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Brucellosis is a worldwide extended zoonosis caused by pathogens of the genus Brucella. While most B. abortus, B. melitensis, and B. suis biovars grow slowly in complex media, they multiply intensely in livestock genitals and placenta indicating high metabolic capacities. Mutant analyses in vitro and in infection models emphasize that erythritol (abundant in placenta and genitals) is a preferred substrate of brucellae, and suggest hexoses, pentoses, and gluconeogenic substrates use in host cells. While Brucella sugar and erythritol catabolic pathways are known, growth on 3-4 carbon substrates persists in Fbp- and GlpX-deleted mutants, the canonical gluconeogenic fructose 1,6-bisphosphate (F1,6bP) bisphosphatases. Exploiting the prototrophic and fast-growing properties of B. suis biovar 5, we show that gluconeogenesis requires fructose-bisphosphate aldolase (Fba); the existence of a novel broad substrate bisphosphatase (Bbp) active on sedoheptulose 1,7-bisphosphate (S1,7bP), F1,6bP, and other phosphorylated substrates; that Brucella Fbp unexpectedly acts on S1,7bP and F1,6bP; and that, while active in B. abortus and B. melitensis, GlpX is disabled in B. suis biovar 5. Thus, two Fba-dependent reactions (dihydroxyacetone-phosphate + glyceraldehyde 3-phosphate ⇌ F1,6bP; and dihydroxyacetone-phosphate + erythrose 4-phosphate ⇌ S1,7bP) can, respectively, yield fructose 6-phosphate and sedoheptulose 7-phosphate for classical gluconeogenesis and the Pentose Phosphate Shunt (PPS), the latter reaction opening a new gluconeogenic route. Since erythritol generates the PPS-intermediate erythrose 4-phosphate, and the Fba/Fbp-Bbp route predicts sedoheptulose 7-phosphate generation from erythrose 4-phosphate, we reexamined the erythritol connections with PPS. Growth on erythritol required transaldolase or the Fba/Fbp-Bbp pathway, strongly suggesting that Fba/Fbp- Bbp works as a PPS entry for both erythritol and gluconeogenic substrates in Brucella. We propose that, by increasing erythritol channeling into PPS through these peculiar routes, brucellae proliferate in livestock genitals and placenta in the high numbers that cause abortion and infertility, and make brucellosis highly contagious. These findings could be the basis for developing attenuated brucellosis vaccines safer in pregnant animals. [ABSTRACT FROM AUTHOR]

Glycogen storage disease type Ib (GSD1b) is due to a defect in the glucose-6-phosphate transporter (G6PT) of the endoplasmic reticulum, which is encoded by the SLC37A4 gene. This transporter allows the glucose-6-phosphate that is made in the cytosol to cross the endoplasmic reticulum (ER) membrane and be hydrolyzed by glucose-6-phosphatase (G6PC1), a membrane enzyme whose catalytic site faces the lumen of the ER. Logically, G6PT deficiency causes the same metabolic symptoms (hepatorenal glycogenosis, lactic acidosis, hypoglycemia) as deficiency in G6PC1 (GSD1a). Unlike GSD1a, GSD1b is accompanied by low neutrophil counts and impaired neutrophil function, which is also observed, independently of any metabolic problem, in G6PC3 deficiency. Neutrophil dysfunction is, in both diseases, due to the accumulation of 1,5-anhydroglucitol-6-phosphate (1,5-AG6P), a potent inhibitor of hexokinases, which is slowly formed in the cells from 1,5-anhydroglucitol (1,5-AG), a glucose analog that is normally present in blood. Healthy neutrophils prevent the accumulation of 1,5-AG6P due to its hydrolysis by G6PC3 following transport into the ER by G6PT. An understanding of this mechanism has led to a treatment aimed at lowering the concentration of 1,5-AG in blood by treating patients with inhibitors of SGLT2, which inhibits renal glucose reabsorption. The enhanced urinary excretion of glucose inhibits the 1,5-AG transporter, SGLT5, causing a substantial decrease in the concentration of this polyol in blood, an increase in neutrophil counts and function and a remarkable improvement in neutropenia-associated clinical signs and symptoms. [ABSTRACT FROM AUTHOR]

