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Brucellosis is a bacterial zoonosis caused by the genus Brucella , which mainly affects domestic animals. In these natural hosts, brucellae display a tropism towards the reproductive organs, such as the placenta, replicating in high numbers and leading to placentitis and abortion, an ability also exerted by the B. melitensis live-attenuated Rev1 strain, the only vaccine available for ovine brucellosis. It is broadly accepted that this tropism is mediated, at least in part, by the presence of certain preferred nutrients in the placenta, particularly erythritol, a polyol that is ultimately incorporated into the Brucella central carbon metabolism via two reactions dependent on transaldolase (Tal) or fructose-bisphosphate aldolase (Fba). In the light of these remarks, we propose that blocking the incorporation of erythritol into the central carbon metabolism of Rev1 by deleting the genes encoding Tal and Fba may impair the ability of the vaccine to proliferate massively in the placenta. Therefore, a Rev1Δ fba Δ tal double mutant was generated and confirmed to be unable to use erythritol. This mutant exhibited a reduced intracellular fitness both in BeWo trophoblasts and THP-1 macrophages. In the murine model, Rev1Δ fba Δ tal provided comparable protection to the Rev1 reference vaccine while inducing fewer adverse reproductive events in pregnant animals. Altogether, these results postulate the Rev1Δ fba Δ tal mutant as a reproductively safer Rev1-derived vaccine candidate to be studied in the natural host.

Trypanosoma brucei is a causative agent of the Human and Animal African Trypanosomiases. The mammalian stage parasites infect various tissues and organs including the bloodstream, central nervous system, skin, adipose tissue and lungs. They rely on ATP produced in glycolysis, consuming large amounts of glucose, which is readily available in the mammalian host. In addition to glucose, glycerol can also be used as a source of carbon and ATP and as a substrate for gluconeogenesis. However, the physiological relevance of glycerol-fed gluconeogenesis for the mammalian-infective life cycle forms remains elusive. To demonstrate its (in)dispensability, first we must identify the enzyme(s) of the pathway. Loss of the canonical gluconeogenic enzyme, fructose-1,6-bisphosphatase, does not abolish the process hence at least one other enzyme must participate in gluconeogenesis in trypanosomes. Using a combination of CRISPR/Cas9 gene editing and RNA interference, we generated mutants for four enzymes potentially capable of contributing to gluconeogenesis: fructose-1,6-bisphoshatase, sedoheptulose-1,7-bisphosphatase, phosphofructokinase and transaldolase, alone or in various combinations. Metabolomic analyses revealed that flux through gluconeogenesis was maintained irrespective of which of these genes were lost. Our data render unlikely a previously hypothesised role of a reverse phosphofructokinase reaction in gluconeogenesis and preclude the participation of a novel biochemical pathway involving transaldolase in the process. The sustained metabolic flux in gluconeogenesis in our mutants, including a triple-null strain, indicates the presence of a unique enzyme participating in gluconeogenesis. Additionally, the data provide new insights into gluconeogenesis and the pentose phosphate pathway, and improve the current understanding of carbon metabolism of the mammalian-infective stages of T. brucei., Competing Interests: The authors have declared that no competing interests exist., (Copyright: © 2024 Kovářová et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.)

In atherosclerosis, macrophage-derived foam cell formation is considered to be a hallmark of the pathological process; this occurs via the uptake of modified lipoproteins. In the present study, we aim to determine the role of transaldolase in foam cell formation and atherogenesis and reveal the mechanisms underlying its role. Bone marrow-derived macrophages (BMDMs) isolated from mice successfully form foam cells after treatment with oxidized low-density lipoprotein (80 μg/mL). Elevated transaldolase levels in the foam cell model are assessed by quantitative polymerase chain reaction and western blot analysis. Transaldolase overexpression and knockdown in BMDMs are achieved via plasmid transfection and small interfering RNA technology, respectively. We find that transaldolase overexpression effectively attenuates, whereas transaldolase knockdown accelerates, macrophage-derived foam cell formation through the inhibition or activation of cholesterol uptake mediated by the scavenger receptor cluster of differentiation 36 (CD36) in a p38 mitogen-activated protein kinase (MAPK) signaling-dependent manner. Transaldolase-mediated glutathione (GSH) homeostasis is identified as the upstream regulator of p38 MAPK-mediated CD36-dependent cholesterol uptake in BMDMs. Transaldolase upregulates GSH production, thereby suppressing p38 activity and reducing the CD36 level, ultimately preventing foam cell formation and atherosclerosis. Thus, our findings indicate that the transaldolase-GSH-p38-CD36 axis may represent a promising therapeutic target for atherosclerosis.

Sulfoquinovose (SQ) is a key component of plant sulfolipids (sulfoquinovosyl diacylglycerols) and a major environmental reservoir of biological sulfur. Breakdown of SQ is achieved by bacteria through the pathways of sulfoglycolysis. The sulfoglycolytic sulfofructose transaldolase (sulfo-SFT) pathway is used by gut-resident firmicutes and soil saprophytes. After isomerization of SQ to sulfofructose (SF), the namesake enzyme catalyzes the transaldol reaction of SF transferring dihydroxyacetone to 3C/4C acceptors to give sulfolactaldehyde and fructose-6-phosphate or sedoheptulose-7-phosphate. We report the 3D cryo-EM structure of SF transaldolase from Bacillus megaterium in apo and ligand bound forms, revealing a decameric structure formed from two pentameric rings of the protomer. We demonstrate a covalent "Schiff base" intermediate formed by reaction of SF with Lys89 within a conserved Asp-Lys-Glu catalytic triad and defined by an Arg-Trp-Arg sulfonate recognition triad. The structural characterization of the signature enzyme of the sulfo-SFT pathway provides key insights into molecular recognition of the sulfonate group of sulfosugars., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The Author(s). Published by Elsevier Ltd.. All rights reserved.)

