Glycerol is metabolized in a complex and strain-dependent manner in Enterococcus faecalis.

Enterococcus faecalis is equipped with two pathways of glycerol dissimilation. Glycerol can either first be phosphorylated by glycerol kinase and then oxidized by glycerol-3-phosphate oxidase (the glpK pathway) or first be oxidized by glycerol dehydrogenase and then phosphorylated by dihydroxyacetone kinase (the dhaK pathway). Both pathways lead to the formation of dihydroxyacetone phosphate, an intermediate of glycolysis. It was assumed that the glpK pathway operates during aerobiosis and that the dhaK pathway operates under anaerobic conditions. Because this had not been analyzed by a genetic study, we constructed mutants of strain JH2-2 affected in both pathways. The growth of these mutants on glycerol under aerobic and anaerobic conditions was monitored. In contrast to the former model, results strongly suggest that glycerol is catabolized simultaneously by both pathways in the E. faecalis JH2-2 strain in the presence of oxygen. In accordance with the former model, glycerol is metabolized by the dhaK pathway under anaerobic conditions. Comparison of different E. faecalis isolates revealed an impressive diversity of growth behaviors on glycerol. Analysis by BLAST searching and real-time reverse transcriptase PCR revealed that this diversity is based not on different gene contents but rather on differences in gene expression. Some strains used preferentially the glpK pathway whereas others probably exclusively the dhaK pathway under aerobic conditions. Our results demonstrate that the species E. faecalis cannot be represented by only one model of aerobic glycerol catabolism.

Structure/function analysis of a critical disulfide bond in the active site of L-xylulose reductase.

L-xylulose reductase (XR) is involved in water re-absorption and cellular osmoregulation. The crystal structure of human XR complemented with site-directed mutagenesis (Cys138Ala) indicated that the disulfide bond in the active site between Cys138 and Cys150 is unstable and may affect the reactivity of the enzyme. The effects of reducing agents on the activities of the wild-type and mutant enzymes indicated the reversibility of disulfide-bond formation, which resulted in three-fold decrease in catalytic efficiency. Furthermore, the addition of cysteine (>2 mM) inactivated human XR and was accompanied by a 10-fold decrease in catalytic efficiency. TOF-MS analysis of the inactivated enzyme showed the S-cysteinylation of Cys138 in the wild-type and Cys150 in the mutant enzymes. Thus, the action of human XR may be regulated by cellular redox conditions through reversible disulfide-bond formation and by S-cysteinylation.

Engineering of Saccharomyces cerevisiae for the production of dihydroxyacetone (DHA) from sugars: a proof of concept.

Dihydroxyacetone (DHA) has numerous industrial applications. In this work, we pursue the idea to produce DHA from sugars in the yeast Saccharomyces cerevisiae, via glycerol as an intermediate. Firstly, three glycerol dehydrogenase (GDH) genes from different microbial sources were expressed in yeast. Among them, the NAD(+)-dependent GDH of Hansenula polymorpha showed the highest glycerol-oxidizing activity. DHA concentration in shake-flask experiments was roughly 100mg/lDHA from 20g/l glucose, i.e. five times the wild-type level. This level was achieved only when cultures were subjected to osmotic stress, known to enhance glycerol production and accumulation in S. cerevisiae. Secondly, DHA kinase activity was abolished to prevent conversion of DHA to dihydroxyacetone phosphate (DHAP). The dak1Deltadak2Delta double-deletion mutant overexpressing H. polymorpha gdh produced 700mg/l DHA under the same conditions. Although current DHA yield and titer still need to be optimized, our approach provides the proof of concept for producing DHA from sugars in yeast.

Aldose reductase, diabetes, and thickening of the retinal inner limiting membrane.

Capillary basement membrane thickening is typical of the diabetic retina, and aldose reductase appears to be involved since a diabetic-like thickening can be induced by galactose feeding and prevented with aldose reductase inhibitors. Because aldose reductase is present in the Mueller's cells, studies were undertaken to determine if thickening of the retinal inner limiting membrane, which is the basement membrane of these cells, can be induced by long-term galactose feeding and be prevented with an aldose reductase inhibitor. Weanling male, Sprague-Dawley rats were given a 50% galactose diet with or without an aldose reductase inhibitor (0.04% tolrestat, Ayerst). Quantitative computer planimetry on electron micrographs demonstrated a significant galactose-induced thickening of the inner limiting membrane which was prevented by the aldose reductase inhibitor. The results were consistent with the notion that basement membrane thickening is involved in diabetic retinopathy and can be delayed or prevented with aldose reductase inhibitors.