Neutropenia and neutrophil dysfunction found in deficiencies in G6PC3 and in the glucose‐6‐phosphate transporter (G6PT/SLC37A4) are due to accumulation of 1,5‐anhydroglucitol‐6‐phosphate (1,5‐AG6P), an inhibitor of hexokinase made from 1,5‐anhydroglucitol (1,5‐AG), an abundant polyol present in blood. Lowering blood 1,5‐AG with an SGLT2 inhibitor greatly improved neutrophil counts and function in G6PC3‐deficient mice and in patients with G6PT‐deficiency. We evaluate this treatment in two G6PC3‐deficient children. While neutropenia was severe in one child (PT1), which was dependent on granulocyte cololony‐stimulating factor (GCSF), it was significantly milder in the other one (PT2), which had low blood 1,5‐AG levels and only required GCSF during severe infections. Treatment with the SGLT2‐inhibitor empagliflozin decreased 1,5‐AG in blood and 1,5‐AG6P in neutrophils and improved (PT1) or normalized (PT2) neutrophil counts, allowing to stop GCSF. On empagliflozin, both children remained infection‐free (>1 year – PT2; >2 years – PT1) and no side effects were reported. Remarkably, sequencing of SGLT5, the gene encoding the putative renal transporter for 1,5‐AG, disclosed a rare heterozygous missense mutation in PT2, replacing the extremely conserved Arg401 by a histidine. The higher urinary clearance of 1,5‐AG explains the more benign neutropenia and the outstanding response to empagliflozin treatment found in this child. Our data shows that SGLT2 inhibitors are an excellent alternative to treat the neutropenia present in G6PC3‐deficiency. [ABSTRACT FROM AUTHOR]

In addition to its role as carbon and energy source, fructose metabolism was reported to affect other cellular processes, such as biofilm formation by streptococci and bacterial pathogenicity in plants. Fructose genes encoding a 1-phosphofructokinase and a phosphotransferase system (PTS) fructose-specific enzyme IIABC component reside commonly in a gene cluster with a DeoR family regulator in various gram-positive bacteria. We present a comprehensive study of fructose metabolism in Lactococcus lactis, including a systematic study of fru mutants, global messenger analysis, and a molecular characterization of its regulation. The fru operon is regulated at the transcriptional level by both FruR and CcpA and at the metabolic level by inducer exclusion. The FruR effector is fructose-1-phosphate (F1P), as shown by combined analysis of transcription and measurements of the intracellular F1P pools in mutants either unable to produce this metabolite or accumulating it. The regulation of the fru operon by FruR requires four adjacent 10-bp direct repeats. The well-conserved organization of the fru promoter region in various low-GC gram-positive bacteria, including CRE boxes as well as the newly defined FruR motif, suggests that the regulation scheme defined in L. lactis could be applied to these bacteria. Transcriptome profiling of fruR and fruC mutants revealed that the effect of F1P and FruR regulation is limited to the fru operon in L. lactis. This result is enforced by the fact that no other targets for FruR were found in the available low-GC gram-positive bacteria genomes, suggesting that additional phenotypical effects due to fructose metabolism do not rely directly on FruR control, but rather on metabolism.

The purpose of this study was to identify the biochemical and genetic defect in L-2-hydroxyglutaric aciduria, a neurometabolic disorder characterized by the presence of elevated concentrations of L-2-hydroxyglutaric acid in urine, plasma, and cerebrospinal fluid. Evidence is provided for the existence in rat tissues of a FAD-dependent enzyme catalyzing specifically the oxidation of L-2-hydroxyglutarate to [alpha]-ketoglutarate. This enzyme is mainly expressed in liver and kidney but also at lower levels in heart, brain, and other tissues. Subcellular fractionation indicates that the liver enzyme is present in mitochondria, where it is bound to membranes. Based on this information, a database search led to the identification of a gene encoding a human hypothetical protein homologous to bacterial FAD-dependent malate dehydrogenases and targeted to mitochondria. The gene encoding this protein, present on chromosome 14q22.1, was found to be in a region homozygous in patients with L-2-hydroxyglutaric aciduria from two consanguineous families. Three mutations that replaced a highly conserved residue (Lys-71-Glu and Glu-176-Asp) or removed exon 9 were identified in homozygous state in patients from three distinct families and were found to cosegregate with the disease. It is concluded that L-2-hydroxyglutarate is normally metabolized to [alpha]-ketoglutarate in mammalian tissues and that L-2-hydroxyglutaric aciduria is caused by mutations in the gene that most likely encodes L-2-hydroxyglutarate dehydrogenase. The pathological findings observed in this metabolic disorder must therefore be due to a toxic effect of L-2-hydroxyglutarate on the central nervous system. inborn error of metabolism | leukoencephalopathy | ataxia