Sulfoquinovose (SQ, 6-deoxy-6-sulfo-D-glucose) is a sulfo-sugar with a ubiquitous distribution in the environment due to its production by plants and other photosynthetic organisms. Bacteria play an important role in degradation of SQ and recycling of its constituent sulfur and carbon. Since its discovery in 1963, SQ was noted to have a structural resemblance to glucose-6-phosphate and proposed to be degraded through a pathway analogous to glycolysis, termed sulfoglycolysis. Studies in recent years have uncovered an unexpectedly diverse array of sulfoglycolytic pathways in different bacteria, including one analogous to the Embden-Meyerhof-Parnas pathway (sulfo-EMP), one analogous to the Entner-Doudoroff pathway (sulfo-ED), and two involving sulfo-sugar cleavage by a transaldolase (sulfo-TAL) and transketolase (sulfo-TK), respectively, analogous to reactions in the pentose phosphate (PP) pathway. In addition, a non-sulfoglycolytic SQ degradation pathway was also reported, involving oxygenolytic C-S cleavage catalyzed by a homolog of alkanesulfonate monooxygenase (sulfo-ASMO). Here, we review the discovery of these new mechanisms of SQ degradation and lessons learnt in the study of new catabolic enzymes and pathways in bacteria., (© 2022 The Author(s).)

Metabolic pathways underpin the growth and virulence of intracellular parasites and are therefore promising antiparasitic targets. The pentose phosphate pathway (PPP) is vital in most organisms, providing a reduced form of nicotinamide adenine dinucleotide phosphate (NADPH) and ribose sugar for nucleotide synthesis; however, it has not yet been studied in Toxoplasma gondii, a widespread intracellular pathogen and a model protozoan organism. Herein, we show that T. gondii has a functional PPP distributed in the cytoplasm and nucleus of its acutely-infectious tachyzoite stage. We produced eight parasite mutants disrupting seven enzymes of the PPP in T. gondii. Our data show that of the seven PPP proteins, the two glucose-6-phosphate dehydrogenases (TgG6PDH1, TgG6PDH2), one of the two 6-phosphogluconate dehydrogenases (Tg6PGDH1), ribulose-5-phosphate epimerase (TgRuPE) and transaldolase (TgTAL) are dispensable in vitro as well as in vivo, disclosing substantial metabolic plasticity in T. gondii. Among these, TgG6PDH2 plays a vital role in defense against oxidative stress by the pathogen. Further, we show that Tg6PGDH2 and ribulose-5-phosphate isomerase (TgRPI) are critical for tachyzoite growth. The depletion of TgRPI impairs the flux of glucose in central carbon pathways, and causes decreased expression of ribosomal, microneme and rhoptry proteins. In summary, our results demonstrate the physiological need of the PPP in T. gondii while unraveling metabolic flexibility and antiparasitic targets., Competing Interests: The authors have declared that no competing interests exist.

In many animals, progression of developmental stages is temporally controlled by steroid hormones. In Drosophila, the level of ecdysone titer oscillates and developmental stage transitions, such as larval molting and metamorphosis, are induced at each of ecdysone peaks. Ecdysone titer also peaks at the stage of mid-embryogenesis and the embryonic ecdysone is necessary for morphogenesis of several organs, although the regulatory mechanisms of embryonic organogenesis dependent on ecdysone signaling are still open questions. In this study, we find that absence or interruption of embryonic ecdysone signaling caused multiple defects in the tracheal system, including decrease in luminal protein deposition, uneven dilation of the dorsal trunk and loss of terminal branches. We also reveal that an ecdysone-inducible gene polished rice (pri) is essential for tip cell fate decision in dorsal branches. As over-expression of pri can restore the defects caused by disturbance of ecdysone biosynthesis, pri functions as one of the major mediators of embryonic ecdysone signal in tracheogenesis. These results demonstrate that ecdysone and its downstream target pri play essential roles in tracheal development by modulating cell fate decision., (© 2021 Molecular Biology Society of Japan and John Wiley & Sons Australia, Ltd.)

Disulfide bonds between cysteine residues are important post-translational modifications in proteins that have critical roles for protein structure and stability, as redox-active catalytic groups in enzymes or allosteric redox switches that govern protein function 1-4 . In addition to forming disulfide bridges, cysteine residues are susceptible to oxidation by reactive oxygen species, and are thus central not only to the scavenging of these but also to cellular signalling and communication in biological as well as pathological contexts 5,6 . Oxidized cysteine species are highly reactive and may form covalent conjugates with, for example, tyrosines in the active sites of some redox enzymes 7,8 . However, to our knowledge, regulatory switches with covalent crosslinks other than disulfides have not previously been demonstrated. Here we report the discovery of a covalent crosslink between a cysteine and a lysine residue with a NOS bridge that serves as an allosteric redox switch in the transaldolase enzyme of Neisseria gonorrhoeae, the pathogen that causes gonorrhoea. X-ray structure analysis of the protein in the oxidized and reduced state reveals a loaded-spring mechanism that involves a structural relaxation upon redox activation, which is propagated from the allosteric redox switch at the protein surface to the active site in the protein interior. This relaxation leads to a reconfiguration of key catalytic residues and elicits an increase in enzymatic activity of several orders of magnitude. The redox switch is highly conserved in related transaldolases from other members of the Neisseriaceae; for example, it is present in the transaldolase of Neisseria meningitides (a pathogen that is the primary cause of meningitis and septicaemia in children). We surveyed the Protein Data Bank and found that the NOS bridge exists in diverse protein families across all domains of life (including Homo sapiens) and that it is often located at catalytic or regulatory hotspots. Our findings will inform strategies for the design of proteins and peptides, as well as the development of new classes of drugs and antibodies that target the lysine-cysteine redox switch 9,10 .