Implications of aldose reductase in cataracts in human diabetes.

Cataracts removed intracapsularly by cryoprobe technique from human diabetics were analyzed for sugars and polyols by gas liquid chromatography. The contents of sorbitol and fructose of lenses followed blood glucose levels at least up to 250 mg/dl. Studies indicate that human lens is capable of synthesizing substantial amounts of polyol pathway metabolites given exposure to high glucose levels such as are prevalent in diabetes. The synthesis of sorbitol was found to be susceptible to quercitrin, an inhibitor of aldose reductase. The implications of these findings in the formation of cataracts in diabetic individuals have been discussed.

Aldose reductase, glomerular metabolism, and diabetic nephropathy.

To explore a possible link between diabetic nephropathy and the enhanced activity of the polyol pathway known to occur in diabetes, we examined several pertinent metabolic parameters in glomeruli isolated from control and streptozotocin-diabetic rats and assessed whether changes observed in diabetic glomeruli could be prevented by the oral administration of the aldose reductase inhibitor sorbinil. We found that glomerular polyol content is significantly increased in diabetes, whereas glomerular myo-inositol content is significantly reduced. The sorbitol accumulation and myo-inositol depletion were both completely prevented by sorbinil, which was given throughout the duration of diabetes. Activity of the membrane-bound sodium/potassium adenosine triphosphatase (Na-K-ATPase) was decreased in diabetic samples; this change was also completely prevented by sorbinil. Erythrocyte deformability is another factor that has been implicated in the pathogenesis of microangiopathic complications. The ability of red blood cells to undergo an adaptation in shape that permits passage through the smallest vessels is impaired in diabetes. Using blood from control, diabetic, and sorbinil-treated diabetic rats, we found that erythrocyte deformability was decreased in diabetic samples and that sorbinil treatment significantly improved this parameter. Thus, if the glomerular consequences of sorbitol accumulation, myo-inositol depletion, reduced Na-K-ATPase activity, and decreased erythrocyte deformability are pathogenetically implicated in diabetic nephropathy, the ability of sorbinil to impact on these changes suggests that it could favorably impact on the nephropathic process.

Studies of aldose reductase using neuronal cell culture and ligated rat sciatic nerve.

Sorbinil (CP 45,634), a potent aldose reductase (AR) inhibitor, has the ability to normalize both sorbitol levels and functional parameters such as orthograde axonal transport and motor nerve conduction velocity in peripheral nerves of diabetic rats, which implicates flux through the polyol pathway in the pathophysiology of diabetic neuropathy. In order to understand more fully the role of this enzyme, it is important to determine the major cellular location of AR in peripheral nerve. Experiments were designed that have taken advantage of the observation that peripheral nerve axons degenerate distal to an injury site, while Schwann cells remain viable. One sciatic nerve in each experimental rat was chronically ligated (up to 6 weeks) before inducing diabetes by an intravenous (iv) injection of streptozotocin (STZ; 65 mg/kg). Two days after the STZ injection, both sciatic nerves were removed from each animal, and the ligated nerve was divided into proximal (Schwann cells and axons) and distal (Schwann cells only) portions before being processed for sorbitol determinations. The intact nerves and the proximal portion of the ligated nerves (having both Schwann cells and axons) retained the ability to accumulate sorbitol after STZ injection, while the distal portion (having Schwann cells only) lost this capacity 4 days after ligation. This lack of ability to accumulate sorbitol was not due to failure of the substrate (glucose) to reach the distal nerve segment. Additionally, homogenates of whole nerves and of proximal portions of ligated nerves were able to form sorbitol from glucose in the presence of NADPH while homogenates of distal portions of ligated nerves had lost approximately 85% of this activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired nerve regeneration in streptozotocin-diabetic rats. Effects of treatment with an aldose reductase inhibitor.