Ethylmalonic acid (EMA) is a major and potentially cytotoxic metabolite associated with short‐chain acyl‐CoA dehydrogenase (SCAD) deficiency, a condition whose status as a disease is uncertain. Unexplained high EMA is observed in some individuals with complex neurological symptoms, who carry the SCAD gene (ACADS) variants, c.625G>A and c.511C>T. The variants have a high allele frequency in the general population, but are significantly overrepresented in individuals with elevated EMA. This has led to the idea that these variants need to be associated with variants in other genes to cause hyperexcretion of ethylmalonic acid and possibly a diseased state. Ethylmalonyl‐CoA decarboxylase (ECHDC1) has been described and characterized as an EMA metabolite repair enzyme, however, its clinical relevance has never been investigated. In this study, we sequenced the ECHDC1 gene (ECHDC1) in 82 individuals, who were reported with unexplained high EMA levels due to the presence of the common ACADS variants only. Three individuals with ACADS c.625G>A variants were found to be heterozygous for ECHDC1 loss‐of‐function variants. Knockdown experiments of ECHDC1, in healthy human cells with different ACADS c.625G>A genotypes, showed that ECHDC1 haploinsufficiency and homozygosity for the ACADS c.625G>A variant had a synergistic effect on cellular EMA excretion. This study reports the first cases of ECHDC1 gene defects in humans and suggests that ECHDC1 may be involved in elevated EMA excretion in only a small group of individuals with the common ACADS variants. However, a direct link between ECHDC1/ACADS deficiency, EMA and disease could not be proven. [ABSTRACT FROM AUTHOR]

Background: Besides major clinical/biochemical features, neutropenia and inflammatory bowel disease (IBD) constitute common complications of Glycogen storage disease type Ib (GSD Ib). However, their management is still challenging. Although previous reports have shown benefit of empagliflozin administration on neutropenia, no follow-up data on bowel (macro/microscopic) morphology are available. We herein present for the first time longitudinal assessment of bowel morphology in a GSD Ib child suffering from Crohn disease-like enterocolitis treated with empagliflozin. Case presentation: A 14-year-old boy with GSD Ib and severe IBD was (off-label) treated with empagliflozin (20 mg/day) after informed oral and written consent was obtained from the patient's parents. No adverse events were noted. Clinical symptoms and stool frequency improved within the first week of treatment. Pediatric Crohn disease activity index (PCDAI) normalised within the first month of treatment. Abdomen magnetic resonance imaging (MRI) performed 3 months after treatment initiation showed dramatic decrease in disease activity and length. Similar findings were reported on histology at 5.5 months. At 7.5 months hemoglobin levels normalised and fecal calprotectin almost normalised. Improved neutrophil count, metabolic control and quality of life were also noted. G-CSF dose was decreased by 33% and the patient was partly weaned from tube feeding. Conclusions: This is the first report presenting extensive gastrointestinal morphology follow-up in a GSD Ib patient receiving empagliflozin. The present case suggests that empagliflozin can be safe and effective in inducing IBD remission in GSD Ib patients and can even postpone surgery. Future studies are required to confirm its effect over time and assess its benefit in various disease stages. The development of an international collaborating networks for systematic data collection is worthy. [ABSTRACT FROM AUTHOR]

It is traditionally assumed that enzymes of intermediary metabolism are extremely specific and that this is sufficient to prevent the production of useless and/or toxic side‐products. Recent work indicates that this statement is not entirely correct. In reality, enzymes are not strictly specific, they often display weak side activities on intracellular metabolites (substrate promiscuity) that resemble their physiological substrate or slowly catalyse abnormal reactions on their physiological substrate (catalytic promiscuity). They thereby produce non‐classical metabolites that are not efficiently metabolised by conventional enzymes. In an increasing number of cases, metabolite repair enzymes are being discovered that serve to eliminate these non‐classical metabolites and prevent their accumulation. Metabolite repair enzymes also eliminate non‐classical metabolites that are formed through spontaneous (ie, not enzyme‐catalysed) reactions. Importantly, genetic deficiencies in several metabolite repair enzymes lead to 'inborn errors of metabolite repair', such as L‐2‐hydroxyglutaric aciduria, D‐2‐hydroxyglutaric aciduria, 'ubiquitous glucose‐6‐phosphatase' (G6PC3) deficiency, the neutropenia present in Glycogen Storage Disease type Ib or defects in the enzymes that repair the hydrated forms of NADH or NADPH. Metabolite repair defects may be difficult to identify as such, because the mutated enzymes are non‐classical enzymes that act on non‐classical metabolites, which in some cases accumulate only inside the cells, and at rather low, yet toxic, concentrations. It is therefore likely that many additional metabolite repair enzymes remain to be discovered and that many diseases of metabolite repair still await elucidation. [ABSTRACT FROM AUTHOR]