The microbial degradation of pentoses in the human gut is a crucial factor for the utilization of plant-based dietary fibers. A vast majority of gut microbes are able to use these C5-sugars as a carbon and energy source. However, the underlying metabolic pathways are not fully understood. Bioinformatic analysis showed that a large number of abundant gut bacteria lack genes encoding a transaldolase as a key enzyme of the pentose phosphate pathway. Among them was the important human gut microbe Prevotella copri, which was able to grow in minimal media containing xylose or hemicelluloses as the sole carbon source. Therefore, we looked for an alternative pathway for pentose conversion in P. copri using bioinformatics, enzyme activity assays, and the detection of intermediates of pentose metabolism. It became evident that the organism converted C5-sugars via the sedoheptulose-1,7-bisphosphate pathway (SBPP) to connect pentose metabolism with glycolysis. To circumvent the transaldolase reaction, P. copri uses the combined catalysis of a pyrophosphate-dependent phosphofructokinase and a fructose-bisphosphate aldolase. Furthermore, we present strong evidence that the SBPP is widely distributed in important gut bacteria, including members of the phyla Bacteroides, Firmicutes, Proteobacteria, Verrucomicrobia, and Lentisphaerae., (© 2020 The Authors. The FEBS Journal published by John Wiley & Sons Ltd on behalf of Federation of European Biochemical Societies.)

Background: During the pentose phosphate pathway (PPP), two important components, NADPH and pentoses, are provided to the cell. Previously it was shown that this metabolic pathway is a source of reducing agent for lipid synthesis from glucose in the yeast Yarrowia lipolytica. Y. lipolytica is an attractive microbial host since it is able to convert untypical feedstocks, such as glycerol, into oils, which subsequently can be transesterified to biodiesel. However, the lipogenesis process is a complex phenomenon, and it still remains unknown which genes from the PPP are involved in lipid synthesis., Results: To address this problem we overexpressed five genes from this metabolic pathway: transaldolase (TAL1, YALI0F15587g), transketolase (TKL1, YALI0E06479g), ribulose-phosphate 3-epimerase (RPE1, YALI0C11880g) and two dehydrogenases, NADP + -dependent glucose-6-phosphate dehydrogenase (ZWF1, YALI0E22649g) and NADP + -dependent 6-phosphogluconate dehydrogenase (GND1, YALI0B15598g), simultaneously with diacylglycerol acyltransferase (DGA1, YALI0E32769g) and verified each resulting strain's ability to synthesize fatty acid growing on both glycerol and glucose as a carbon source. Our results showed that co-expression of DGA1 and TKL1 results in higher SCO synthesis, increasing lipid content by 40% over the control strain (DGA1 overexpression)., Conclusions: Simultaneous overexpression of DGA1 and TKL1 genes results in a higher lipid titer independently from the fermentation conditions, such as carbon source, pH and YE supplementation.

β-Hydroxy-α-amino acids (βH-AAs) are key components of many bioactive molecules as well as exist as specialised metabolites. Among these βH-AAs, 4-fluorothreonine (4-FT) is the only naturally occurring fluorinated AA discovered thus far. Here we report overexpression and biochemical characterisation of 4-fluorothreonine transaldolase from Streptomyces sp. MA37 (FTaseMA), a homologue of FTase previously identified in the biosynthesis of 4-FT in S. cattleya. FTaseMA displays considerable substrate plasticity to generate 4-FT as well as other β-hydroxy-α-amino acids with various functionalities at C4 position, giving the prospect of new chemo-enzymatic applications. The enzyme has a hybrid of two catalytic domains, serine hydroxymethyltransferase (S) and aldolase (A). Site-directed mutagenesis allowed the identification of the key residues of FTases, suggesting that the active site of A domain has a historical reminiscent feature in metal-dependent aldolases. Elemental analysis demonstrated that FTaseMA is indeed a Zn 2+ -dependent enzyme, the first example of pyridoxal phosphate (PLP) enzyme family fused with a metal-binding domain carrying out a distinct catalytic role. Finally, FTaseMA showed divergent evolutionary origin with other PLP dependent enzymes.

Background: The Gram-positive facultative methylotrophic bacterium Bacillus methanolicus uses the sedoheptulose-1,7-bisphosphatase (SBPase) variant of the ribulose monophosphate (RuMP) cycle for growth on the C 1 carbon source methanol. Previous genome sequencing of the physiologically different B. methanolicus wild-type strains MGA3 and PB1 has unraveled all putative RuMP cycle genes and later, several of the RuMP cycle enzymes of MGA3 have been biochemically characterized. In this study, the focus was on the characterization of the transaldolase (Ta) and its possible role in the RuMP cycle in B. methanolicus., Results: The Ta genes of B. methanolicus MGA3 and PB1 were recombinantly expressed in Escherichia coli, and the gene products were purified and characterized. The PB1 Ta protein was found to be active as a homodimer with a molecular weight of 54 kDa and displayed K M of 0.74 mM and V max of 16.3 U/mg using Fructose-6 phosphate as the substrate. In contrast, the MGA3 Ta gene, which encodes a truncated Ta protein lacking 80 amino acids at the N-terminus, showed no Ta activity. Seven different mutant genes expressing various full-length MGA3 Ta proteins were constructed and all gene products displayed Ta activities. Moreover, MGA3 cells displayed Ta activities similar as PB1 cells in crude extracts., Conclusions: While it is well established that B. methanolicus can use the SBPase variant of the RuMP cycle this study indicates that B. methanolicus possesses Ta activity and may also operate the Ta variant of the RuMP.

