Gluconic acid production by gad mutant of Klebsiella pneumoniae.

Klebsiella pneumoniae produces many economically important chemicals. Using glucose as a carbon source, the main metabolic product in K. pneumoniae is 2,3-butanediol. Gluconic acid is an intermediate of the glucose oxidation pathway. In the current study, a metabolic engineering strategy was used to develop a gluconic acid-producing K. pneumoniae strain. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. Gluconic acid accumulation by K. pneumoniae Δgad was an acid-dependent aerobic process, with accumulation observed at pH 5.5 or lower, and at higher levels of oxygen supplementation. Under all other conditions tested, 2,3-butanediol was the main metabolic product of the process. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by K. pneumoniae Δgad, and the conversion ratio of glucose to gluconic acid reached 1 g/g. The K. pneumoniae Δgad described in this study is the first genetically modified strain used for gluconic acid production, and this optimized method for gluconic acid production may have important industrial applications. Gluconic acid is an intermediate of this glucose oxidation pathway. Deletion of gad, resulting in loss of gluconate dehydrogenase activity, led to the accumulation of gluconic acid in the culture broth. In fed batch fermentation, a final concentration of 422 g/L gluconic acid was produced by the K. pneumoniae Δgad strain, and the conversion ratio of glucose to gluconic acid reached 1 g/g.

Biomarkers of hypothalamic-pituitary-adrenal axis activity in mice lacking 11ß-HSD1 and H6PDH.

Glucocorticoid concentrations are a balance between production under the negative feedback control and diurnal rhythm of the hypothalamic-pituitary-adrenal (HPA) axis and peripheral metabolism, for example by the enzyme 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1), which catalyses the reduction of inactive cortisone (11-dehydrocorticosterone (11-DHC) in mice) to cortisol (corticosterone in mice). Reductase activity is conferred upon 11ß-HSD1 by hexose-6-phosphate dehydrogenase (H6PDH). 11ß-HSD1 is implicated in the development of obesity, and selective 11ß-HSD1 inhibitors are currently under development. We sought to address the concern regarding potential up-regulation of the HPA axis associated with inhibition of 11ß-HSD1. We assessed biomarkers for allele combinations of 11ß-HSD1 and H6PDH derived from double heterozygous mouse crosses. H6PDH knock out (KO) adrenals were 69% larger than WT while 11ß-HSD1 KO and double KO (DKO) adrenals were ~30% larger than WT - indicative of increased HPA axis drive in KO animals. ACTH-stimulated circulating corticosterone concentrations were 2.2-fold higher in H6PDH KO animals and ~1.5-fold higher in 11ß-HSD1 KO and DKO animals compared with WT, proportional to the observed adrenal hypertrophy. KO of H6PDH resulted in a substantial increase in urinary DHC metabolites in males (65%) and females (61%). KO of 11ß-HSD1 alone or in combination with H6PDH led to significant increases (36 and 42% respectively) in urinary DHC metabolites in females only. Intermediate 11ß-HSD1/H6PDH heterozygotes maintained a normal HPA axis. Urinary steroid metabolite profile by gas chromatography/mass spectrometry as a biomarker assay may be beneficial in assaying HPA axis status clinically in cases of congenital and acquired 11ß-HSD1/H6PDH deficiency.

Biological pretreatment with a cellobiose dehydrogenase-deficient strain of Trametes versicolor enhances the biofuel potential of canola straw.

The use of Trametes versicolor as a biological pretreatment for canola straw was explored in the context of biofuel production. Specifically, the effects on the straw of a wild-type strain (52J) and a cellobiose dehydrogenase (CDH)-deficient strain (m4D) were investigated. The xylose and glucose contents of the straw treated with 52J were significantly reduced, while only the xylose content was reduced with m4D treatment. Lignin extractability was greatly improved with fungal treatments compared to untreated straw. Saccharification of the residue of the m4D-treated straw led to a significant increase in proportional glucose yield, which was partially attributed to the lack of cellulose catabolism by m4D. Overall, the results of this study indicate that CDH facilitates cellulose access by T. versicolor. Furthermore, treatment of lignocellulosic material with m4D offers improvements in lignin extractability and saccharification efficacy compared to untreated biomass without loss of substrate due to fungal catabolism.

Biochemistry and physiology of hexose-6-phosphate knockout mice.

