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Transaminases play key roles in central metabolism, transferring the amino group from a donor substrate to an acceptor. These enzymes can often act, with low efficiency, on compounds different from the preferred substrates. To understand what might have shaped the substrate specificity of this class of enzymes, we examined the reactivity of six human cytosolic transaminases towards amino acids whose main degradative pathways do not include any transamination. We also tested whether sugars and sugar phosphates could serve as alternative amino group acceptors for these cytosolic enzymes. Each of the six aminotransferases reacted appreciably with at least three of the alternative amino acid substrates in vitro, albeit at usually feeble rates. Reactions with L-Thr, L-Arg, L-Lys and L-Asn were consistently very slow—a bias explained in part by the structural differences between these amino acids and the preferred substrates of the transaminases. On the other hand, L-His and L-Trp reacted more efficiently, particularly with GTK (glutamine transaminase K; also known as KYAT1). This points towards a role of GTK in the salvage of L-Trp (in cooperation with ω-amidase and possibly with the cytosolic malate dehydrogenase, MDH1, which efficiently reduced the product of L-Trp transamination). Finally, the transaminases were extremely ineffective at utilizing sugars and sugar derivatives, with the exception of the glycolytic intermediate dihydroxyacetone phosphate, which was slowly but appreciably transaminated by some of the enzymes to yield serinol phosphate. Evidence for the formation of this compound in a human cell line was also obtained. We discuss the biological and evolutionary implications of our results.

The recent identification of NAA80/NAT6 as the enzyme that acetylates actins generated new insight into the process of post-translational actin modifications; however, the role of NAA80 in human physiology and pathology has not been clarified yet. We report two individuals from a single family harbouring a homozygous c.389T>C, p.(Leu130Pro) NAA80 genetic variant. Both individuals show progressive high-frequency sensorineural hearing loss, craniofacial dysmorphisms, developmental delay and mild proximal and axial muscle weakness. Based on the molecular structure, we predicted and confirmed the NAA80 c.389T>C, p.(Leu130Pro) variant to result in protein destabilization, causing severely decreased NAA80 protein availability. Concurrently, individuals exhibited a ∼50% decrease of actin acetylation. NAA80 individual derived fibroblasts and peripheral blood mononuclear cells showed increased migration, increased filopodia counts and increased levels of polymerized actin, in agreement with previous observations in NAA80 knock-out cells. Furthermore, the significant clinical overlap between NAA80 individuals and individuals with pathogenic variants in several actin subtypes reflects the general importance of controlled actin dynamics for the inner ear, brain and muscle. Taken together, we describe a new syndrome, caused by NAA80 genetic variants leading to decreased actin acetylation and disrupted associated molecular functions. Our work suggests a crucial role for NAA80-mediated actin dynamics in neuronal health, muscle health and hearing., Muffels et al. report two brothers harbouring a homozygous missense NAA80 genetic variant, with sensorineural hearing loss, craniofacial dysmorphisms, developmental delay and muscle weakness. At the cellular level, they show that the NAA80 variant leads to NAA80 deficiency, causing decreased actin acetylation and disrupted actin cytoskeletal functions., Graphical Abstract Graphical Abstract

The cytosolic enzyme ethylmalonyl-CoA decarboxylase (ECHDC1) decarboxylates ethyl- or methyl-malonyl-CoA, two side-products of acetyl-CoA carboxylase. These CoA derivatives can be used to synthesize a subset of branched-chain fatty acids (FAs). We previously found that ECHDC1 limits the synthesis of these abnormal FAs in cell lines, but its effects in vivo are unknown. To further evaluate the effects of ECHDC1 deficiency, we generated knock-out mice. These mice were viable, fertile, showed normal postnatal growth, and lacked obvious macroscopic and histologic changes. Surprisingly, tissues from wild-type mice already contained methyl-branched FAs due to methylmalonyl-CoA incorporation, but these FAs were only increased in the intraorbital glands of ECHDC1 knock-out mice. In contrast, ECHDC1 knock-out mice accumulated 16- to 20-carbon FAs carrying ethyl-branches in all tissues, which were undetectable in wild-type mice. Ethyl-branched FAs were incorporated into different lipids, including acylcarnitines, phosphatidylcholines, plasmanylcholines, and triglycerides. Interestingly, we found a variety of unusual glycine-conjugates in the urine of knock-out mice, which included adducts of ethyl-branched compounds in different stages of oxidation. This suggests that the excretion of potentially toxic intermediates of branched-chain FA metabolism might prevent a more dramatic phenotype in these mice. Curiously, ECHDC1 knock-out mice also accumulated 2,2-dimethylmalonyl-CoA. This indicates that the broad specificity of ECHDC1 might help eliminate a variety of potentially dangerous branched-chain dicarboxylyl-CoAs. We conclude that ECHDC1 prevents the formation of ethyl-branched FAs, and that urinary excretion of glycine-conjugates allows mice to eliminate potentially deleterious intermediates of branched-chain FA metabolism.

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 methyl-branches. 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 ethyl-branched 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.

Repair of a certain type of oxidative DNA damage leads to the release of phosphoglycolate, which is an inhibitor of triose phosphate isomerase and is predicted to indirectly inhibit phosphoglycerate mutase activity. Thus, we hypothesized that phosphoglycolate might play a role in a metabolic DNA damage response. Here, we determined how phosphoglycolate is formed in cells, elucidated its effects on cellular metabolism and tested whether DNA damage repair might release sufficient phosphoglycolate to provoke metabolic effects. Phosphoglycolate concentrations were below 5 µM in wild-type U2OS and HCT116 cells and remained unchanged when we inactivated phosphoglycolate phosphatase (PGP), the enzyme that is believed to dephosphorylate phosphoglycolate. Treatment of PGP knockout cell lines with glycolate caused an up to 500-fold increase in phosphoglycolate concentrations, which resulted largely from a side activity of pyruvate kinase. This increase was much higher than in glycolate-treated wild-type cells and was accompanied by metabolite changes consistent with an inhibition of phosphoglycerate mutase, most likely due to the removal of the priming phosphorylation of this enzyme. Surprisingly, we found that phosphoglycolate also inhibits succinate dehydrogenase with a Ki value of

N-acetylneuraminate (Neu5Ac), an abundant sugar present in glycans in vertebrates and some bacteria, can be used as an energy source by several prokaryotes, including Escherichia coli. In solution, more than 99% of Neu5Ac is in cyclic form (≈ 92% beta-anomer and ≈ 7% alpha-anomer), whereas < 0.5% is in the open form. The aldolase that initiates Neu5Ac metabolism in E. coli, NanA, has been reported to act on the alpha-anomer. Surprisingly, when we performed this reaction at pH 6 to minimize spontaneous anomerization, we found NanA and its human homolog NPL preferentially metabolize the open form of this substrate. We tested whether the E. coli Neu5Ac anomerase NanM could promote turnover, finding it stimulated the utilization of both beta and alpha-anomers by NanA in vitro. However, NanM is localized in the periplasmic space and cannot facilitate Neu5Ac metabolism by NanA in the cytoplasm in vivo. We discovered that YhcH, a cytoplasmic protein encoded by many Neu5Ac catabolic operons and belonging to a protein family of unknown function (DUF386), also facilitated Neu5Ac utilization by NanA and NPL, and displayed Neu5Ac anomerase activity in vitro. YhcH contains Zn, and its accelerating effect on the aldolase reaction was inhibited by metal chelators. Remarkably, several transition metals accelerated Neu5Ac anomerization in the absence of enzyme. Experiments with E. coli mutants indicated that YhcH expression provides a selective advantage for growth on Neu5Ac. In conclusion, YhcH plays the unprecedented role of providing an aldolase with the preferred unstable open form of its substrate.