The genomes of most cellulolytic clostridia do not contain genes annotated as transaldolase. Therefore, for assimilating pentose sugars or for generating C 5 precursors (such as ribose) during growth on other (non-C 5 ) substrates, they must possess a pathway that connects pentose metabolism with the rest of metabolism. Here we provide evidence that for this connection cellulolytic clostridia rely on the sedoheptulose 1,7-bisphosphate (SBP) pathway, using pyrophosphate-dependent phosphofructokinase (PP i -PFK) instead of transaldolase. In this reversible pathway, PFK converts sedoheptulose 7-phosphate (S7P) to SBP, after which fructose-bisphosphate aldolase cleaves SBP into dihydroxyacetone phosphate and erythrose 4-phosphate. We show that PP i -PFKs of Clostridium thermosuccinogenes and C lostridium thermocellum indeed can convert S7P to SBP, and have similar affinities for S7P and the canonical substrate fructose 6-phosphate (F6P). By contrast, (ATP-dependent) PfkA of Escherichia coli , which does rely on transaldolase, had a very poor affinity for S7P. This indicates that the PP i -PFK of cellulolytic clostridia has evolved the use of S7P. We further show that C. thermosuccinogenes contains a significant SBP pool, an unusual metabolite that is elevated during growth on xylose, demonstrating its relevance for pentose assimilation. Last, we demonstrate that a second PFK of C. thermosuccinogenes that operates with ATP and GTP exhibits unusual kinetics toward F6P, as it appears to have an extremely high degree of cooperative binding, resulting in a virtual on/off switch for substrate concentrations near its K ½ value. In summary, our results confirm the existence of an SBP pathway for pentose assimilation in cellulolytic clostridia., (© 2020 Koendjbiharie et al.)

In this work, we analyzed several genes participating in the rearrangement pathway for xylulose 5-phosphate (Xu5P) in the methylotrophic yeast Pichia pastoris (syn. Komagataella phaffii). P. pastoris has two set of genes for fructose-1,6-bisphosphate aldolase (FBA1 and FBA2) and transaldolase (TAL1 and TAL2), although there are single-copy genes for fructose-1,6-bisphosphatase (FBP1) and transketolase (TKL1), respectively. Expressions of FBP1 and TAL2 were upregulated by non-fermentative carbon sources, especially methanol was the best inducer for them, and FBA2 was induced only by methanol. On the other hand, FBA1, TAL1 and TKL1 showed constitutive expression. Strain fbp1Δ showed severe growth defect on methanol and non-fermentable carbon sources, and growth rate of strain fba2Δ in methanol medium was slightly decreased. Moreover, Fba2p and Tal2p possessed peroxisome targeting signal type 1 (PTS1), and EGFP-Fba2p and EGFP-Tal2p were found to be localized in peroxisomes. From these findings, it was suggested that Fba2p, Fbp1p and Tal2p participate in the rearrangement pathway for Xu5P in peroxisomes, and that the altered Calvin cycle and non-oxidative pentose phosphate pathway involving Tal2p function in a complementary manner., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The polycistronic and non-canonical gene tarsal-less (tal, known as pri) was reported to be required for embryonic and imaginal development in Drosophila; however, there are few reports of the tal gene in the silkworm Bombyx mori. Here, we cloned a tal-like (Bmtal) gene, and a sequence analysis showed that the Bmtal cDNA (1661 bp) contains five small open reading frames (smORFs) (A1, A2, A3, A4, and B) that encode short peptides of 11-12 (A1-A4) amino acid residues containing an LDPTG(E)L(Q)(V)Y motif that is conserved in Drosophila Tal, as well as a 32-amino-acid B peptide. Reverse transcription-quantitative polymerase chain reaction showed that the expression of the Bmtal gene was highest in the trachea, followed by the silk gland and Malpighian tubule, in day 3 fifth-instar larvae. Subcellular localization showed that BmTal localized in the nucleus. By regulating the expression of the full-length Bmtal gene and the functional smORFs of Bmtal, we showed that the expression levels of the Bmovo gene and genes related to the Notch, transforming growth factor-β, and Hippo signaling pathways changed with changes in BmTal peptide expression. A co-immunoprecipitation assay showed that BmTal interacts with polyubiquitin, which triggered degradation and/or processing of the 14-3-3 protein zeta. A comparative transcriptome analysis showed that 2843 (2045) genes were up- (down)-regulated after Bmtal gene expression was up-regulated. The up- (down)-regulated differentially expressed genes were enriched in 326 (278) gene ontology terms (P ≤ 0.05) and 54 (59) Kyoto Encyclopedia of Genes and Genomes pathways (P ≤ 0.05), and the results indicated that the BmTal peptides could function as mediators of hormone levels or the activities of multiple pathways, including the peroxisome proliferator-activated receptor, Hedgehog, mitogen-activated protein kinase, adipocytokine, and gonadotropin-releasing hormone signaling pathways, as well as the innate immune response. These results increase our understanding of the function and mechanism of BmTal at the genome-wide level.

Xylose-negative Corynebacterium glutamicum has been engineered to utilize xylose as the sole carbon source via either the xylose isomerase (XI) pathway or the Weimberg pathway. Heterologous expression of xylose isomerase and overexpression of a gene encoding for xylulose kinase enabled efficient xylose utilization. In this study, we show that two functionally-redundant transcriptional regulators (GntR1 and GntR2) present on xylose repress the pentose phosphate pathway genes. For efficient xylose utilization, pentose phosphate pathway genes and a phosphoketolase gene were overexpressed with the XI pathway in C. glutamicum. Overexpression of the genes encoding for transaldolase (Tal), 6-phosphogluconate dehydrogenase (Gnd), or phosphoketolase (XpkA) enhanced the growth and xylose consumption rates compared to the wild-type with the XI pathway alone. However, co-expression of these genes did not have a synergetic effect on xylose utilization. For the succinate production from xylose, overexpression of the tal gene with the XI pathway in a succinate-producing strain improved xylose utilization and increased the specific succinate production rate by 2.5-fold compared to wild-type with the XI pathway alone. Thus, overexpression of the tal, gnd, or xpkA gene could be helpful for engineering C. glutamicum toward production of value-added chemicals with efficient xylose utilization., (Copyright © 2017 Elsevier B.V. All rights reserved.)