Hexose-6-phosphate dehydrogenase (H6PDH) has emerged as an important factor in setting the redox status of the endoplasmic reticulum (ER) lumen. An important role of H6PDH is to generate a high NADPH/NADP(+) ratio which permits 11ß-hydroxysteroid dehydrogenase type 1 (11ß-HSD1) to act as an oxo-reductase, catalyzing the activation of glucocorticoids (GCs). In H6PDH knockout mice 11ß-HSD1 assumes dehydrogenase activity and inactivates GCs, rendering the target cell relatively GC insensitive. Consequently, H6PDHKO mice have a phenotype consistent with defects in the permissive and adaptive actions of GCs upon physiology. H6PDHKO mice have also offered an insight into muscle physiology as they also present with a severe vacuolating myopathy, abnormalities of glucose homeostasis and activation of the unfolded protein response due to ER stress, and a number of mechanisms driving this phenotype are thought to be involved. This article will review what we understand of the redox control of GC hormone metabolism regulated by H6PDH, and how H6PDHKO mice have allowed an in-depth understanding of its potentially novel, GC-independent roles in muscle physiology.

Mutations of the hexose-6-phosphate dehydrogenase gene rarely cause hyperandrogenemic polycystic ovary syndrome.

BACKGROUND/AIM: Hexose-6-phosphate dehydrogenase (H6PD) inactivating mutations cause cortisone reductase deficiency, which manifests with hyperandrogenism unexplained by commonly used tests and, thus, mimics polycystic ovary syndrome (PCOS). The aim of this study was to screen for mutations of H6PD gene in PCOS patients with biochemical hyperandrogenemia. METHODS: Direct DNA sequencing of the entire H6PD coding sequence was performed in 74 PCOS patients and 31 healthy controls. Results were confirmed by PCR-restriction fragment length polymorphism assay to determine the genotypic frequency of the variants. RESULTS: Multiple novel missense variants were detected in the study. Two exon 2 variants (acccaggc deletion proximal to the start codon and D151A) and two exon 5 variants (R453Q and P554L) were common, occurring in 23.8%, 17.1%, 35.2%, and 16.1%, respectively. There was significant linkage disequilibrium between the exon 2 and exon 5 variants. No significant differences were observed in the genotype, allele distributions, or adrenal function tests of the variants between cases and control groups. We did not detect any reported inactivating mutations in our study. CONCLUSION: Although the H6PD gene is very polymorphic and missense variants are common, coding variants rarely (<1.5%) are responsible for hyperandrogenemic PCOS. We suggest that genetic studies be reserved for patients with dexamethasone-suppressible adrenal hyperandrogenism who have a discrepancy between urinary 17α-hydroxycorticoid and cortisol excretion.

Redirecting reductant flux into hydrogen production via metabolic engineering of fermentative carbon metabolism in a cyanobacterium.

Some aquatic microbial oxygenic photoautotrophs (AMOPs) make hydrogen (H(2)), a carbon-neutral, renewable product derived from water, in low yields during autofermentation (anaerobic metabolism) of intracellular carbohydrates previously stored during aerobic photosynthesis. We have constructed a mutant (the ldhA mutant) of the cyanobacterium Synechococcus sp. strain PCC 7002 lacking the enzyme for the NADH-dependent reduction of pyruvate to D-lactate, the major fermentative reductant sink in this AMOP. Both nuclear magnetic resonance (NMR) spectroscopy and liquid chromatography-mass spectrometry (LC-MS) metabolomic methods have shown that autofermentation by the ldhA mutant resulted in no D-lactate production and higher concentrations of excreted acetate, alanine, succinate, and hydrogen (up to 5-fold) compared to that by the wild type. The measured intracellular NAD(P)(H) concentrations demonstrated that the NAD(P)H/NAD(P)(+) ratio increased appreciably during autofermentation in the ldhA strain; we propose this to be the principal source of the observed increase in H(2) production via an NADH-dependent, bidirectional [NiFe] hydrogenase. Despite the elevated NAD(P)H/NAD(P)(+) ratio, no decrease was found in the rate of anaerobic conversion of stored carbohydrates. The measured energy conversion efficiency (ECE) from biomass (as glucose equivalents) converted to hydrogen in the ldhA mutant is 12%. Together with the unimpaired photoautotrophic growth of the ldhA mutant, these attributes reveal that metabolic engineering is an effective strategy to enhance H(2) production in AMOPs without compromising viability.

Deletion of hexose-6-phosphate dehydrogenase activates the unfolded protein response pathway and induces skeletal myopathy.