Most fatty acids (FAs) are straight chains and are synthesized by fatty acid synthase (FASN) using acetyl-CoA and malonyl-CoA units. Yet, FASN is known to be promiscuous as it may use methylmalonyl-CoA instead of malonyl-CoA and thereby introduce methylbranches. We have recently found that the cytosolic enzyme ECHDC1 degrades ethylmalonyl- CoA and methylmalonyl-CoA, which presumably result from promiscuous reactions catalyzed by acetyl-CoA carboxylase on butyryl- and propionyl-CoA. Here, we tested the hypothesis that ECHDC1 is a metabolite repair enzyme that serves to prevent the formation of methyl- or ethyl-branched FAs by FASN. Using the purified enzyme, we found that FASN can incorporate not only methylmalonyl-CoA but also ethylmalonyl-CoA, producing methyl- or ethyl-branched FAs. Using a combination of gas-chromatography and liquid chromatography coupled to mass spectrometry, we observed that inactivation of ECHDC1 in adipocytes led to an increase in several methyl-branched FAs (present in different lipid classes), while its overexpression reduced them below wild-type levels. In contrast, the formation of ethyl-branched FAs was observed almost exclusively in ECHDC1 knockout cells, indicating that ECHDC1 and the low activity of FASN toward ethylmalonyl-CoA efficiently prevent their formation. We conclude that ECHDC1 performs a typical metabolite repair function by destroying methyl- and ethylmalonyl-CoA. This reduces the formation of methyl-branched FAs and prevents the formation of ethylbranched FAs by FASN. The identification of ECHDC1 as a key modulator of the abundance of methyl-branched FAs opens the way to investigate their function. [ABSTRACT FROM AUTHOR]

Cellular membrane lateral organization, in particular the assembly of lipids in domains, is difficult to evaluate at high resolution. Here, we used atomic force microscopy (AFM) to investigate at high-resolution lipid membranes containing variable amounts of sphingomyelin (SM) and cholesterol (Chol), two abundant membrane lipids. To this end, we developed new AFM tip functionalization strategies to specifically probe SM and Chol. Multiparametric AFM imaging allowed us to highlight the lateral submicrometric organization of these two lipids within lipid bilayers through the simultaneous topographic evidence of different phase regimes together with the extraction of their nanomechanical properties and the specific detection of lipid moieties by functionalized AFM probes. The combination of AFM topography and nanomechanical mapping with specific probes for molecular recognition of lipids represents a novel approach to identify lipid-enriched domains in supported bilayers and offers a unique perspective to directly observe lipid assemblies in living cells. [ABSTRACT FROM AUTHOR]

Key points Using recombinant DNA technology, the present study provides the first strong and direct evidence indicating that β-alanine is an efficient substrate for the mammalian transaminating enzymes 4-aminobutyrate-2-oxoglutarate transaminase and alanine-glyoxylate transaminase., The concentration of carnosine and anserine in murine skeletal and heart muscle depends on circulating availability of β-alanine, which is in turn controlled by degradation of β-alanine in liver and kidney., Chronic oral β-alanine supplementation is a popular ergogenic strategy in sports because it can increase the intracellular carnosine concentration and subsequently improve the performance of high-intensity exercises. The present study can partly explain why the β-alanine supplementation protocol is so inefficient, by demonstrating that exogenous β-alanine can be effectively routed toward oxidation., Abstract The metabolic fate of orally ingested β-alanine is largely unknown. Chronic β-alanine supplementation is becoming increasingly popular for improving high-intensity exercise performance because it is the rate-limiting precursor of the dipeptide carnosine (β-alanyl- l-histidine) in muscle. However, only a small fraction (3-6%) of the ingested β-alanine is used for carnosine synthesis. Thus, the present study aimed to investigate the putative contribution of two β-alanine transamination enzymes, namely 4-aminobutyrate-2-oxoglutarate transaminase (GABA-T) and alanine-glyoxylate transaminase (AGXT2), to the homeostasis of carnosine and its methylated analogue anserine. We found that, when transfected into HEK293T cells, recombinant mouse and human GABA-T and AGXT2 are able to transaminate β-alanine efficiently. The reaction catalysed by GABA-T is inhibited by vigabatrin, whereas both GABA-T and AGXT2 activity is inhibited by aminooxyacetic acid (AOA). Both GABA-T and AGXT2 are highly expressed in the mouse liver and kidney and the administration of the inhibitors effectively reduced their enzyme activity in liver (GABA-T for vigabatrin; GABA-T and AGXT2 for AOA). In vivo, injection of AOA in C57BL/6 mice placed on β-alanine (0.1% w/v in drinking water) for 2 weeks lead to a 3-fold increase in circulating β-alanine levels and to significantly higher levels of carnosine and anserine in skeletal muscle and heart. By contrast, specific inhibition of GABA-T by vigabatrin did not affect carnosine and anserine levels in either tissue. Collectively, these data demonstrate that homeostasis of carnosine and anserine in mammalian skeletal muscle and heart is controlled by circulating β-alanine levels, which are suppressed by hepatic and renal β-alanine transamination upon oral β-alanine intake. [ABSTRACT FROM AUTHOR]