β-Lactone natural products occur infrequently in nature but possess a variety of potent and valuable biological activities. They are commonly derived from β-hydroxy-α-amino acids, which are themselves valuable chiral building blocks for chemical synthesis and precursors to numerous important medicines. However, despite a number of excellent synthetic methods for their asymmetric synthesis, few effective enzymatic tools exist for their preparation. Here we report cloning of the biosynthetic gene cluster for the β-lactone antibiotic obafluorin and delineate its biosynthetic pathway. We identify a nonribosomal peptide synthetase with an unusual domain architecture and an L-threonine:4-nitrophenylacetaldehyde transaldolase responsible for (2S,3R)-2-amino-3-hydroxy-4-(4-nitrophenyl)butanoate biosynthesis. Phylogenetic analysis sheds light on the evolutionary origin of this rare enzyme family and identifies further gene clusters encoding L-threonine transaldolases. We also present preliminary data suggesting that L-threonine transaldolases might be useful for the preparation of L-threo-β-hydroxy-α-amino acids.

We have studied the effect of hypoxia on the expression level of mRNA of the basic enzymes of pentose-phosphate cycle (G6PD, TKT, TALDO1, PGLS and RPIA) and glucose-6-phosphate isomerase (GPI) in U87 glioma cells in relation to inhibition of IRE1 (inositol requiring enzyme 1). It was shown that hypoxia leads to up-regulation of the expression of GPI and PGLS genes and to down-regulation of TALDO1 and RPIA genes in control glioma cells. Changes for GPI gene were more significant than for other genes. At the same time, inhibition of IRE1 modified the effect of hypoxia on the expression of all studied genes. In particular, it increased sensitivity to hypoxia of G6PD and TKT genes expression and suppressed the effect of hypoxia on the expression of GPI and RPIA genes. Additionally, inhibition of IRE1 eliminated hypoxic regulation of PGLS gene and did not change significantly effect of hypoxia on the expression of TALDO1 gene in glioma cells. Present study demonstrated that hypoxia, which often contributes to tumor growth, affects the expression of most studied genes and inhibition of IRE1 modified the hypoxic regulation of pentose-phosphate cycle gene expressions in a gene specific manner and thus possibly contributes to slower glioma growth, but several aspects of this regulation warrant further investigation.

Data on fimbrial and afimbrial adhesion factors of bifidobacteria are presented. Pili-like structures, their composition and conditions of formation in various species of bifidobacteria are described. Several sugar-lytic enzymes serve as afimbrial adhesins in bifidobacteria. Transaldolase and enolase are detected in bifidobacteria on cells' surface. Transaldolase ensures binding of bifi- dobacteria with mucin and their auto-aggregation. Surface enolase has an affinity to plasminogen, thus.bifidobacteria obtain a surface-bound protein with proteolytic activity. Molecular structures giving bifidobacteria hydrophobic properties are described - surface lipoprotein Bop A and lipoteichoic acids.

Background: Succinate has been identified by the U.S. Department of Energy as one of the top 12 building block chemicals, which can be used as a specialty chemical in the agricultural, food, and pharmaceutical industries. Escherichia coli are now one of the most important succinate producing candidates. However, the stoichiometric maximum succinate yield under anaerobic conditions through the reductive branch of the TCA cycle is restricted by NADH supply in E. coli., Results: In the present work, we report a rational approach to increase succinate yield by regulating NADH supply via pentose phosphate (PP) pathway and enhancing flux towards succinate. The deregulated genes zwf243 (encoding glucose-6-phosphate dehydrogenase) and gnd361 (encoding 6-phosphogluconate dehydrogenase) involved in NADPH generation from Corynebacterium glutamicum were firstly introduced into E. coli for succinate production. Co-expression of beneficial mutated dehydrogenases, which removed feedback inhibition in the oxidative part of the PP pathway, increased succinate yield from 1.01 to 1.16 mol/mol glucose. Three critical genes, pgl (encoding 6-phosphogluconolactonase), tktA (encoding transketolase) and talB (encoding transaldolase) were then overexpressed to redirect more carbon flux towards PP pathway and further improved succinate yield to 1.21 mol/mol glucose. Furthermore, introducing Actinobacillus succinogenes pepck (encoding phosphoenolpyruvate carboxykinase) together with overexpressing sthA (encoding soluble transhydrogenase), further increased succinate yield to 1.31 mol/mol glucose. In addition, removing byproduct formation through inactivating acetate formation genes ackA-pta and heterogenously expressing pyc (encoding pyruvate carboxylase) from C. glutamicum led to improved succinate yield to 1.4 mol/mol glucose. Finally, synchronously overexpressing dcuB and dcuC encoding succinate exporters enhanced succinate yield to 1.54 mol/mol glucose, representing 52 % increase relative to the parent strain and amounting to 90 % of the strain-specific stoichiometric maximum (1.714 mol/mol glucose)., Conclusions: It's the first time to rationally regulate pentose phosphate pathway to improve NADH supply for succinate synthesis in E. coli. 90 % of stoichiometric maximum succinate yield was achieved by combining further flux increase towards succinate and engineering its export. Regulation of NADH supply via PP pathway is therefore recommended for the production of products that are NADH-demanding in E. coli.

Epithelial ovarian cancer is the most lethal gynecological malignancies for readily metastasis. Exosomes have played an influential role in carcinogenicity and cancer progression. Our aim is to discover exosome-related mechanisms in ovarian cancer progress and explore potential diagnostic biomarkers and therapeutic targets of ovarian cancer. We initially presented the proteomic profiles of exosomes derived from two late-stage ovarian cell lines, OVCA429 and HO8910PM. A total of 2940 exosomal proteins were recorded by MS. FunRich appropriately processed these exosomal proteins, manifesting some superiority in contrast to Blast2go. Moreover, we demonstrated the pentose phosphate pathway was a dominant mechanism in exosome mediated intracellular communication. Glucose-6-phosphate dehydrogenase, transketolase and transaldolase 1, three key enzymes regulated pentose phosphate pathway, were all marked in the same exosomal parts of proteins between two ovarian cell lines. Moreover, these key proteins might become diagnostic, prognostic biomarkers and therapeutic targets of ovarian cancer.

