrees C, and displays a half-life of 42 and 26 min at 85 and 90 degrees C, respectively, indicating that the enzyme is thermostable at physiological operating temperatures of 80 degrees C. The structure represents the first crystal structure of an NADP+-dependent member of the medium-chain dehydrogenase/reductase (MDR) superfamily from Archaea.     

The role of Sdh4p Tyr-89 in ubiquinone reduction by the Saccharomyces cerevisiae succinate dehydrogenase

Succinate dehydrogenase (complex II or succinate:ubiquinone oxidoreductase) is a tetrameric, membrane-bound enzyme that catalyzes the oxidation of succinate and the reduction of ubiquinone in the mitochondrial respiratory chain. Two electrons from succinate are transferred one at a time through a flavin cofactor and a chain of iron-sulfur clusters to reduce ubiquinone to an ubisemiquinone intermediate and to ubiquinol. Residues that form the proximal quinone-binding site (Q(P)) must recognize ubiquinone, stabilize the ubisemiquinone intermediate, and protonate the ubiquinone to ubiquinol, while minimizing the production of reactive oxygen species. We have investigated the role of the yeast Sdh4p Tyr-89, which forms a hydrogen bond with ubiquinone in the Q(P) site. This tyrosine residue is conserved in all succinate:ubiquinone oxidoreductases studied to date. In the human SDH, mutation of this tyrosine to cysteine results in paraganglioma, tumors of the parasympathetic ganglia in the head and neck. We demonstrate that Tyr-89 is essential for ubiquinone reductase activity and that mutation of Tyr-89 to other residues does not increase the production of reactive oxygen species. Our results support a role for Tyr-89 in the protonation of ubiquinone and argue that the generation of reactive oxygen species is not causative of tumor formation.     

A mutation affecting lipoamide dehydrogenase, pyruvate dehydrogenase and 2-oxoglutarate dehydrogenase activities in Saccharomyces cerevisiae

Summary In Saccharomyces cerevisiae a nuclear recessive mutation, lpd1, which simultaneously abolishes the activities of lipoamide dehydrogenase, 2-oxoglutarate dehydrogenase and pyruvate dehydrogenase has been identified. Strains carrying this mutation can grow on glucose or poorly on ethanol, but are unable to grow on media with glycerol or acetate as carbon source. The mutation does not prevent the formation of other tricarboxylic acid cycle enzymes such as fumarase, NAD⁺-linked isocitrate dehydrogenase or succinate-cytochrome c oxidoreductase, but these are produced at about 50%–70% of the wild-type levels. The mutation probably affects the structural gene for lipoamide dehydrogenase since the amount of this enzyme in the cell is subject to a gene dosage effect; heterozygous lpd1 diploids produce half the amount of a homozygous wild-type strain. Moreover, a yeast sequence complementing this mutation when present in the cell on a multicopy plasmid leads to marked overproduction of lipoamide dehydrogenase. Homozygous lpd1 diploids were unable to sporulate indicating that some lipoamide dehydrogenase activity is essential for sporulation to occur on acetate.     

Metabolism of D-arabinose by Escherichia coli B-r

The pathway for d-arabinose metabolism in Escherichia coli B/r has been determined. Evidence is presented to support the following metabolic scheme: d-arabinose d-ribulose right harpoon-up d-ribulose-5-phosphate d-xylulose-5-phosphate.     

Inactivation of Saccharomyces cerevisiae glucose-6-phosphate dehydrogenase by diethylpyrocarbonate

Glucose-6-phosphate dehydrogenase purified from Saccharomyces cerevisiae is rapidly inactivated by diethylpyrocarbonate at pH 6.8 and 30 degrees C with a concomitant increase in absorbance at 242 nm. The second-order rate constant for inactivation was calculated to be 487.8 M-1 min-1. The pH dependence of inactivation suggests the involvement of an amino acid residue having a pKa of 6.77. These results indicate that the inactivation is due to the modification of a histidine residue(s). In the presence of substrate, glucose-6-phosphate or NADP+, the rate of inactivation is decreased, indicating that the essential histidine residue(s) is located at the active site, possibly at the region of overlap of substrates at the binding site.     

The reduction of aromatic alpha-keto acids by cytoplasmic malate dehydrogenase and lactate dehydrogenase

This study demonstrates that cytoplasmic malate dehydrogenase (MDH-s) catalyzes the reduction of aromatic alpha-keto acids in the presence of NADH, that the enzyme which has been described in the literature as aromatic alpha-keto acid reductase (KAR; EC 1.1.1.96) is identical to MDH-s, and that the reduction of aromatic alpha-keto acids is due predominantly to a previously unrecognized secondary activity of MDH-s and the remainder is due to the previously recognized activity of lactate dehydrogenase (LDH) toward aromatic keto-acids. MDH-s and KAR have the same molecular weight, subunit structure, and tissue distribution. Starch gel electrophoresis followed by histochemical staining using either p-hydroxy-phenylpyruvic acid (HPPA) or malate as the substrate shows that KAR activity comigrates with MDH-s in all species studied except some marine species. Inhibition with malate, the end product of the MDH reaction, substantially reduces or totally eliminates KAR activity. Genetically determined electrophoretic variants of MDH-s seen in the fresh water bony fish of the genus Xiphophorus and the amphibian Rana pipiens exhibited identical variation for KAR, and the two traits cosegregated in the offspring from one R. pipiens heterozygote studied. Both enzymes comigrate with no electrophoretic variation among several inbred strains of mice. Antisera raised against purified chicken MDH-s totally inhibited both MDH-s and KAR activity in chicken liver homogenates. There is no evidence to suggest that any protein besides MDH-s and LDH catalyzes this reaction with the possible exception of the situation in Xiphophorus, in which a third independent zone of HPPA reduction is observed. In most species the activity formerly described as KAR appears to be due to a previously unsuspected activity of MDH-s toward aromatic monocarboxylic alpha-keto acids. In all species examined the KAR activity is associated only with MDH-s; in tissue homogenates the mitochondrial form of MDH (MDH-m) is not detected after electrophoresis using HPPA as a substrate.     

Stereoselective reduction of 2-substituted cyclohexanones by Saccharomyces cerevisiae

A comparative study of two modifications of enzymic reduction of ethyl N-{2-{4-[(2-oxo-cyclohexyl)methyl]phe- noxy}ethyl} carbamate (1), an insect juvenile hormone bioanalog, was performed using Saccharomyces cerevisiae in two bioreactors of different size, 250-ml shake-flask and 1-l fermenter. The two major products of this reduction were obtained in 45–49% (w/w) yields but with > 99% enantiomeric purity. Their absolute configurations were assigned as ethyl (1S,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (2a) and ethyl (1R,2S)-N-{2-{4-[(2-hydroxycyclohexyl)methyl]phenoxy}ethyl}carbamate (3a).