Homologues of the putative dehydrogenase YjhC are found in operons involved in the metabolism of N-acetylneuraminate (Neu5Ac) or related compounds. We observed that purified recombinant YjhC forms Neu5Ac from two dehydrated forms of this compound, 2,7-anhydro-N-acetylneuraminate (2,7-AN) and 2-deoxy-2,3-didehydro-N-acetylneuraminate (2,3-EN) that are produced during the degradation of sialoconjugates by some sialidases. The conversion of 2,7-AN into Neu5Ac is reversible and reaches its equilibrium when the ratio of 2,7-AN to Neu5Ac is ≈1/6. The conversion of 2,3-EN is irreversible, leading to a mixture of Neu5Ac and 2,7-AN. NMR analysis of the reaction catalysed by YjhC on 2,3-EN indicated that Neu5Ac was produced as the α-anomer. All conversions require NAD+ as a cofactor, which is regenerated in the reaction. They appear to involve the formation of keto (presumably 4-keto) intermediates of 2,7-AN, 2,3-EN and Neu5Ac, which were detected by liquid chromatography-mass spectrometry (LC-MS). The proposed reaction mechanism is reminiscent of the one catalysed by family 4 β-glycosidases, which also use NAD+ as a cofactor. Both 2,7-AN and 2,3-EN support the growth of Escherichia coli provided the repressor NanR, which negatively controls the expression of the yjhBC operons, has been inactivated. Inactivation of either YjhC or YjhB in NanR-deficient cells prevents the growth on 2,7-AN and 2,3-EN. This confirms the role of YjhC in 2,7-AN and 2,3-EN metabolism and indicates that transport of 2,7-AN and 2,3-EN is carried out by YjhB, which is homologous to the Neu5Ac transporter NanT.

Neutropenia and neutrophil dysfunction cause serious infections and inflammatory bowel disease in glycogen storage disease type Ib (GSD-Ib). Our discovery that accumulating 1,5-anhydroglucitol-6-phosphate (1,5AG6P) caused neutropenia in a glucose-6-phosphatase 3 (G6PC3)–deficient mouse model and in 2 rare diseases (GSD-Ib and G6PC3 deficiency) led us to repurpose the widely used antidiabetic drug empagliflozin, an inhibitor of the renal glucose cotransporter sodium glucose cotransporter 2 (SGLT2). Off-label use of empagliflozin in 4 GSD-Ib patients with incomplete response to granulocyte colony-stimulating factor (GCSF) treatment decreased serum 1,5AG and neutrophil 1,5AG6P levels within 1 month. Clinically, symptoms of frequent infections, mucosal lesions, and inflammatory bowel disease resolved, and no symptomatic hypoglycemia was observed. GCSF could be discontinued in 2 patients and tapered by 57% and 81%, respectively, in the other 2. The fluctuating neutrophil numbers in all patients were increased and stabilized. We further demonstrated improved neutrophil function: normal oxidative burst (in 3 of 3 patients tested), corrected protein glycosylation (2 of 2), and normal neutrophil chemotaxis (1 of 1), and bactericidal activity (1 of 1) under treatment. In summary, the glucose-lowering SGLT2 inhibitor empagliflozin, used for type 2 diabetes, was successfully repurposed for treating neutropenia and neutrophil dysfunction in the rare inherited metabolic disorder GSD-Ib without causing symptomatic hypoglycemia. We ascribe this to an improvement in neutrophil function resulting from the reduction of the intracellular concentration of 1,5AG6P.

6-NADH and 6-NADPH are strong inhibitors of several dehydrogenases that may form spontaneously from NAD(P)H. They are known to be oxidized to NAD(P)+ by mammalian renalase, an FAD-linked enzyme mainly present in heart and kidney, and by related bacterial enzymes. We partially purified an enzyme oxidizing 6-NADPH from rat liver, and, surprisingly, identified it as pyridoxamine-phosphate oxidase (PNPO). This was confirmed by the finding that recombinant mouse PNPO oxidized 6-NADH and 6-NADPH with catalytic efficiencies comparable to those observed with pyridoxine- and pyridoxamine-5′-phosphate. PNPOs from Escherichia coli, Saccharomyces cerevisiae and Arabidopsis thaliana also displayed 6-NAD(P)H oxidase activity, indicating that this ‘side-activity’ is conserved. Remarkably, ‘pyridoxamine-phosphate oxidase-related proteins’ (PNPO-RP) from Nostoc punctiforme, A. thaliana and the yeast S. cerevisiae (Ygr017w) were not detectably active on pyridox(am)ine-5′-P, but oxidized 6-NADH, 6-NADPH and 2-NADH suggesting that this may be their main catalytic function. Their specificity profiles were therefore similar to that of renalase. Inactivation of renalase and of PNPO in mammalian cells and of Ygr017w in yeasts led to the accumulation of a reduced form of 6-NADH, tentatively identified as 4,5,6-NADH3, which can also be produced in vitro by reduction of 6-NADH by glyceraldehyde-3-phosphate dehydrogenase or glucose-6-phosphate dehydrogenase. As 4,5,6-NADH3 is not a substrate for renalase, PNPO or PNPO-RP, its accumulation presumably reflects the block in the oxidation of 6-NADH. These findings indicate that two different classes of enzymes using either FAD (renalase) or FMN (PNPOs and PNPO-RPs) as a cofactor play an as yet unsuspected role in removing damaged forms of NAD(P).

Steady-state enzyme kinetics typically relies on the measurement of ‘initial rates’, obtained when the substrate is not significantly consumed and the amount of product formed is negligible. Although initial rates are usually faster than those measured later in the reaction time-course, sometimes the speed of the reaction appears instead to increase with time, reaching a steady level only after an initial delay or ‘lag phase’. This behavior needs to be interpreted by the experimentalists. To assist interpretation, this article analyzes the many reasons why, during an enzyme assay, the observed rate can be slow in the beginning and then progressively accelerate. The possible causes range from trivial artifacts to instances in which deeper mechanistic or biophysical factors are at play. We provide practical examples for most of these causes, based firstly on experiments conducted with ornithine δ-aminotransferase and with other pyridoxal-phosphate dependent enzymes that have been studied in our laboratory. On the side to this survey, we provide evidence that the product of the ornithine δ-aminotransferase reaction, glutamate 5-semialdehyde, cyclizes spontaneously to pyrroline 5-carboxylate with a rate constant greater than 3 s−1.