Internal standard based absolute quantitation of glycolytic intermediates was performed to characterize the thermodynamic states of Saccharomyces cerevisiae metabolism. A mixture of (13)C-labeled glycolytic intermediates was prepared via extraction from S. cerevisiae cells cultivated using a synthetic medium containing [U-(13)C] glucose as the sole carbon source. The (13)C-labeled metabolite mixture was used as an internal standard for the analysis of S. cerevisiae cultivated in a medium containing natural glucose. The methodology was employed for the absolute quantitation of glycolytic intermediates of BY4742, pfk1Δ, and zwf1Δ strains of S. cerevisiae. Fructose-1,6-bisphosphate was the most abundant intermediate in the BY4742 strains in the log phase of growth. Estimation of the Gibbs free energy change (ΔG) from the absolute concentration revealed that several reactions, such as those catalyzed by ribose-5-phosphate keto-isomerase and phosphoglucose isomerase, were commonly at near-equilibrium in all three strains. A significant shift in thermodynamic state was also observed for the transketolase-transaldolase reaction, for which ΔG was -6.6 ± 0.5 kJ mol(-1) in the BY4742 strain and 5.4 ± 0.3 kJ mol(-1) in the zwf1Δ strain., (Copyright © 2015. Published by Elsevier B.V.)

A-500359s, A-503083s, and A-102395 are capuramycin-type nucleoside antibiotics that were discovered using a screen to identify inhibitors of bacterial translocase I, an essential enzyme in peptidoglycan cell wall biosynthesis. Like the parent capuramycin, A-500359s and A-503083s consist of three structural components: a uridine-5'-carboxamide (CarU), a rare unsaturated hexuronic acid, and an aminocaprolactam, the last of which is substituted by an unusual arylamine-containing polyamide in A-102395. The biosynthetic gene clusters for A-500359s and A-503083s have been reported, and two genes encoding a putative non-heme Fe(II)-dependent α-ketoglutarate:UMP dioxygenase and an l-Thr:uridine-5'-aldehyde transaldolase were uncovered, suggesting that C-C bond formation during assembly of the high carbon (C6) sugar backbone of CarU proceeds from the precursors UMP and l-Thr to form 5'-C-glycyluridine (C7) as a biosynthetic intermediate. Here, isotopic enrichment studies with the producer of A-503083s were used to indeed establish l-Thr as the direct source of the carboxamide of CarU. With this knowledge, the A-102395 gene cluster was subsequently cloned and characterized. A genetic system in the A-102395-producing strain was developed, permitting the inactivation of several genes, including those encoding the dioxygenase (cpr19) and transaldolase (cpr25), which abolished the production of A-102395, thus confirming their role in biosynthesis. Heterologous production of recombinant Cpr19 and CapK, the transaldolase homolog involved in A-503083 biosynthesis, confirmed their expected function. Finally, a phosphotransferase (Cpr17) conferring self-resistance was functionally characterized. The results provide the opportunity to use comparative genomics along with in vivo and in vitro approaches to probe the biosynthetic mechanism of these intriguing structures., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)

Background: Studying the survival of yeast in stationary phase, known as chronological lifespan, led to the identification of molecular ageing factors conserved from yeast to higher organisms. To identify functional interactions among yeast chronological ageing genes, we conducted a haploproficiency screen on the basis of previously identified long-living mutants. For this, we created a library of heterozygous Saccharomyces cerevisiae double deletion strains and aged them in a competitive manner., Results: Stationary phase survival was prolonged in a double heterozygous mutant of the metabolic enzyme non-quiescent mutant 1 (NQM1), a paralogue to the pentose phosphate pathway enzyme transaldolase (TAL1), and the transcription factor vitamin H response transcription factor 1 (VHR1). We find that cells deleted for the two genes possess increased clonogenicity at late stages of stationary phase survival, but find no indication that the mutations delay initial mortality upon reaching stationary phase, canonically defined as an extension of chronological lifespan. We show that both genes influence the concentration of metabolites of glycolysis and the pentose phosphate pathway, central metabolic players in the ageing process, and affect osmolality of growth media in stationary phase cultures. Moreover, NQM1 is glucose repressed and induced in a VHR1 dependent manner upon caloric restriction, on non-fermentable carbon sources, as well as under osmotic and oxidative stress. Finally, deletion of NQM1 is shown to confer resistance to oxidizing substances., Conclusions: The transaldolase paralogue NQM1 and the transcription factor VHR1 interact haploproficiently and affect yeast stationary phase survival. The glucose repressed NQM1 gene is induced under various stress conditions, affects stress resistance and this process is dependent on VHR1. While NQM1 appears not to function in the pentose phosphate pathway, the interplay of NQM1 with VHR1 influences the yeast metabolic homeostasis and stress tolerance during stationary phase, processes associated with yeast ageing.