The purpose of the present work was to progress in our understanding of the pathophysiology of L-2-hydroxyglutaric aciduria, due to a defect in L-2-hydroxyglutarate dehydrogenase, by creating and studying a mouse model of this disease. L-2-hydroxyglutarate dehydrogenase-deficient mice (l2hgdh-/-) accumulated L-2-hydroxyglutarate in tissues, most particularly in brain and testis, where the concentration reached ≈ 3.5 μmol/g. Male mice showed a 30% higher excretion of L-2-hydroxyglutarate compared to female mice, supporting that this dicarboxylic acid is partially made in males by lactate dehydrogenase C, a poorly specific form of this enzyme exclusively expressed in testes. Involvement of mitochondrial malate dehydrogenase in the formation of L-2-hydroxyglutarate was supported by the commensurate decrease in the formation of this dicarboxylic acid when down-regulating this enzyme in mouse l2hgdh-/- embryonic fibroblasts. The concentration of lysine and arginine was markedly increased in the brain of l2hgdh-/- adult mice. Saccharopine was depleted and glutamine was decreased by ≈ 40%. Lysine-α-ketoglutarate reductase, which converts lysine to saccharopine, was inhibited by L-2-hydroxyglutarate with a Ki of ≈ 0.8 mM. As low but significant activities of the bifunctional enzyme lysine-α-ketoglutarate reductase/saccharopine dehydrogenase were found in brain, these findings suggest that the classical lysine degradation pathway also operates in brain and is inhibited by the high concentrations of L-2-hydroxyglutarate found in l2hgdh-/- mice. Pathological analysis of the brain showed significant spongiosis. The vacuolar lesions mostly affected oligodendrocytes and myelin sheats, as in other dicarboxylic acidurias, suggesting that the pathophysiology of this model of leukodystrophy may involve irreversible pumping of a dicarboxylate in oligodendrocytes. Neurobehavioral testing indicated that the mice mostly suffered from a deficit in learning capacity. In conclusion, the findings support the concept that L-2-hydroxyglutaric aciduria is a disorder of metabolite repair. The accumulation of L-2-hydroxyglutarate exerts toxic effects through various means including enzyme inhibition and glial cell swelling. [ABSTRACT FROM AUTHOR]

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (Υ-aminobutyryl- histidine) synthesis in skeletal muscle and brain, respectively. This enzyme uses, also at substantial rates, lysine, ornithine, and arginine instead of histidine, yet the resulting dipeptides are virtually absent from muscle or brain, suggesting that they are removed by a "metabolite repair" enzyme. Using a radiolabeled substrate, we found that rat skeletal muscle, heart, and brain contained a cytosolic β-alanyl-lysine dipeptidase activity. This enzyme, which has the characteristics of a metalloenzyme, was purified ≈200-fold from rat skeletal muscle. Mass spectrometry analysis of the fractions obtained at different purification stages indicated parallel enrichment of PM20D2, a peptidase of unknown function belonging to the metallopeptidase 20 family. Western blotting showed coelution of PM20D2 with β-alanyl-lysine dipeptidase activity. Recombinant mouse PM20D2 hydrolyzed β-alanyl-lysine, β-alanyl-ornithine, Υ-aminobutyryl-lysine, and Υ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and Υ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or Υ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like β-alanyl-lysine and α-lysyl-lysine. The same activity profile was observed with human PM20D2, yet this enzyme was ~ 100-200-fold less active on all substrates tested than the mouse enzyme. Cotransfection in HEK293T cells of mouse or human PM20D2 together with carnosine synthase prevented the accumulation of abnormal dipeptides (β-alanyl-lysine, β-alanyl-ornithine, Υ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2. [ABSTRACT FROM AUTHOR]