All forms of mammalian actin comprise at their N-terminus a negatively charged region consisting of an N-acetylated aspartate or glutamate followed by two or three acidic residues. This structural feature is unique to actins and important for their interaction with other proteins. The enzyme catalyzing the acetylation of the N-terminal acidic residue is thought to be NAA10, an enzyme that acetylates multiple intracellular proteins. We report here that this acetylation is essentially carried out by NAT6 (Fus2), a protein of unknown function. Tests of the activity of human recombinant NAT6 on a series of purified proteins showed that the best substrate had several acidic residues near its N-terminus. Accordingly NAT6 was particularly active on highly acidic peptides with sequences corresponding to the N-terminus of different forms of mammalian actins. Knocking out of NAT6 in two human cell lines led to absence of acetylation of the first residue of mature beta-actin (Asp2) and gamma-actin-1 (Glu2). Complete acetylation of these two actins was restored by re-expression of NAT6, or by incubation of extracts of NAT6-deficient cells with low concentrations of recombinant NAT6, while NAA10 showed much less or no activity in such assays. Alpha-actin-1 expressed in NAT6-knockout cells was not acetylated at its N-terminus, indicating that the requirement of NAT6 for acetylation of actin N-termini also applies to the skeletal muscle actin isoform. Taken together, our findings reveal that NAT6 plays a critical role in the maturation of actins by carrying out the acetylation of their N-terminal acidic residue.

Carnosine synthase is the ATP-dependent ligase responsible for carnosine (β-alanyl-histidine) and homocarnosine (γ-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, γ-aminobutyryl-lysine, and γ-aminobutyryl-ornithine as its best substrates. It also acted at lower rates on β-alanyl-arginine and γ-aminobutyryl-arginine but virtually not on carnosine or homocarnosine. Although acting preferentially on basic dipeptides derived from β-alanine or γ-aminobutyrate, PM20D2 also acted at lower rates on some "classic dipeptides" like α-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, γ-aminobutyryl-lysine), thus favoring the synthesis of carnosine and homocarnosine and confirming the metabolite repair role of PM20D2.

Mammalian ACSF4-U26 (Acyl-CoA Synthetase Family Member 4), a protein of unknown function, comprises a putative adenylation-domain (AMP-binding domain) similar to those of bacterial non-ribosomal peptide synthetases, a putative phosphopantetheine attachment site and a C-terminal PQQDH (pyrroloquinoline quinone dehydrogenase)-related domain. Orthologues comprising these three domains are present in many eukaryotes including plants. Remarkably, the adenylation domain of plant ACSF4-U26 shares more identity with Ebony, the insect enzyme that ligates β-alanine to several amines, than with vertebrate or insect ACSF4-U26 and prediction of its specificity suggests that it also serves to activate β-alanine. In the presence of ATP, purified mouse recombinant ACSF4-U26 progressively formed a covalent bond with radiolabelled β-alanine. The bond was alkali labile, suggesting a (thio)ester, and was not formed in a point-mutant devoid of the phosphopantetheine attachment site. Competition experiments with various amino acids indicated that the reaction was nearly specific for β-alanine, for which a KM of ≈ 5 µM was computed. The loaded enzyme was used to study the formation of a potential end-product. Among the twenty standard amino acids, only cysteine was able to cause unloading of the enzyme. This effect was mimicked by cysteamine and by dithiothreitol, and was unaffected by the absence of the PQQDH-related domain, suggesting that the β-alanine transfer onto thiols is catalyzed by the ACSF4-U26 adenylation domain, but is physiologically irrelevant. We conclude that ACSF4-U26 is a β-alanine activating enzyme, and hypothesize that it is involved in a rare intracellular reaction, possibly an infrequent post-translational or post-transcriptional modification. This article is protected by copyright. All rights reserved.

Defects in the human protein TMEM165 are known to cause a subtype of Congenital Disorders of Glycosylation. Transmembrane protein 165 (TMEM165) belongs to an uncharacterized family of membrane proteins called Uncharacterized Protein Family 0016, which are well conserved throughout evolution and share characteristics reminiscent of the cation/Ca 2+ exchanger superfamily. Gcr1 dependent translation factor 1 (Gdt1p), the budding yeast member of this family, contributes to Ca 2+ homeostasis via an uncharacterized Ca 2+ transport pathway localized in the Golgi apparatus. The gdt1Δ mutant was found to be sensitive to high concentrations of Ca 2+ , and interestingly, this sensitivity was suppressed by expression of TMEM165, the human ortholog of Gdt1p, indicating conservation of function among the members of this family. Patch-clamp analyses on human cells indicated that TMEM165 expression is linked to Ca 2+ ion transport. Furthermore, defects in TMEM165 affected both Ca 2+ and pH homeostasis. Based on these results, we propose that Gdt1p and TMEM165 could be members of a unique family of Golgi-localized Ca 2+ /H + antiporters and that modification of the Golgi Ca 2+ and pH balance could explain the glycosylation defects observed in TMEM165-deficient patients.

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 tri-functional 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.

Aspartate N-acetyltransferase (NAT8L, N-acetyltransferase 8-like), the enzyme that synthesizes N-acetylaspartate, is membrane-bound and is at least partially associated with the ER (endoplasmic reticulum). The aim of the present study was to determine which regions of the protein are important for its catalytic activity and its subcellular localization. Transfection of truncated forms of NAT8L into HEK (human embryonic kidney)-293T cells indicated that the 68 N-terminal residues (regions 1 and 2) have no importance for the catalytic activity and the subcellular localization of this enzyme, which was exclusively associated with the ER. Mutation of conserved residues that precede (Arg81 and Glu101, in region 3) or follow (Asp168 and Arg220, in region 5) the putative membrane region (region 4) markedly affected the kinetic properties, suggesting that regions 3 and 5 form the catalytic domain and that the membrane region has a loop structure. Evidence is provided for the membrane region comprising α-helices and the catalytic site being cytosolic. Transfection of chimaeric proteins in which GFP (green fluorescent protein) was fused to different regions of NAT8L indicated that the membrane region (region 4) is necessary and sufficient to target NAT8L to the ER. Thus NAT8L is targeted to the ER membrane by a hydrophobic loop that connects two regions of the catalytic domain.

The reduced forms of NAD and NADP, two major nucleotides playing a central role in metabolism, are continuously damaged by enzymatic or heat-dependent hydration. We report the molecular identification of the eukaryotic dehydratase that repairs these nucleotides and show that this enzyme (Carkd in mammals, YKL151C in yeast) catalyzes the dehydration of the S form of NADHX and NADPHX, at the expense of ATP, which is converted to ADP. Surprisingly, the Escherichia coli homolog, YjeF, a bidomain protein, catalyzes a similar reaction, but using ADP instead of ATP. The latter reaction is ascribable to the C-terminal domain of YjeF. This represents an unprecedented example of orthologous enzymes using either ADP or ATP as phosphoryl donor. We also show that eukaryotic proteins homologous to the N-terminal domain of YjeF (apolipoprotein A-1-binding protein (AIBP) in mammals, YNL200C in yeast) catalyze the epimerization of the S and R forms of NAD(P)HX, thereby allowing, in conjunction with the energy-dependent dehydratase, the repair of both epimers of NAD(P)HX. Both enzymes are very widespread in eukaryotes, prokaryotes, and archaea, which together with the ADP dependence of the dehydratase in some species indicates the ancient origin of this repair system.