Rats with streptozotocin-induced diabetes have a decreased rate of sciatic nerve regeneration. We studied the effects on this defect of treatment with the aldose reductase inhibitor, ponalrestat (25 mg/kg per day via an endogastric tube). The nerves of diabetic rats were crush-injured at 5 weeks of diabetes and regeneration evaluated 7 days later with the pinch-reflex test. Ponalrestat treatment was started at day 3 after streptozotocin injection and was continued for the whole experimental period, i.e. until 6 weeks of diabetes. The treatment prevented effectively the accumulation of sorbitol and fructose in the nerves of diabetic rats, but was without effect on the sciatic nerve regeneration (controls 21.8 +/- 1.2 mm/7 days (mean +/- SEM, n = 6), untreated diabetics 15.8 +/- 1.8 (n = 7), ponalrestat-treated diabetics 16.2 +/- 1.0 (n = 10]. The results indicate that there is no connection between increased sorbitol pathway flux and impaired regeneration in streptozotocin diabetic rats.

Metabolic engineering of mannitol production in Lactococcus lactis: influence of overexpression of mannitol 1-phosphate dehydrogenase in different genetic backgrounds.

To obtain a mannitol-producing Lactococcus lactis strain, the mannitol 1-phosphate dehydrogenase gene (mtlD) from Lactobacillus plantarum was overexpressed in a wild-type strain, a lactate dehydrogenase(LDH)-deficient strain, and a strain with reduced phosphofructokinase activity. High-performance liquid chromatography and (13)C nuclear magnetic resonance analysis revealed that small amounts (<1%) of mannitol were formed by growing cells of mtlD-overexpressing LDH-deficient and phosphofructokinase-reduced strains, whereas resting cells of the LDH-deficient transformant converted 25% of glucose into mannitol. Moreover, the formed mannitol was not reutilized upon glucose depletion. Of the metabolic-engineering strategies investigated in this work, mtlD-overexpressing LDH-deficient L. lactis seemed to be the most promising strain for mannitol production.

The contributions of Jin H. Kinoshita to aldose reductase research.

Sorbitol is a sugar alcohol formed from the aldose reductase catalyzed reduction of glucose. Mounting experimental evidence links the abnormal intracellular accumulation of sorbitol to the onset and severity of diabetes-associated pathology which results in a variety of tissue and/or functional changes in the cornea, lens, retina, iris, peripheral nerves, and kidney. Animal studies indicate that aldose reductase inhibitors, by inhibiting the formation of sorbitol in target tissues affected by diabetes, can either prevent or significantly delay the onset of many of these diabetes-associated changes. The pioneering studies of Dr Jin Kinoshita have been instrumental in defining the pathophysiological role of aldose reductase and excess sorbitol production under diabetic conditions. These studies provide a firm scientific groundwork for investigating the premise that inhibition of sorbitol formation is a new, pharmacologically direct treatment for diabetic complications that is independent of the control of blood sugar levels.

The effect of oxidants on biomembranes and cellular metabolism.

During the reductive process in the tissues, the aerobes generate a number of oxidants. Unless these oxidants are reduced, oxidative damage and cell death would occur. Oxidation of plasma membrane lipids leads to autocatalytic chain reactions which eventually alter the permeability of the cell. The role of oxidative damage in the pathophysiology of diabetic complications and ischemic reperfusion injury of myocardium, especially the changes in the channel activity which may lead to arrhythmia have been studied. Hyperglycemia activates aldose reductase which could efficiently reduce glucose to sorbitol in the presence of NADPH. Since NADPH is also aldose required by glutathione reductase for reducing oxidants, its diversion would lead to membrane lipid oxidation and permeability changes which are probably responsible for diabetic complications such as cataractogenesis, retinopathy, neuropathy etc. Antioxidants such as butylated hydroxy toluene (BHT) and also reductase inhibitors prevent or delay some of these complications. By using patch-clamp technique in isolated frog myocytes, we have shown that hydroxy radicals generated by ferrous sulfate and ascorbate as well as lipid peroxides such as t-butyl hydroperoxide facilitate the entry of Na+ by oxidizing Na+-channels. Increased intracellular Na+ leads to an increase in Na+/Ca2+ exchange. The increased Na+ concentration by itself may produce electrical disturbance which would result in arrhythmia. Increased Ca2+ may affect proteases and may help in the conversion of xanthine dehydrogenase to xanthine oxidase, consequently increased production of super oxide radicals. Increased membrane lipid peroxidation and other oxygen free-radical associated membrane damage in myocytes has been demonstrated.

The effect of iron on metabolism of vitamin C in chickens.