Nature has evolved different strategies for the reversible cleavage of ketose phosphosugars as essential metabolic reactions in all domains of life. Prominent examples are the Schiff-base forming class I FBP and F6P aldolase as well as transaldolase, which all exploit an active center lysine to reversibly cleave the C3-C4 bond of fructose-1,6-bisphosphate or fructose-6-phosphate to give two 3-carbon products (aldolase), or to shuttle 3-carbon units between various phosphosugars (transaldolase). In contrast, transketolase and phosphoketolase make use of the bioorganic cofactor thiamin diphosphate to cleave the preceding C2-C3 bond of ketose phosphates. While transketolase catalyzes the reversible transfer of 2-carbon ketol fragments in a reaction analogous to that of transaldolase, phosphoketolase forms acetyl phosphate as final product in a reaction that comprises ketol cleavage, dehydration and phosphorolysis. In this review, common and divergent catalytic principles of these enzymes will be discussed, mostly, but not exclusively, on the basis of crystallographic snapshots of catalysis. These studies in combination with mutagenesis and kinetic analysis not only delineated the stereochemical course of substrate binding and processing, but also identified key catalytic players acting at the various stages of the reaction. The structural basis for the different chemical fates and lifetimes of the central enamine intermediates in all five enzymes will be particularly discussed, in addition to the mechanisms of substrate cleavage, dehydration and ring-opening reactions of cyclic substrates. The observation of covalent enzymatic intermediates in hyperreactive conformations such as Schiff-bases with twisted double-bond linkages in transaldolase and physically distorted substrate-thiamin conjugates with elongated substrate bonds to be cleaved in transketolase, which probably epitomize a canonical feature of enzyme catalysis, will be also highlighted., (Copyright © 2014 Elsevier Inc. All rights reserved.)

After exposure to [U-(13)C3]glycerol, the liver produces primarily [1,2,3-(13)C3]- and [4,5,6-(13)C3]glucose in equal proportions through gluconeogenesis from the level of trioses. Other (13)C-labeling patterns occur as a consequence of alternative pathways for glucose production. The pentose phosphate pathway (PPP), metabolism in the citric acid cycle, incomplete equilibration by triose phosphate isomerase, or the transaldolase reaction all interact to produce complex (13)C-labeling patterns in exported glucose. Here, we investigated (13)C labeling in plasma glucose in rats given [U-(13)C3]glycerol under various nutritional conditions. Blood was drawn at multiple time points to extract glucose for NMR analysis. Because the transaldolase reaction and incomplete equilibrium by triose phosphate isomerase cannot break a (13)C-(13)C bond within the trioses contributing to glucose, the appearance of [1,2-(13)C2]-, [2,3-(13)C2]-, [5,6-(13)C2]-, and [4,5-(13)C2]glucose provides direct evidence for metabolism of glycerol in the citric acid cycle or the PPP but not an influence of either triose phosphate isomerase or the transaldolase reaction. In all animals, [1,2-(13)C2]glucose/[2,3-(13)C2]glucose was significantly greater than [5,6-(13)C2]glucose/[4,5-(13)C2]glucose, a relationship that can only arise from gluconeogenesis followed by passage of substrates through the PPP. In summary, the hepatic PPP in vivo can be detected by (13)C distribution in blood glucose after [U-(13)C3]glycerol administration., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)

In the metabolic network of the cell, many intermediary products are shared between different pathways. d-Glyceraldehyde-3-phosphate, a glycolytic intermediate, is a substrate of GAPDH but is also utilized by transaldolase and transketolase in the scrambling reactions of the nonoxidative pentose phosphate pathway. Recent efforts to engineer baker's yeast strains capable of utilizing pentose sugars present in plant biomass rely on increasing the carbon flux through this pathway. However, the competition between transaldolase and GAPDH for d-glyceraldehyde-3-phosphate produced in the first transketolase reaction compromises the carbon balance of the pathway, thereby limiting the product yield. Guided by the hypothesis that reduction in GAPDH activity would increase the availability of d-glyceraldehyde-3-phosphate for transaldolase and thereby improve ethanol production during fermentation of pentoses, we performed a comprehensive characterization of the three GAPDH isoenzymes in baker's yeast, Tdh1, Tdh2, and Tdh3 and analyzed the effect of their deletion on xylose utilization by engineered strains. Our data suggest that overexpression of transaldolase is a more promising strategy than reduction in GAPDH activity to increase the flux through the nonoxidative pentose phosphate pathway., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

Background: Fusarium oxysporum is among the few filamentous fungi that have been reported of being able to directly ferment biomass to ethanol in a consolidated bioprocess. Understanding its metabolic pathways and their limitations can provide some insights on the genetic modifications required to enhance its growth and subsequent fermentation capability. In this study, we investigated the hypothesis reported previously that phosphoglucomutase and transaldolase are metabolic bottlenecks in the glycolysis and pentose phosphate pathway of the F. oxysporum metabolism., Results: Both enzymes were homologously overexpressed in F. oxysporum F3 using the gpdA promoter of Aspergillus nidulans for constitutive expression. Transformants were screened for their phosphoglucomutase and transaldolase genes expression levels with northern blot. The selected transformant exhibited high mRNA levels for both genes, as well as higher specific activities of the corresponding enzymes, compared to the wild type. It also displayed more than 20 and 15% higher specific growth rate upon aerobic growth on glucose and xylose, respectively, as carbon sources and 30% higher biomass to xylose yield. The determination of the relative intracellular amino and non-amino organic acid concentrations at the end of growth on glucose revealed higher abundance of most determined metabolites between 1.5- and 3-times in the recombinant strain compared to the wild type. Lower abundance of the determined metabolites of the Krebs cycle and an 68-fold more glutamate were observed at the end of the cultivation, when xylose was used as carbon source., Conclusions: Homologous overexpression of phosphoglucomutase and transaldolase in F. oxysporum was shown to enhance the growth characteristics of the strain in both xylose and glucose in aerobic conditions. The intracellular metabolites profile indicated how the changes in the metabolome could have resulted in the observed growth characteristics.