Pseudouridine, the fifth-most abundant nucleoside in RNA, is not metabolized in mammals, but is excreted intact in urine. The purpose of the present work was to search for an enzyme that would dephosphorylate pseudouridine 5′-phosphate, a potential intermediate in RNA degradation. We show that human erythrocytes contain a pseudouridine-5′-phosphatase displaying a Km ≤ 1 μM for its substrate. The activity of the partially purified enzyme was dependent on Mg2+, and was inhibited by Ca2+ and vanadate, suggesting that it belonged to the ‘haloacid dehalogenase’ family of phosphatases. Its low molecular mass (26 kDa) suggested that this phosphatase could correspond to the protein encoded by the HDHD1 (haloacid dehalogenase-like hydrolase domain-containing 1) gene, present next to the STS (steroid sulfatase) gene on human chromosome Xp22. Purified human recombinant HDHD1 dephosphorylated pseudouridine 5′-phosphate with a kcat of 1.6 s−1, a Km of 0.3 μM and a catalytic efficiency at least 1000-fold higher than that on which it acted on other phosphate esters, including 5′-UMP. The molecular identity of pseudouridine-5′-phosphatase was confirmed by the finding that its activity was negligible (

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 E(4), two cysteine conjugates, but were inactive on other physiological amines or amino acids. Confocal microscopy indicated that NAT8 was associated with the endoplasmic 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 byor=2-fold, whereas a mutation (R149K) replacing an extremely conserved arginine suppressed the activity. Sequencing of genomic DNA and EST clones corresponding to the NAT8B 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.

The brain-specific compound NAA (N-acetylaspartate) occurs almost exclusively in neurons, where its concentration reaches approx. 20 mM. Its abundance is determined in patients by MRS (magnetic resonance spectroscopy) to assess neuronal density and health. The molecular identity of the NAT (N-acetyltransferase) that catalyses NAA synthesis has remained unknown, because the enzyme is membrane-bound and difficult to purify. Database searches indicated that among putative NATs (i.e. proteins homologous with known NATs, but with uncharacterized catalytic activity) encoded by the human and mouse genomes two were almost exclusively expressed in brain, NAT8L and NAT14. Transfection studies in HEK-293T [human embryonic kidney-293 cells expressing the large T-antigen of SV40 (simian virus 40)] indicated that NAT8L, but not NAT14, catalysed the synthesis of NAA from L-aspartate and acetyl-CoA. The specificity of NAT8L, its Km for aspartate and its sensitivity to detergents are similar to those described for brain Asp-NAT. Confocal microscopy analysis of CHO (Chinese-hamster ovary) cells and neurons expressing recombinant NAT8L indicates that it is associated with the ER (endoplasmic reticulum), but not with mitochondria. A mutation search in the NAT8L gene of the only patient known to be deficient in NAA disclosed the presence of a homozygous 19 bp deletion, resulting in a change in reading frame and the absence of production of a functional protein. We conclude that NAT8L, a neuron-specific protein, is responsible for NAA synthesis and is mutated in primary NAA deficiency (hypoacetylaspartia). The molecular identification of this enzyme will lead to new perspectives in the clarification of the function of this most abundant amino acid derivative in neurons and for the diagnosis of hypoacetylaspartia in other patients.

Glucose 1,6-bisphosphate (Glc-1,6-P(2)) 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-P(2) 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 (K(a) approximately 20 mum) 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 approximately 20-fold increase in the concentration of Glc-1,6-P(2). Transfection with PMM1 caused a profound decrease (>5-fold) in Glc-1,6-P(2) in cells that were or were not cotransfected with PGM2L1. Furthermore, the concentration of Glc-1,6-P(2) 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-P(2) in brain. In addition, the role of PGM2L1 as the enzyme responsible for the synthesis of the elevated concentrations of Glc-1,6-P(2) in brain is established.

Our aim was to identify the product formed by sedoheptulokinase and to understand the mechanism of formation of erythritol in patients with sedoheptulokinase deficiency. Mouse recombinant sedoheptulokinase was found to be virtually specific for sedoheptulose and its reaction product was identified as sedoheptulose 7-phosphate. Assays of sedoheptulose in plant extracts disclosed that this sugar is present in carrots ( approximately 7mumol/g) and in several fruits. Sedoheptulose 1-phosphate is shown to be a substrate for aldolase B, which cleaves it to dihydroxyacetone-phosphate and erythrose. This suggests that, in patients deficient in sedoheptulose-7-kinase, sedoheptulose is phosphorylated by fructokinase to sedoheptulose 1-phosphate. Cleavage of the latter by aldolase B would lead to the formation of erythrose, which would then be reduced to erythritol.

The molecular identity of mammalian phosphopentomutase has not yet been established unequivocally. That of glucose-1,6-bisphosphate synthase, the enzyme that synthesizes a cofactor for phosphomutases and putative regulator of glycolysis, is completely unknown. In the present work, we have purified phosphopentomutase from human erythrocytes and found it to copurify with a 68-kDa polypeptide that was identified by mass spectrometry as phosphoglucomutase 2 (PGM2), a protein of the alpha-d-phosphohexomutase family and sharing about 20% identity with mammalian phosphoglucomutase 1. Data base searches indicated that vertebrate genomes contained, in addition to PGM2, a homologue (PGM2L1, for PGM2-like 1) sharing about 60% sequence identity with this protein. Both PGM2 and PGM2L1 were overexpressed in Escherichia coli, purified, and their properties were studied. Using catalytic efficiency as a criterion, PGM2 acted more than 10-fold better as a phosphopentomutase (both on deoxyribose 1-phosphate and on ribose 1-phosphate) than as a phosphoglucomutase. PGM2L1 showed only low (

The purpose of this work was to identify the function of bacterial homologues of fructosamine 3-kinase (FN3K), a mammalian enzyme responsible for the removal of fructosamines from proteins. FN3K homologues were identified in approximately 200 (i.e. approximately 27%) of the sequenced bacterial genomes. In 11 of these genomes, from phylogenetically distant bacteria, the FN3K homologue was immediately preceded by a low-molecular-weight protein-tyrosine-phosphatase (LMW-PTP) homologue, which is therefore probably functionally related to the FN3K homologue. Five bacterial FN3K homologues (from Escherichia coli, Enterococcus faecium, Lactobacillus plantarum, Staphylococcus aureus and Thermus thermophilus) were overexpressed in E. coli, purified and their kinetic properties investigated. Four were ribulosamine/erythrulosamine 3-kinases acting best on free lysine and cadaverine derivatives, but not on ribulosamines bound to the alpha amino group of amino acids. They also phosphorylated protein-bound ribulosamines or erythrulosamines, but not protein-bound fructosamines, therefore having properties similar to those of mammalian FN3K-related protein. The E. coli FN3K homologue (YniA) was inactive on all tested substrates. The LMW-PTP of T. thermophilus, which forms an operon with an FN3K homologue, and an LMW-PTP of S. aureus (PtpA) were overexpressed in E. coli, purified and shown to dephosphorylate not only protein tyrosine phosphates, but protein ribulosamine 5-phosphates as well as free ribuloselysine 5-phosphate and erythruloselysine 4-phosphate. These LMW-PTPs were devoid of ribulosamine 3-phosphatase activity. It is concluded that most bacterial FN3K homologues are ribulosamine/erythrulosamine 3-kinases. They may serve, in conjunction with a phosphatase, to deglycate products of glycation formed from ribose 5-phosphate or erythrose 4-phosphate.