Hexose-6-phosphate dehydrogenase (H6PD) is the initial component of a pentose phosphate pathway inside the endoplasmic reticulum (ER) that generates NADPH for ER enzymes. In liver H6PD is required for the 11-oxoreductase activity of 11beta-hydroxysteroid dehydrogenase type 1, which converts inactive 11-oxo-glucocorticoids to their active 11-hydroxyl counterparts; consequently, H6PD null mice are relatively insensitive to glucocorticoids, exhibiting fasting hypoglycemia, increased insulin sensitivity despite elevated circulating levels of corticosterone, and increased basal and insulin-stimulated glucose uptake in muscles normally enriched in type II (fast) fibers, which have increased glycogen content. Here, we show that H6PD null mice develop a severe skeletal myopathy characterized by switching of type II to type I (slow) fibers. Running wheel activity and electrically stimulated force generation in isolated skeletal muscle are both markedly reduced. Affected muscles have normal sarcomeric structure at the electron microscopy level but contain large intrafibrillar membranous vacuoles and abnormal triads indicative of defects in structure and function of the sarcoplasmic reticulum (SR). SR proteins involved in calcium metabolism, including the sarcoplasmic/endoplasmic reticulum calcium ATPase (SERCA), calreticulin, and calsequestrin, show dysregulated expression. Microarray analysis and real-time PCR demonstrate overexpression of genes encoding proteins in the unfolded protein response pathway. We propose that the absence of H6PD induces a progressive myopathy by altering the SR redox state, thereby impairing protein folding and activating the unfolded protein response pathway. These studies thus define a novel metabolic pathway that links ER stress to skeletal muscle integrity and function.

Abnormalities of glucose homeostasis and the hypothalamic-pituitary-adrenal axis in mice lacking hexose-6-phosphate dehydrogenase.

Hexose-6-phosphate dehydrogenase (EC 1.1.1.47) catalyzes the conversion of glucose 6-phosphate to 6-phosphogluconolactone within the lumen of the endoplasmic reticulum, thereby generating reduced nicotinamide adenine dinucleotide phosphate. Reduced nicotinamide adenine dinucleotide phosphate is a necessary cofactor for the reductase activity of 11beta-hydroxysteroid dehydrogenase type 1 (EC 1.1.1.146), which converts hormonally inactive cortisone to active cortisol (in rodents, 11-dehydrocorticosterone to corticosterone). Mice with targeted inactivation of hexose-6-phosphate dehydrogenase lack 11beta-hydroxysteroid dehydrogenase type 1 reductase activity, whereas dehydrogenase activity (corticosterone to 11-dehydrocorticosterone) is increased. We now report that both glucose output and glucose use are abnormal in these mice. Mutant mice have fasting hypoglycemia. In mutant primary hepatocytes, glucose output does not increase normally in response to glucagon. Mutant animals have lower hepatic glycogen content when fed and cannot mobilize it normally when fasting. As assessed by RT-PCR, responses of hepatic enzymes to fasting are blunted; enzymes involved in gluconeogenesis (phosphoenolpyruvate carboxykinase, tyrosine aminotransferase) are not appropriately up-regulated, and expression of glucokinase, an enzyme required for glycolysis, is not suppressed. Corticosterone has attenuated effects on expression of these enzymes in cultured mutant primary hepatocytes. Mutant mice have increased sensitivity to insulin, as assessed by homeostatic model assessment values and by increased glucose uptake by the muscle. The hypothalamic-pituitary-adrenal axis is also abnormal. Circulating ACTH, deoxycorticosterone, and corticosterone levels are increased in mutant animals, suggesting decreased negative feedback on the hypothalamic-pituitary-adrenal axis. Comparison with other animal models of adrenal insufficiency suggests that many of the observed abnormalities can be explained by blunted intracellular corticosterone actions, despite elevated circulating levels of this hormone.

Hexose-6-phosphate dehydrogenase knock-out mice lack 11 beta-hydroxysteroid dehydrogenase type 1-mediated glucocorticoid generation.

The local generation of active glucocorticoid by NADPH-dependent, 11beta-hydroxysteroid dehydrogenase type 1 (11beta-HSD1) oxoreductase activity, has emerged as an important factor in regulating hepatic glucose output and visceral adiposity. We have proposed that this NADPH is generated within the endoplasmic reticulum by the enzyme hexose-6-phosphate dehydrogenase. To address this hypothesis, we generated mice with a targeted inactivation of the H6PD gene. These mice were unable to convert 11-dehydrocorticosterone (11-DHC) to corticosterone but demonstrated increased corticosterone to 11-DHC conversion consistent with lack of 11beta-HSD1 oxoreductase and a concomitant increase in dehydrogenase activity. This increased corticosterone clearance in the knock-out mice resulted in a reduction in circulating corticosterone levels. Our studies define the critical requirement of hexose-6-phosphate dehydrogenase for 11beta-HSD1 oxoreductase activity and add a new dimension to the investigation of 11beta-HSD1 as a therapeutic target in patients with the metabolic syndrome.