The objective of the paper was to show the effect of supplementation of iron on vitamin C synthesis in chickens. Iron administered per as caused than increase of vitamin C synthesis in chickens. The increase of vitamin C synthesis following iron supplementation is due to the alteration of L-gulono-gamma-oxidase activity in tissues; the enzyme taking part in last phase of vitamin C formation, and due to increase of D-glucuronic acid level, an intermediate product during vitamin C synthesis. High doses of iron cause the increase of vitamin C metabolism in chickens.

The missing link in the fungal L-arabinose catabolic pathway, identification of the L-xylulose reductase gene.

The fungal L-arabinose pathway consists of five enzymes, aldose reductase, L-arabinitol 4-dehydrogenase, L-xylulose reductase, xylitol dehydrogenase, and xylulokinase. All the genes encoding the enzymes of this pathway are known except for that of L-xylulose reductase (EC 1.1.1.10). We identified a gene encoding this enzyme from the filamentous fungus Trichoderma reesei (Hypocrea jecorina). The gene was named lxr1. It was overexpressed in the yeast Saccharomyces cerevisiae, and the enzyme activity was confirmed in a yeast cell extract. Overexpression of all enzymes of the L-arabinose pathway in S. cerevisiae led to growth of S. cerevisiae on L-arabinose; i.e., we could show that the pathway is active in a heterologous host. The lxr1 gene encoded a protein with 266 amino acids and a calculated molecular mass of 28 428 Da. The LXRI protein is an NADPH-specific reductase. It has activity with L-xylulose, D-xylulose, D-fructose, and L-sorbose. The highest affinity is toward L-xylulose (K(m) = 16 mM). In the reverse direction, we found activity with xylitol, D-arabinitol, D-mannitol, and D-sorbitol. It requires a bivalent cation for activity. It belongs to the protein family of short chain dehydrogenases. The enzyme is catalytically similar and homologous in sequence to a D-mannitol:NADP 2-dehydrogenase (EC 1.1.1.138).

D-Erythroascorbic acid is an important antioxidant molecule in Saccharomyces cerevisiae.

D-Arabinono-1,4-lactone oxidase catalysing the final step of D-erythroascorbic acid biosynthesis was purified from the mitochondrial fraction of Saccharomyces cerevisiae. Based on the amino acid sequence analysis of the enzyme, an unknown open reading frame (ORF), YML086C, was identified as the ALO1 gene encoding the enzyme. The ORF of ALO1 encoded a polypeptide consisting of 526 amino acids with a calculated molecular mass of 59493Da. The deduced amino acid sequence of the enzyme shared 32% and 21% identity with that of L-gulono-1,4-lactone oxidase from rat and L-galactono-1,4-lactone dehydrogenase from cauliflower, respectively, and contained a putative transmembrane segment and a covalent FAD binding site. Blot hybridization analyses showed that a single copy of the gene was present in the yeast genome and that mRNA of the ALO1 gene was 1.8kb in size. In the alo1 mutants, D-erythroascorbic acid and the activity of D-arabinono-1,4-lactone oxidase could not be detected. The intracellular concentration of D-erythroascorbic acid and the enzyme activity increased up to 6.9-fold and 7.3-fold, respectively, in the transformant cells carrying ALO1 in multicopy plasmid. The alo1 mutants showed increased sensitivity towards oxidative stress, but overexpression of ALO1 made the cells more resistant to oxidative stress.

Gene transfer for targeted modification of salmonid fish metabolism.

The reviewed studies addressed the possibility of using gene transfer for correction of L-ascorbic acid biosynthesis and carbohydrate utilization in rainbow trout. Analyses of enzymatic activities in the L-AAB pathway indicated that reasons for the lack of L-AA production can be common in fish and scurvy-prone animals. Rat gulonolactone oxidase cDNA was transferred into trout. Regardless of the fact that rGLO transcription occurred in embryos, neither GLO protein, nor enzyme activity were detected. There was no production of L-AA in transgenic fish raised on vitamin C-free diets or injected with L-gulonolactone. These results indicated that the conditions required for translation or stability of rGLO were not present in trout tissues. To augment carbohydrates utilization, human glucose transporter 1 and rat hexokinase II cDNAs were tested. In the transfected embryos. HK activity, rates of hexose uptake and glucose oxidation were increased. The effect of hGLUT1 on glucose metabolism was greater than that of rHKII. Trout carrying hGLUT1 and rHKII with viral or piscine promoters were created. Though interpretation of the metabolic effects of the transgenes was complicated with mosaicism, a tendency to improved carbohydrate utilization was revealed in some of the transgenic individuals.