Metabolic enzymes are very specific. However, most of them show weak side activities toward compounds that are structurally related to their physiological substrates, thereby producing side products that may be toxic. In some cases, 'metabolite repair enzymes' eliminating side products have been identified. We show that mammalian glyceraldehyde 3-phosphate dehydrogenase and pyruvate kinase, two core glycolytic enzymes, produce 4-phosphoerythronate and 2-phospho-L-lactate, respectively. 4-Phosphoerythronate strongly inhibits an enzyme of the pentose phosphate pathway, whereas 2-phospho-L-lactate inhibits the enzyme producing the glycolytic activator fructose 2,6-bisphosphate. We discovered that a single, widely conserved enzyme, known as phosphoglycolate phosphatase (PGP) in mammals, dephosphorylates both 4-phosphoerythronate and 2-phospho-L-lactate, thereby preventing a block in the pentose phosphate pathway and glycolysis. Its yeast ortholog, Pho13, similarly dephosphorylates 4-phosphoerythronate and 2-phosphoglycolate, a side product of pyruvate kinase. Our work illustrates how metabolite repair enzymes can make up for the limited specificity of metabolic enzymes and permit high flux in central metabolic pathways.

Vitamin C, a reducing agent and antioxidant, is a cofactor in reactions catalyzed by Cu(+)-dependent monooxygenases and Fe(2+)-dependent dioxygenases. It is synthesized, in vertebrates having this capacity, from d-glucuronate. The latter is formed through direct hydrolysis of uridine diphosphate (UDP)-glucuronate by enzyme(s) bound to the endoplasmic reticulum membrane, sharing many properties with, and most likely identical to, UDP-glucuronosyltransferases. Non-glucuronidable xenobiotics (aminopyrine, metyrapone, chloretone and others) stimulate the enzymatic hydrolysis of UDP-glucuronate, accounting for their effect to increase vitamin C formation in vivo. Glucuronate is converted to l-gulonate by aldehyde reductase, an enzyme of the aldo-keto reductase superfamily. l-Gulonate is converted to l-gulonolactone by a lactonase identified as SMP30 or regucalcin, whose absence in mice leads to vitamin C deficiency. The last step in the pathway of vitamin C synthesis is the oxidation of l-gulonolactone to l-ascorbic acid by l-gulonolactone oxidase, an enzyme associated with the endoplasmic reticulum membrane and deficient in man, guinea pig and other species due to mutations in its gene. Another fate of glucuronate is its conversion to d-xylulose in a five-step pathway, the pentose pathway, involving identified oxidoreductases and an unknown decarboxylase. Semidehydroascorbate, a major oxidation product of vitamin C, is reconverted to ascorbate in the cytosol by cytochrome b(5) reductase and thioredoxin reductase in reactions involving NADH and NADPH, respectively. Transmembrane electron transfer systems using ascorbate or NADH as electron donors serve to reduce semidehydroascorbate present in neuroendocrine secretory vesicles and in the extracellular medium. Dehydroascorbate, the fully oxidized form of vitamin C, is reduced spontaneously by glutathione, as well as enzymatically in reactions using glutathione or NADPH. The degradation of vitamin C in mammals is initiated by the hydrolysis of dehydroascorbate to 2,3-diketo-l-gulonate, which is spontaneously degraded to oxalate, CO(2) and l-erythrulose. This is at variance with bacteria such as Escherichia coli, which have enzymatic degradation pathways for ascorbate and probably also dehydroascorbate.

FN3K (fructosamine 3-kinase) is a mammalian enzyme that catalyses the phosphorylation of fructosamines, which thereby becomes unstable and detaches from proteins. The homologous mammalian enzyme, FN3K-RP (FN3K-related protein), does not phosphorylate fructosamines but ribulosamines, which are probably formed through a spontaneous reaction of amines with ribose 5-phosphate, an intermediate of the pentose-phosphate pathway and the Calvin cycle. We show in the present study that spinach leaf extracts display a substantial ribulosamine kinase activity (approx. 700 times higher than the specific activity of FN3K in erythrocytes). The ribulosamine kinase was purified approx. 400 times and shown to phosphorylate ribulose-epsilon-lysine, protein-bound ribulosamines and also, with higher affinity, erythrulose-epsilon-lysine and protein-bound erythrulosamines. Evidence is presented for the fact that the third carbon of the sugar portion is phosphorylated by this enzyme and that this leads to the formation of unstable compounds decomposing with half-lives of approx. 30 min at 37 degrees C (ribulosamine 3-phosphates) and 5 min at 30 degrees C (erythrulosamine 3-phosphates). This decomposition results in the formation of a 2-oxo-3-deoxyaldose and inorganic phosphate, with regeneration of the free amino group. The Arabidopsis thaliana homologue of FN3K/FN3K-RP was overexpressed in Escherichia coli and shown to have properties similar to those of the enzyme purified from spinach leaves. These results indicate that the plant FN3K/FN3K-RP homologue, which appears to be targeted to the chloroplast in many species, is a ribulosamine/erythrulosamine 3-kinase. This enzyme may participate in a protein deglycation process removing Amadori products derived from ribose 5-phosphate and erythrose 4-phosphate, two Calvin cycle intermediates that are potent glycating agents.

Fructosamine 3-kinase (FN3K) and FN3K-related protein (FN3K-RP) catalyze the phosphorylation of the Amadori products ribulosamines, psicosamines, and, in the case of FN3K, fructosamines. BLAST searches in chordate genomes revealed two genes encoding proteins homologous to FN3K or FN3K-RP in various mammals and in chicken but only one gene, encoding a protein more similar to FN3K-RP than to FN3K, in fishes and the sea squirt Ciona intestinalis. This suggests that a gene duplication event occurred after the fish radiation and that the FN3K gene evolved more rapidly than the FN3K-RP gene. In agreement with this distribution, only one enzyme, phosphorylating ribulosamines and psicosamines but not fructosamines, was found in the tissues from a fish (Clarias gariepinus), whereas two enzymes with specificities similar to either FN3K or FN3K-RP were found in mouse, rat, and chicken tissues. FN3K is particularly active in brain, heart, kidney, and skeletal muscle. Its activity is also relatively elevated in erythrocytes from man, rat, and mouse but barely detectable in erythrocytes from chicken and pig, which correlates well with the low intracellular concentration of glucose in erythrocytes from these species. This is in keeping with the specific role of FN3K to repair protein damage caused by glucose. FN3K-RP was more evenly distributed in tissues, except for skeletal muscle where its activity was particularly low. This may be related to low activity of the pentose phosphate pathway in this tissue, as suggested by assays of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase. This finding, together with the high affinity of FN3K-RP for ribulosamines, suggests that this enzyme may serve to repair damage caused by the powerful glycating agent, ribose 5-phosphate.