Enzymes of intermediary metabolism are less specific than what is usually assumed: they often act on metabolites that are not their 'true' substrate, making abnormal metabolites that may be deleterious if they accumulate. Some of these abnormal metabolites are reconverted to normal metabolites by repair enzymes, which play therefore a role akin to the proofreading activities of DNA polymerases and aminoacyl-tRNA synthetases. An illustrative example of such repair enzymes is L-2-hydroxyglutarate dehydrogenase, which eliminates a metabolite abnormally made by a Krebs cycle enzyme. Mutations in L-2-hydroxyglutarate dehydrogenase lead to L-2-hydroxyglutaric aciduria, a leukoencephalopathy. Other examples are the epimerase and the ATP-dependent dehydratase that repair hydrated forms of NADH and NADPH; ethylmalonyl-CoA decarboxylase, which eliminates an abnormal metabolite formed by acetyl-CoA carboxylase, an enzyme of fatty acid synthesis; L-pipecolate oxidase, which repairs a metabolite formed by a side activity of an enzyme of L-proline biosynthesis. Metabolite proofreading enzymes are likely quite common, but most of them are still unidentified. A defect in these enzymes may account for new metabolic disorders. [ABSTRACT FROM AUTHOR]

Protein deglycation, a new form of protein repair, involves several enzymes. Fructosamine-3-kinase (FN3K), an enzyme found in mammals and birds, phosphorylates fructosamines on the third carbon of their sugar moiety, making them unstable and causing them to detach from proteins. This enzyme acts particularly well on fructose-epsilon-lysine, both in free form and in the accessible regions of proteins. Mice deficient in FN3K accumulate protein-bound fructosamines and free fructoselysine, indicating that the deglycation mechanism initiated by FN3K is operative in vivo. Mammals and birds also have an enzyme designated 'FN3K-related protein' (FN3KRP), which shares ≈65% sequence identity with FN3K. Unlike FN3K, FN3KRP does not phosphorylate fructosamines, but acts on ribulosamines and erythrulosamines. As with FN3K, the third carbon is phosphorylated and this leads to destabilization of the ketoamines. Experiments with intact erythrocytes indicate that FN3KRP is also a protein-repair enzyme. Its physiological substrates are most likely formed from ribose 5-phosphate and erythrose 4-phosphate, which give rise to ketoamine 5- or 4-phosphates. The latter are dephosphorylated by 'low-molecular-weight protein-tyrosine-phosphatase-A' (LMW-PTP-A) before FN3KRP transfers a phosphate on the third carbon. The specificity of FN3K homologues present in plants and bacteria is similar to that of mammalian FN3KRP, suggesting that deglycation of ribulosamines and/or erythrulosamines is an ancient mechanism. Mammalian cells contain also a phosphatase acting on fructosamine 6-phosphates, which result from the reaction of proteins with glucose 6-phosphate. [ABSTRACT FROM AUTHOR]

The purpose of the present work was to identify the catalytic activity of AGXT2L1 and AGXT2L2, two closely related, putative pyridoxal-phosphate-dependent enzymes encoded by vertebrate genomes. The existence of bacterial homologues (40- 50% identity with AGXT2L1 and AGXT2L2) forming bi- or trifunctional proteins with a putative kinase belonging to the family of aminoglycoside phosphotransferases suggested that AGXT2L1 and AGXT2L2 acted on phosphorylated and aminated compounds. Vertebrate genomes were found to encode a homologue (AGPHD1) of these putative bacterial kinases, which was therefore likely to phosphorylate an amino compound bearing a hydroxyl group. These and other considerations led us to hypothesize that AGPHD1 corresponded to 5-hydroxy-L-lysine kinase and that AGXT2L1 and AGXT2L2 catalyzed the pyridoxal-phosphate-dependent breakdown of phosphoethanolamine and 5-phosphohydroxy-L-lysine. The three recombinant human proteins were produced and purified to homogeneity. AGPHD1 was indeed found to catalyze the GTP-dependent phosphorylation of 5-hydroxy-L-lysine. The phosphorylation product made by this enzyme was metabolized by AGXT2L2, which converted it to ammonia, inorganic phosphate, and 2-aminoadipate semialdehyde. AGXT2L1 catalyzed a similar reaction on phosphoethanolamine, converting it to ammonia, inorganic phosphate, and acetaldehyde. AGPHD1 and AGXT2L2 are likely to be the mutated enzymes in 5-hydroxylysinuria and 5-phosphohydroxylysinuria, respectively. The high level of expression of AGXT2L1 in human brain, as well as data in the literature linking AGXT2L1 to schizophrenia and bipolar disorders, suggest that these diseases may involve a perturbation of brain phosphoethanolamine metabolism. AGXT2L1 and AGXT2L2, the first ammoniophospholyases to be identified, belong to a family of aminotransferases acting on ω-amines. [ABSTRACT FROM AUTHOR]