Prenatal and early postnatal treatment in 3-phosphoglycerate-dehydrogenase deficiency.

3-phosphoglycerate-dehydrogenase (3-PGDH) deficiency is an L-serine biosynthesis disorder, characterised by congenital microcephaly, severe psychomotor retardation, and intractable seizures. We report prenatal diagnosis of an affected fetus by DNA mutation analysis. Ultrasound assessment showed a reduction in fetal head circumference from the 75th percentile at 20 weeks' gestation to the 29th percentile at 26 weeks. L-serine was then given to the mother, which resulted in an enlarged fetal head circumference to the 76th percentile at 31 weeks. At birth, the girl's head circumference was normal, and at 48 months' follow-up, her psychomotor development has been unremarkable. 3-PGDH deficiency is an inborn metabolic error that can be successfully treated antenatally.

Serine-deficiency syndromes.

PURPOSE OF REVIEW: Serine-deficiency disorders comprise a new group of neurometabolic diseases and are caused by defects in the biosynthesis of the amino acid L-serine. In contrast to most neurometabolic disorders, serine-deficiency disorders are potentially treatable. Furthermore, the severe neurological symptoms observed in patients underscore the important roles of the serine biosynthetic pathway in brain tissue. An overview of the patients with serine-deficiency disorders reported to date, the biochemical findings and the results of treatment with amino acids is presented. RECENT FINDINGS: L-Serine biosynthesis plays an important role in multiple cellular reactions, particularly in the brain, as L-serine is a precursor of important metabolites such as nucleotides, phospholipids and the neurotransmitters glycine and D-serine. Disturbances of serine-glycine metabolism in relation to N-methyl-D-aspartate-receptor activation are supposed to play a role in psychiatric disease as well. Recent findings concerning these roles of L-serine-derived phospholipids and neurotransmitters are presented. SUMMARY: Congenital microcephaly, seizures and severe psychomotor retardation are symptoms of serine deficiency and can be treated with supplementation of L-serine, sometimes combined with glycine. The symptoms observed in serine deficiency confirm that L-serine and L-serine-derived metabolites play important roles in the central nervous system.

Congenital microcephaly and seizures due to 3-phosphoglycerate dehydrogenase deficiency: outcome of treatment with amino acids.

Congenital microcephaly, intractable seizures and severe psychomotor retardation characterize 3-phosphoglycerate dehydrogenase (3-PGDH) deficiency, a disorder of L-serine biosynthesis. The enzyme defect results in low concentrations of serine and to a variable degree of glycine in plasma and cerebrospinal fluid. Short-term beneficial effects have been reported of oral treatment with the deficient amino acids. In this paper, we report the first follow-up data of amino acid therapy in five patients treated for 3-7.5 years. Different treatment regimes were used, but a favourable response to amino acids was observed in all patients. A major reduction in seizure frequency occurred in all patients; two patients became free of seizures. Amino acids were well tolerated and no adverse effects were documented. A progress of psychomotor development was only observed in one patient, diagnosed early and treated with a high dosage of L-serine. A favourable outcome of 3-PGDH deficiency depends on early diagnosis and treatment.

V490M, a common mutation in 3-phosphoglycerate dehydrogenase deficiency, causes enzyme deficiency by decreasing the yield of mature enzyme.

A deficiency of 3-phosphoglycerate dehydrogenase (PHGDH) is a disorder of serine biosynthesis identified in children with congenital microcephaly, seizures, and severe psychomotor retardation. We report here the identification of the 1468G-->A (V490M) mutation of this gene in two siblings of an Ashkenazi Jewish family, providing further evidence that the V490M mutation is a common, panethnic cause of this deficiency. Using a novel, DNA-based diagnostic test, the mutation was not detected in 400 non-Jewish controls; one heterozygote was found among 400 persons of Ashkenazi Jewish ethnicity. Extensive biochemical studies were undertaken to characterize the effect of this mutation on enzyme activity, turnover, and stability. The V490M PHGDH yielded less than 35% of the activity observed for the wild-type enzyme when overexpressed by transient transfection or when comparing the endogenous activity in fibroblast cells from the patients with controls. Immunoblotting studies showed a comparable reduction in the level of immunoreactive PHGDH in cells expressing the mutant enzyme. Pulse-chase experiments with metabolically labeled PHGDH indicated that this resulted from an increased rate of degradation of the mutant enzyme following its synthesis. Thermolability analyses of mutant and wild-type enzyme activity revealed no significant differences. While others have proposed that the V490M mutation decreases the V(max) of the enzyme, we conclude that this mutation impairs the folding and/or assembly of PHGDH but has minimal effects on the activity or stability of that portion of the V490M mutant that reaches a mature conformation.