Fructosamine-3-kinase (FN3K) phosphorylates fructosamine residues, leading to their destabilization and their shedding from protein. Support for the occurrence of this deglycation mechanism in intact cells has been obtained by showing that hemoglobin is significantly more glycated when human erythrocytes are incubated with an elevated glucose concentration in the presence of 1-deoxy-1-morpholinofructose (DMF), a cell-permeable inhibitor of FN3K, than in its absence. The aim of this work was to identify the fructosamine residues on hemoglobin that are removed as a result of the action of FN3K in intact erythrocytes. Highly glycated hemoglobin derived from intact human erythrocytes incubated for 48 h with 200 mm glucose and DMF was incubated in vitro with FN3K and [gamma-(32)P]ATP. After reduction of fructosamine 3-phosphates with borohydride, the protein was digested with trypsin. Peptides were separated by reversed-phase high-performance liquid chromatography, and the radioactive peaks were analyzed by mass spectrometry. Nine different modified residues were identified. These were Lys-alpha-16, Lys-alpha-61, Lys-alpha-139, Val-beta-1, Lys-beta-17, Lys-beta-59, Lys-beta-66, Lys-beta-132, and Lys-beta-144. Some (e.g. Lys-alpha-139) were readily phosphorylated to a maximal extent by FN3K in vitro whereas others (e.g. Val-beta-1) were slowly and only very partially phosphorylated. The radiolabeled peptides containing reduced fructosamine 3-phosphates bound to Lys-alpha-16, Lys-alpha-139, and Lys-beta-17 were much less abundant if the hemoglobin substrate used for the in vitro phosphorylation with FN3K and [gamma-(32)P]ATP came from erythrocytes incubated with an elevated glucose concentration in the absence of DMF, indicating that these lysine residues had been substantially deglycated in intact cells when FN3K action was unrefrained. Other residues (e.g. Val-beta-1, Lys-alpha-61) seemed to be insignificantly deglycated in intact cells.

The frl (fructoselysine) operon encodes fructoselysine 6-kinase and fructoselysine 6-phosphate deglycase, allowing the conversion of fructoselysine into glucose 6-phosphate and lysine. We now show that a third enzyme encoded by this operon catalyses the metal-dependent reversible interconversion of fructoselysine with its C-3 epimer, psicoselysine. The enzyme can be easily assayed through the formation of tritiated water from [3-3H]fructoselysine. Psicoselysine supports the growth of Escherichia coli, causing the induction of the three enzymes of the frl operon. No growth on fructoselysine or psicoselysine was observed with Tn5 mutants in which the putative transporter (FrlA) or fructoselysine 6-phosphate deglycase (FrlB) had been inactivated, indicating the importance of the frl operon for the metabolism of both substrates. The ability of E. coli to grow on psicoselysine suggests the occurrence of this unusual Amadori compound in Nature.

Vitamin C synthesis in rat liver is enhanced by several xenobiotics, including aminopyrine and chloretone. The effect of these agents has been linked to induction of enzymes potentially involved in the formation of glucuronate, a precursor of vitamin C. Using isolated rat hepatocytes as a model, we show that a series of agents (aminopyrine, antipyrine, chloretone, clotrimazole, metyrapone, proadifen, and barbital) induced in a few minutes an up to 15-fold increase in the formation of glucuronate, which was best observed in the presence of sorbinil, an inhibitor of glucuronate reductase. They also caused an approximately 2-fold decrease in the concentration of UDP-glucuronate but little if any change in the concentration of UDP-glucose. Depletion of UDP-glucuronate with resorcinol or d-galactosamine markedly decreased the formation of glucuronate both in the presence and in the absence of aminopyrine, confirming the precursor-product relationship between UDP-glucuronate and free glucuronate. Most of the agents did not induce the formation of detectable amounts of glucuronides, indicating that the formation of glucuronate is not due to a glucuronidation-deglucuronidation cycle. With the exception of barbital (which inhibits glucuronate reductase), all of the above mentioned agents also caused an increase in the concentration of ascorbic acid. They had little effect on glutathione concentration, and their effect on glucuronate and vitamin C formation was not mimicked by glutathione-depleting agents such as diamide and buthionine sulfoximine. It is concluded that the stimulation of vitamin C synthesis exerted by some xenobiotics is mediated through a rapid increase in the conversion of UDP-glucuronate to glucuronate, which does not apparently involve a glucuronidation-deglucuronidation cycle.

The existence of glucose-6-phosphate transport across the liver microsomal membrane is still controversial. In this paper, we show that S3483, a chlorogenic acid derivative known to inhibit glucose-6-phosphatase in intact microsomes, caused the intravesicular accumulation of glucose-6-phosphate when the latter was produced by glucose-6-phosphatase from glucose and carbamoyl-phosphate. S3483 also inhibited the conversion of glucose-6-phosphate to 6-phosphogluconate occurring inside microsomes in the presence of electron acceptors (NADP or metyrapone). These data indicate that liver microsomal membranes contain a reversible glucose-6-phosphate transporter, which furnishes substrate not only to glucose-6-phosphatase, but also to hexose-6-phosphate dehydrogenase.

Glucokinase is inhibited in the liver by a regulatory protein (GKRP) whose effects are increased by Fru-6-P and suppressed by Fru-1-P. To identify the binding site of these phosphate esters, we took advantage of the homology of GKRP to the isomerase domain of GlmS (glucosamine-6-phosphate synthase) and created 12 different mutants of rat GKRP. Mutations of three residues predicted to bind to Fru-6-P resulted in proteins that were approximately 5-fold (S110A) and 50-fold (S179A and K514A) less potent as inhibitors of glucokinase and had an at least 100-fold reduced affinity for the effectors. Mutation of another residue of the putative binding site (T109A) resulted in a 10-fold decrease in the inhibitory power and an inversion of the effect of sorbitol-6-P, a Fru-6-P analog. The replacement of Gly(107), a residue close to the binding site, by cysteine (as in GlmS and Xenopus GKRP) resulted in a protein that had 20 times more affinity for Fru-6-P and 30 times less affinity for Fru-1-P. These results are consistent with GKRP having one single binding site for phosphate esters. They also show that a missense mutation of GKRP can lead to a gain of function.

Hydration of NAD(P)H to NAD(P)HX, which inhibits several dehydrogenases, is corrected by an ATP-dependent dehydratase and an epimerase recently identified as the products of the vertebrate Carkd (carbohydrate kinase domain) and Aibp (apolipoprotein AI-binding protein) genes respectively. The purpose of the present study was to assess the presence of these enzymes in mammalian tissues and determine their subcellular localization. The Carkd gene encodes proteins with a predicted mitochondrial propeptide (mCARKD), a signal peptide (spCARKD) or neither of them (cCARKD). Confocal microscopy analysis of transfected CHO (Chinese-hamster ovary) cells indicated that cCARKD remains in the cytosol, whereas mCARKD and spCARKD are targeted to the mitochondria and the endoplasmic reticulum respectively. Unlike the other two forms, spCARKD is N-glycosylated, supporting its targeting to the endoplasmic reticulum. The Aibp gene encodes two different proteins, which we show to be targeted to the mitochondria (mAIBP) and the cytosol (cAIBP). Quantification of the NAD(P)HX dehydratase and epimerase activities in rat tissues, performed after partial purification, indicated that both enzymes are widely distributed, with total activities of ≈3–10 nmol/min per g of tissue. Liver fractionation by differential centrifugation confirmed the presence of the dehydratase and the epimerase in the cytosol and in mitochondria. These data support the notion that NAD(P)HX repair is extremely widespread.