Our goal was to identify the reaction catalyzed by NAT8 (N-acetyltransferase 8), a putative N-acetyltransferase homologous to the enzyme (NAT8L) that produces N-acetylaspartate in brain. The almost exclusive expression of NAT8 in kidney and liver and its predicted association with the endoplasmic reticulum suggested that it was cysteinyl-S-conjugate N-acetyltransferase, the microsomal enzyme that catalyzes the last step of mercapturic acid formation. In agreement, HEK293T extracts of cells overexpressing NAT8 catalyzed the N-acetylation of S-benzyl-L-cysteine and leukotriene E4, two cysteine conjugates, but were inactive on other physiological amines or amino acids. Confocal microscopy indicated that NAT8 was associated with the eudoplasmic reticulum. Neither of the two frequent single nucleotide polymorphisms found in NAT8, E104K nor F143S, changed the enzymatic activity or the expression of the protein by ≥-fold, whereas a mutation (R149K) replacing an extremely conserved arginine suppressed the activity. Sequencing of genomic DNA and EST clones corresponding to the NATSB gene, which resulted from duplication of the NAT8 gene in the primate lineage, disclosed the systematic presence of a premature stop codon at codon 16. Furthermore, truncated NAT8B and NAT8 proteins starting from the following methionine (Met-25) showed no cysteinyl-S-conjugate N-acetyltransferase activity when transfected in HEK293T cells. Taken together, these findings indicate that NAT8 is involved in mercapturic acid formation and confirm that NAT8B is an inactive gene in humans. NAT8 homologues are found in all vertebrate genomes, where they are often encoded by multiple, tandemly repeated genes as many other genes encoding xenobiotic metabolism enzymes. [ABSTRACT FROM AUTHOR]

Carnosine (β-alanyl-L-histidine) and homocarnosine (γ-aminobutyryl-L-histidine) are abundant dipeptides in skeletal muscle and brain of most vertebrates and some invertebrates. The formation of both compounds is catalyzed by carnosine synthase, which is thought to convert ATP to AMP and inorganic pyrophosphate, and whose molecular identity is unknown. In the present work, we have purified carnosine synthase from chicken pectoral muscle about 1500-fold until only two major polypeptides of 100 and 90 kDa were present in the preparation. Mass spectrometry analysis of these polypeptides did not yield any meaningful candidate. Carnosine formation catalyzed by the purified enzyme was accompanied by a stoichiometric formation, not of AMP, but of ADP, suggesting that carnosine synthase belongs to the "ATP-grasp family" of ligases. A data base mining approach identified ATPGD1 as a likely candidate. As this protein was absent from chicken protein data bases, we reconstituted its sequence from a PCR-amplified cDNA and found it to fit with the 100-kDa polypeptide of the chicken carnosine synthase preparation. Mouse and human ATPGD1 were expressed in HEK293T cells, purified to homogeneity, and shown to catalyze the formation of carnosine, as confirmed by mass spectrometry, and of homocarnosine. Specificity studies carried out on all three enzymes were in agreement with published data. In particular, they acted with 15-25-fold higher catalytic efficiencies on β-alanine than on γ-aminobutyrate. The identification of the gene encoding carnosine synthase will help for a better understanding of the biological functions of carnosine and related dipeptides, which still remain largely unknown. [ABSTRACT FROM AUTHOR]

Abstract: Our purpose was to identify the sequence of ω-amidase, which hydrolyses the amide group of α-ketoglutaramate, a product formed by glutamine transaminases. In the Bacillus subtilis genome, the gene encoding a glutamine transaminase (mtnV) is flanked by a gene encoding a putative ‘carbon-nitrogen hydrolase’. The closest mammalian homolog of this putative bacterial ω-amidase is ‘nitrilase 2’, whose size and amino acid composition were in good agreement with those reported for purified rat liver ω-amidase. Mouse nitrilase 2 was expressed in Escherichia coli, purified and shown to catalyse the hydrolysis of α-ketoglutaramate and other known substrates of ω-amidase. No such activity was observed with mouse nitrilase 1. We conclude that mammalian nitrilase 2 is ω-amidase. [Copyright &y& Elsevier]