Phenotypic heterogeneity and adverse effects of serine treatment in 3-phosphoglycerate dehydrogenase deficiency: report on two siblings.

Clinical experience with the treatment of 3-phosphoglycerate dehydrogenase deficiency, a rare inherited disorder of serine synthesis, is scarce. We report on two sisters with phenotypic heterogeneity and a favourable response to combined serine and glycine supplementation. The elder sibling was found to be normocephalic at birth and showed moderate delay of white matter myelinisation, while her seizures arrested spontaneously even without treatment. In the younger sister with the classical phenotype, feeding difficulties with recurrent gastro-oesophageal reflux prompted us to treat her temporarily with high-dose serine (1400 mg/kg/day). An arrest of head growth then occurred but could be reversed by reducing the serine supply. In both children serine therapy was associated with decreased concentrations of methionine, isoleucine, and ornithine in the cerebrospinal fluid, attributed to competitive inhibition of neutral amino acid transport across the blood-brain barrier. In contrast to reports in the literature, these findings demonstrate that congenital microcephaly, intractable seizures, and dysmyelinisation are not invariably present in patients with 3-phosphoglycerate dehydrogenase deficiency. An adverse effect of high-dose serine therapy on head growth and on the transport of neutral amino acids across the blood-brain barrier should be considered and requires adjustment of treatment.

3-phosphoglycerate dehydrogenase deficiency in a patient with West syndrome.

3-phosphoglycerate dehydrogenase deficiency is a severe but treatable disorder of serine synthesis, first described in 1996 (Jaeken et al. 1996a). The patient presented with West syndrome, severe psychomotor delay, failure to thrive, microcephaly, atypical ocular movements, and pyramidal signs. Treatment with oral L-serine abolished seizures and improved psychomotor development, hyperexcitability, head growth, cortical and subcortical hypotrophy, and hypomyelination of the brain on MRI scans. 3-phosphoglycerate dehydrogenase deficiency is a treatable congenital error that probably leads to West syndrome.

Molecular characterization of 3-phosphoglycerate dehydrogenase deficiency--a neurometabolic disorder associated with reduced L-serine biosynthesis.

3-phosphoglycerate dehydrogenase (PHGDH) deficiency is a disorder of L-serine biosynthesis that is characterized by congenital microcephaly, psychomotor retardation, and seizures. To investigate the molecular basis for this disorder, the PHGDH mRNA sequence was characterized, and six patients from four families were analyzed for sequence variations. Five patients from three different families were homozygous for a single nucleotide substitution predicted to change valine at position 490 to methionine. The sixth patient was homozygous for a valine to methionine substitution at position 425; both mutations are located in the carboxyterminal part of PHGDH. In vitro expression of these mutant proteins resulted in significant reduction of PHGDH enzyme activities. RNA-blot analysis indicated abundant expression of PHGDH in adult and fetal brain tissue. Taken together with the severe neurological impairment in our patients, the data presented in this paper suggest an important role for PHGDH activity and L-serine biosynthesis in the metabolism, development, and function of the central nervous system.

Hypomyelination and reversible white matter attenuation in 3-phosphoglycerate dehydrogenase deficiency.

White matter abnormalities are a feature of many inborn errors of metabolism and magnetic resonance imaging (MRI) of the brain has become an important tool in the diagnostic work-up of these disorders. Recently, patients were reported with a potentially treatable disorder of serine biosynthesis. They presented with congenital microcephaly, severe psychomotor retardation and intractable seizures. Low concentrations of the amino acids serine, glycine as well as 5-methyltetrahydrofolate were found in plasma and CSF and were due to a deficiency of the enzyme 3-phosphoglycerate dehydrogenase (3-PGDH). We studied four patients aged 10 months to 7 years by MRI before and after treatment with amino acids with a follow-up of 16 months to 6 years. Magnetic resonance spectroscopy (MRS) was performed in two patients at 4 and 16 months of treatment. Pre-treatment MRI demonstrated hypomyelination and profound white matter attenuation in all patients. During treatment, a significant increase in white matter volume was found and a progress of myelination in two patients. The most striking finding on MRS during treatment was an elevated level of white matter choline. Serine biosynthesis defects have to be considered in the differential diagnosis of patients with mental retardation, microcephaly, seizures, and on MRI hypomyelination and white matter attenuation.