The p53-induced protein TIGAR [TP53 (tumour protein 53)-induced glycolysis and apoptosis regulator] is considered to be a F26BPase (fructose-2,6-bisphosphatase) with an important role in cancer cell metabolism. The reported catalytic efficiency of TIGAR as an F26BPase is several orders of magnitude lower than that of the F26BPase component of liver or muscle PFK2 (phosphofructokinase 2), suggesting that F26BP (fructose 2,6-bisphosphate) might not be the physiological substrate of TIGAR. We therefore set out to re-evaluate the biochemical function of TIGAR. Phosphatase activity of recombinant human TIGAR protein was tested on a series of physiological phosphate esters. The best substrate was 23BPG (2,3-bisphosphoglycerate), followed by 2PG (2-phosphoglycerate), 2-phosphoglycolate and PEP (phosphoenolpyruvate). In contrast the catalytic efficiency for F26BP was approximately 400-fold lower than that for 23BPG. Using genetic and shRNA-based cell culture models, we show that loss of TIGAR consistently leads to an up to 5-fold increase in the levels of 23BPG. Increases in F26BP levels were also observed, albeit in a more limited and cell-type dependent manner. The results of the present study challenge the concept that TIGAR acts primarily on F26BP. This has significant implications for our understanding of the metabolic changes downstream of p53 as well as for cancer cell metabolism in general. It also suggests that 23BPG might play an unrecognized function in metabolic control.

Using overexpressed Escherichia coli sorbitol-6-phosphate dehydrogenase to monitor fructose 6-phosphate formation, we found that the stimulation of fructose phosphorylation by glucose was reduced in two human beta-cell glucokinase mutants with a low Hill coefficient or when the activity of wild type glucokinase was decreased by replacing ATP with poorer nucleotide substrates. Mutation of two other residues, neighboring glucose-binding residues in the catalytic site, also reduced the affinity for glucose as a stimulator of fructose phosphorylation. Among a series of glucose analogs, only 3, all substrates of glucokinase, stimulated fructose phosphorylation; other analogs were either inactive or inhibited glucokinase. Glucose increased the apparent affinity for inhibitors that are glucose analogs but not for the glucokinase regulatory protein or palmitoyl-CoA. These data indicate that the stimulatory effect of glucose on fructose phosphorylation reflects the positive cooperativity for glucose and is mediated by binding of glucose to the catalytic site. They support models involving the existence of two slowly interconverting conformations of glucokinase that differ through their affinity for glucose and for glucose analogs. We show by computer simulation that such a model can account for the kinetic properties of glucokinase, including the differential ability of mannoheptulose and N-acetylglucosamine to suppress cooperativity (Agius, L., and Stubbs, M. (2000) Biochem. J. 346, 413-421).

Intact human erythrocytes catalyse the conversion of fructose into fructose 3-phosphate with an apparent K(m) of 30 mM [Petersen, Kappler, Szwergold and Brown (1992) Biochem. J. 284, 363-366]. The physiological significance of this process is still unknown. In the present study we report that the formation of fructose 3-phosphate from 50 mM fructose in intact erythrocytes is inhibited by 1-deoxy-1-morpholinofructose (DMF), a synthetic fructosamine, with an apparent K(i) of 100 microM. (31)P NMR analysis of cell extracts incubated with DMF indicated the presence of an additional phosphorylated compound, which was partially purified and shown to be DMF 3-phosphate by tandem MS. Radiolabelled DMF was phosphorylated by intact erythrocytes with an apparent K(m) ( approximately 100 microM) approx. 300-fold lower than the value reported for fructose phosphorylation on its third carbon. These results indicate that the physiological function of the enzyme that is able to convert fructose into fructose 3-phosphate in intact erythrocytes is probably to phosphorylate fructosamines. This suggests that fructosamines, which are produced non-enzymically from glucose and amino compounds, may be metabolized in human erythrocytes.

Phosphoserine phosphatase belongs to a new class of phosphotransferases forming an acylphosphate during catalysis and sharing three motifs with P-type ATPases and haloacid dehalogenases. The phosphorylated residue was identified as the first aspartate in the first motif (DXDXT) by mass spectrometry analysis of peptides derived from the phosphorylated enzyme treated with NaBH(4) or alkaline [(18)O]H(2)O. Incubation of native phosphoserine phosphatase with phosphoserine in [(18)O]H(2)O did not result in (18)O incorporation in residue Asp-20, indicating that the phosphoaspartate is hydrolyzed, as in P-type ATPases, by attack of the phosphorus atom. Mutagenesis studies bearing on conserved residues indicated that four conservative changes either did not affect (S109T) or caused a moderate decrease in activity (G178A, D179E, and D183E). Other mutations inactivated the enzyme by >80% (S109A and G180A) or even by >/=99% (D179N, D183N, K158A, and K158R). Mutations G178A and D179N decreased the affinity for phosphoserine, suggesting that these residues participate in the binding of the substrate. Mutations of Asp-179 decreased the affinity for Mg(2+), indicating that this residue interacts with the cation. Thus, investigated residues appear to play an important role in the reaction mechanism of phosphoserine phosphatase, as is known for equivalent residues in P-type ATPases and haloacid dehalogenases.

We report the sequence of a human cDNA encoding a protein homologous to devB (a bacterial gene often found in proximity to the gene encoding glucose-6-phosphate dehydrogenase in bacterial genomes) and to the C-terminal part of human hexose-6-phosphate dehydrogenase. The protein was expressed in Escherichia coli, purified and shown to be 6-phosphogluconolactonase, the enzyme catalyzing the second step of the pentose phosphate pathway. Sequence analysis indicates that bacterial devB proteins, the C-terminal part of hexose-6-phosphate dehydrogenase and yeast Sol1-4 proteins are most likely also 6-phosphogluconolactonases and that these proteins are related to glucosamine-6-phosphate isomerases.

We report the sequence of a human cDNA that encodes a 46 kDa transmembrane protein homologous to bacterial transporters for phosphate esters. This protein presents at its carboxy terminus the consensus motif for retention in the endoplasmic reticulum. Northern blots of rat tissues indicate that the corresponding mRNA is mostly expressed in liver and kidney. In two patients with glycogen storage disease type Ib, mutations were observed that either replaced a conserved Gly to Cys or introduced a premature stop codon. The encoded protein is therefore most likely the glucose 6-phosphate translocase that is functionally associated with glucose-6-phosphatase.

Carbohydrate-deficient glycoprotein syndrome type I (CDGI) is most often due to phosphomannomutase deficiency; paradoxically, the human phosphomannomutase gene PMM1 is located on chromosome 22, whereas the CDGI locus is on chromosome 16. We show that phosphomannomutases present in rat or human liver share with homogeneous recombinant PMM1 several kinetic properties and the ability to form an alkali- and NH2OH-sensitive phosphoenzyme with a subunit mass of approximately 30,000 Mr. However, they have a higher affinity for the activator mannose-1,6-bisphosphate than PMM1 and are not recognized by anti-PMM1 antibodies, indicating that they represent a related but different isozyme. Phosphomannomutases belong to a novel mutase family in which the active residue is a phosphoaspartyl or a phosphoglutamyl.