Glucose 1,6-bisphosphate (Glc-1,6-P2) concentration in brain is much higher than what is required for the functioning of phosphoglucomutase, suggesting that this compound has a role other than as a cofactor of phosphomutases. In cell-free systems, Glc-1,6-P2 is formed from 1,3-bisphosphoglycerate and Glc-6-P by two related enzymes: PGM2L1 (phosphoglucomutase 2-like 1) and, to a lesser extent, PGM2 (phosphoglucomutase 2). It is hydrolyzed by the IMP-stimulated brain Glc-1,6-bisphosphatase of still unknown identity. Our aim was to test whether Glc-1,6-bisphosphatase corresponds to the phosphomannomutase PMM1,an enzyme of mysterious physiological function sharing several properties with Glc-1,6-bisphosphatase. We show that IMP, but not other nucleotides, stimulated by >100-fold (Ka ≈ 20 μM) the intrinsic Glc-1,6-bisphosphatase activity of recombinant PMM1 while inhibiting its phosphoglucomutase activity. No such effects were observed with PMM2, an enzyme paralogous to PMM1 that physiologically acts as a phosphomannomutase in mammals. Transfection of HEK293T cells with PGM2L1, but not the related enzyme PGM2, caused an ≈20-fold increase in the concentration of Glc-1,6-P2. Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P2 in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P2 in wild type mouse brain decreased with time after ischemia, whereas it did not change in PMM1-deficient mouse brain. Taken together, these data show that PMM1 corresponds to the IMP-stimulated Glc-1,6-bisphosphatase and that this enzyme is responsible for the degradation of Glc-1,6-P2 in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P2 in brain is established. [ABSTRACT FROM AUTHOR]

We report the identification of the mutations in the only known case of L-3-phosphoserine phosphatase deficiency, a recessively inherited condition. The two mutations correspond to the replacement of the semiconserved Asp32 residue by an asparagine and of the extremely conserved Met52 by a threonine. The effects of both mutations were studied on the human recombinant enzyme, expressed in Escherichia coli. Met52Thr almost abolished the enzymatic activity, whereas the Asp32Asn mutation caused a 50% decrease in Vmax. In addition, L-serine, which inhibits the conversion of [14C] phosphoserine to serine when catalysed by the wild-type enzyme, had a lesser inhibitory effect on the Asp32Asn mutant, indicating a reduction in the rate of phosphoenzyme hydrolysis. These modifications in the properties of the enzyme are consistent with the modification in the kinetic properties observed in fibroblasts from the patient.European Journal of Human Genetics (2004) 12, 163-166. doi:10.1038/sj.ejhg.5201083 Published online 3 December 2003 [ABSTRACT FROM AUTHOR]

Focuses on the sequencing of the serotype-specific, 5.9-kb region II of the Haemophilus influenzae type a capsulation locus. Presence of open reading frames termed acs1 to acs4; Primer extension and DNA sequencing analyses; Regional organization of the cap locus; Alignment of predicted amino acid sequences.

The purpose of this work was to test the hypothesis that mutations in the putative glucose 6-phosphate translocase gene would account for most of the cases of GSD I that are not explained by mutations in the phosphohydrolase gene, ie that are not type Ia. Twenty-three additional families diagnosed as having GSDI non-a (GSD Ib, Ic or Id) have now been analysed. The 9 exons of the gene were amplified by PCR and mutations searched both by SSCP and heteroduplex analysis. Except for one family in which only one mutation was found, all patients had two allelic mutations in the gene encoding the putative glucose 6-phosphate translocase. Sixteen of the mutations are new and they are all predicted to lead to nonfunctional proteins. All investigated patients had some degree of neutropenia or neutrophil dysfunction and the clinical phenotype of the four new patients who had been diagnosed as GSD Ic and the one diagnosed as GSD Id was no different from the GSD Ib patients. Since these patients, and the four type Ic patients from two families previously studied, shared several mutations with GSD Ib patients, we conclude that their basic defect is in the putative glucose 6-phosphate translocase and that they should be reclassified as GSD Ib. Isolated defects in microsomal Pi transporter or in microsomal glucose transporter must be very rare or have phenotypes that are not recognised as GSD I, so that in practice there are only two subtypes of GSD I (GSD Ia and GSD Ib). [ABSTRACT FROM AUTHOR]