Rat liver d-3-phosphoglycerate dehydrogenase was purified to homogeneity and digested with trypsin, and the sequences of two peptides were determined. This sequence information was used to screen a rat hepatoma cDNA library. Among 11 positive clones, two covered the whole coding sequence. The deduced amino acid sequence (533 residues; Mr 56493) shared closer similarity with Bacillus subtilis 3-phosphoglycerate dehydrogenase than with the enzymes from Escherichia coli, Haemophilus influenzae and Saccharomyces cerevisiae. In all cases the similarity was most apparent in the substrate- and NAD+-binding domains, and low or insignificant in the C-terminal domain. A corresponding 2.1 kb mRNA was present in rat tissues including kidney, brain and testis, whatever the dietary status, and also in livers of animals fed a protein-free, carbohydrate-rich diet, but not in livers of control rats, suggesting transcriptional regulation. The full-length rat 3-phosphoglycerate dehydrogenase was expressed in E. coli and purified. The recombinant enzyme and the protein purified from liver displayed hyperbolic kinetics with respect to 3-phosphoglycerate, NAD+ and NADH, but substrate inhibition by 3-phosphohydroxypyruvate was observed; this inhibition was antagonized by salts. Similar properties were observed with a truncated form of 3-phosphoglycerate dehydrogenase lacking the C-terminal domain, indicating that the latter is not implicated in substrate inhibition or in salt effects. By contrast with the bacterial enzyme, rat 3-phosphoglycerate dehydrogenase did not catalyse the reduction of 2-oxoglutarate, indicating that this enzyme is not involved in human d- or l-hydroxyglutaric aciduria.

To delineate the regions of liver glucokinase that are involved in the binding of its regulatory protein and have therefore been conserved throughout evolution, we have cloned the cDNA of the Xenopus laevis enzyme. It contains an open reading frame of 1374 nucleotides and encodes a protein of 458 amino acids, which displays 78 and 79% overall identity to rat and human liver glucokinases, respectively. The conserved regions are predicted to be present mainly in the small domain and the hinge region of glueokinase, and the nonconserved regions in the large domain of the enzyme. We constructed five mutants of Xenopus glucokinase by replacing sets of 2-5 glucokinase-specific residues with their counterparts in the C-terminal half of rat hexokinase I. The affinity for the regulatory protein was not markedly changed for mutants B, D, and E despite a decreased affinity for glucose in mutants B and D. Two other mutants (A and C) were 9- and 250-fold less sensitive to the rat regulator and 40- and 770-fold less sensitive to the Xenopus regulator, respectively, but presented a normal affinity for glucose. The double mutant (A-C) was completely insensitive to inhibition by the regulatory protein. A control mutant (F), obtained by replacing 3 residues that were not conserved in all glucokinases, had a normal affinity for glucose and for the regulatory protein. The property of glucokinase to be inhibited by palmitoyl-CoA was not affected by the mutations described. It is concluded that His-141 to Leu-144, which are located close to the tip of the small domain, as well as Glu-51 and Glu-52, which are present in the large domain of the enzyme close to the hinge region, or nearby residues participate in the binding of the regulatory protein.

It is increasingly evident that metabolites suffer various kinds of damage, that such damage happens in all organisms and that cells have dedicated systems for damage repair and containment. First, chemical biology is demonstrating that diverse metabolites are damaged by side reactions of 'promiscuous' enzymes or by spontaneous chemical reactions, that the products are useless or toxic and that the unchecked buildup of these products can be devastating. Second, genetic and genomic evidence from prokaryotes and eukaryotes is implicating a network of new, conserved enzymes that repair damaged metabolites or somehow pre-empt damage. Metabolite (that is, small-molecule) repair is analogous to macromolecule (DNA and protein) repair and seems from comparative genomic evidence to be equally widespread. Comparative genomics also implies that metabolite repair could be the function of many conserved protein families lacking known activities. How--and how well--cells deal with metabolite damage affects fields ranging from medical genetics to metabolic engineering.

A limited number of enzymes are known that play a role analogous to DNA proofreading by eliminating non-classical metabolites formed by side activities of enzymes of intermediary metabolism. Because few such "metabolite proofreading enzymes" are known, our purpose was to search for an enzyme able to degrade ethylmalonyl-CoA, a potentially toxic metabolite formed at a low rate from butyryl-CoA by acetyl-CoA carboxylase and propionyl-CoA carboxylase, two major enzymes of lipid metabolism. We show that mammalian tissues contain a previously unknown enzyme that decarboxylates ethylmalonyl-CoA and, at lower rates, methylmalonyl-CoA but that does not act on malonyl-CoA. Ethylmalonyl-CoA decarboxylase is particularly abundant in brown adipose tissue, liver, and kidney in mice, and is essentially cytosolic. Because Escherichia coli methylmalonyl-CoA decarboxylase belongs to the family of enoyl-CoA hydratase (ECH), we searched mammalian databases for proteins of uncharacterized function belonging to the ECH family. Combining this database search approach with sequencing data obtained on a partially purified enzyme preparation, we identified ethylmalonyl-CoA decarboxylase as ECHDC1. We confirmed this identification by showing that recombinant mouse ECHDC1 has a substantial ethylmalonyl-CoA decarboxylase activity and a lower methylmalonyl-CoA decarboxylase activity but no malonyl-CoA decarboxylase or enoyl-CoA hydratase activity. Furthermore, ECHDC1-specific siRNAs decreased the ethylmalonyl-CoA decarboxylase activity in human cells and increased the formation of ethylmalonate, most particularly in cells incubated with butyrate. These findings indicate that ethylmalonyl-CoA decarboxylase may correct a side activity of acetyl-CoA carboxylase and suggest that its mutation may be involved in the development of certain forms of ethylmalonic aciduria.

β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.

The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate, a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress in the understanding of the function of NAAG and β-citrylglutamate.

Carnosine (beta-alanyl-L-histidine) and homocarnosine (gamma-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 beta-alanine than on gamma-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.

International audience; Mammalian glucokinase regulatory protein (GKRP), a fructose 6-phosphate and fructose 1-phosphate sensitive inhibitor of glucokinase (GK), appears to have resulted from the duplication of a gene similar to bacterial N-acetylmuramate 6-phosphate etherase (MurQ). In the present work, we show that several genomes of primitive eukaryotes encode a GKRP-like protein with two MurQ repeats. Recombinant Haemophilus influenzae MurQ and the GKRP homologue of the amoebaflagellate Naegleria gruberi both behaved as excellent N-acetylmuramate 6-phosphate etherases, with Kcat values (83 and 20 sec-1) at least as high as that reported for Escherichia coli MurQ. By contrast, rat and xenopus GKRP displayed much lower etherase activities (Kcat = 0.08 and 0.05 sec-1, respectively). The etherase activity of rat GKRP was inhibited by ligands (fructose 6-phosphate, fructose 1-phosphate, sorbitol 6-phosphate) known to regulate its interaction with GK and by mutations affecting the binding of these phosphate esters. This indicated that these phosphate esters all bind to a single regulatory site, which evolved from the original catalytic site. Sorbitol 6-phosphate and other phosphate esters also inhibited the etherase activity of xenopus GKRP, but did not affect its ability to inhibit GK. Thus, unlike what was previously thought, xenopus GKRP has a binding site for phosphate esters, but this site is uncoupled from the GK binding site. Taken together, these data indicate that duplication of the murQ gene led to a eukaryotic type etherase, which subsequently evolved to GKRP by acquiring a new binding specificity whilst losing most of its etherase activity.