Arabinose 5-phosphate (A5P) is the aldopentose version of the ketohexose fructose 6-phosphate (F6P), having identical stereochemistry but lacking atoms corresponding to the 1-carbon and 1-hydroxyl. Despite structural similarity and conservation of the reactive portion of F6P, F6P acts as a substrate whereas A5P is reported to be an inhibitor of transaldolase. To address the lack of A5P reactivity we determined a crystal structure of the Francisella tularensis transaldolase in complex with A5P. This structure reveals that like F6P, A5P forms a covalent Schiff base with active site Lys135. Unlike F6P, A5P binding fails to displace an ordered active site water molecule. Retaining this water necessitates conformational changes at the A5P-protein linkage that possibly hinder reactivity. The findings presented here show the basis of A5P inhibition and suggest an unusual mechanism of competitive, reversible-covalent transaldolase regulation.

Lignocellulosic biomass dedicated to bioethanol production usually contains pentoses and inhibitory compounds such as furfural that are not well tolerated by Saccharomyces cerevisiae. Thus, S. cerevisiae strains with the capability of utilizing both glucose and xylose in the presence of inhibitors such as furfural are very important in industrial ethanol production. Under the synergistic conditions of transaldolase (TAL) and alcohol dehydrogenase (ADH) overexpression, S. cerevisiae MT8-1X/TAL-ADH was able to produce 1.3-fold and 2.3-fold more ethanol in the presence of 70 mM furfural than a TAL-expressing strain and a control strain, respectively. We also tested the strains' ability by mimicking industrial ethanol production from hemicellulosic hydrolysate containing fermentation inhibitors, and ethanol production was further improved by 16% when using MT8-1X/TAL-ADH compared to the control strain. Transcript analysis further revealed that besides the pentose phosphate pathway genes TKL1 and TAL1, ADH7 was also upregulated in response to furfural stress, which resulted in higher ethanol production compared to the TAL-expressing strain. The improved capability of our modified strain was based on its capacity to more quickly reduce furfural in situ resulting in higher ethanol production. The co-expression of TAL/ADH genes is one crucial strategy to fully utilize undetoxified lignocellulosic hydrolysate, leading to cost-competitive ethanol production., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

A recombinant xylose-utilizing Saccharomyces cerevisiae strain carrying one copy of heterologous XYL1 and XYL2 from Pichia stipitis and endogenous XKS1 under the control of the TDH3 promoter in the chromosomal DNA was constructed from the industrial haploid yeast strain NAM34-4C, which showed thermotolerance and acid tolerance. The recombinant S. cerevisiae strain SCB7 grew in minimal medium containing xylose as the sole carbon source, and its shortest generation time (G(short)) was 5 h. From this strain, four mutants showing rapid growth (G(short) = 2.5 h) in the minimal medium were isolated. The mutants carried four mutations that were classified into three linkage groups. Three mutations were dominant and one mutation was recessive to the wild type allele. The recessive mutation was in the PHO13 gene encoding para-nitrophenyl phosphatase. The other mutant genes were not linked to TAL1 gene encoding transaldolase. When the mutants and their parental strain were used for the batch fermentation in a complex medium at pH 4.0 containing 30 g/L xylose at 35 °C with shaking (60 rpm) and an initial cell density (Absorbance at 660 nm) of 1.0, all mutants showed efficient ethanol production and xylose consumption from the early stage of the fermentation culture. In two mutants, within 24 h, 4.8 g/L ethanol was produced, and the ethanol yield was 47%, which was 1.4 times higher than that achieved with the parental strain. The xylose concentration in the medium containing the mutant decreased linearly at a rate of 1 g/L/h until 24 h., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Objectives: To study the effects of both balneotherapy and mud-bath therapy treatments in patients affected by primary fibromyalgia (FM) using rheumatological, psychiatric, biochemical and proteomic approaches., Methods: Forty-one FM patients (39 females, 2 males), who fulfilled the American College of Rheumatology criteria received a 2-week thermal therapy programme consisting of therapy once daily for 6 days/week. Twenty-one patients received mud-bath treatment, while the other twenty balneotherapy. Pain, symptoms, and quality of life were assessed. Oxytocin, brain-derived neurotrophic factor (BDNF), ATP and serotonin transporter levels during therapy were assayed. Comparative whole saliva (WS) proteomic analysis was performed using a combination of two-dimensional electrophoresis (2DE) and mass spectrometry techniques., Results: We observed a reduction in pain, FIQ values and improvement of SF36 in both groups of patients treated with mud-bath or balneotherapy. The improvement of the outcome measures occurred with different timing and duration in the two spa treatments. A significant decrease in BDNF concentrations was observed either after balneotherapy or mud-bath therapy when assayed after twelve weeks, while no significant change in oxytocin levels, ATP levels and serotonin transporter were detected. Significant differences were observed for phosphoglycerate mutase1 (PGAM1) and zinc alpha-2-glycoprotein 1 (AZGP1) protein expression., Conclusions: Our results showed that the thermal treatment might have a beneficial effect on the specific symptoms of the disease. In particular, while balneotherapy gives results that in most patients occur after the end of the treatment but which are no longer noticeable after 3 months, the mud-bath treatment gives longer lasting results.

Coxiella burnetii is an obligate intracellular bacterial pathogen and the causative agent of Q fever. Chronic Q fever can produce debilitating fatigue and C. burnetii is considered a significant bioterror threat. C. burnetii occupies the monocyte phagolysosome and although prior work has explained features of the host-pathogen interaction, many aspects are still poorly understood. We have conducted a proteomic investigation of human Monomac I cells infected with the Nine Mile Phase II strain of C. burnetii and used the results as a framework for a systems biology model of the host response. Our principal methodology was multiplex differential 2D gel electrophoresis using ZDyes, a new generation of covalently linked fluorescent protein detection dyes under development at Montana State University. The 2D gel analysis facilitated the detection of changes in posttranslational modifications on intact proteins in response to infection. The systems model created from our data a framework for the design of experiments to seek a deeper understanding of the host-pathogen interactions.

Transaldolase (TA) exchange overestimates gluconeogenesis measured with deuterated water (²H₂O). However, it is unknown whether TA differs in people with type 2 diabetes (T2DM). ²H₂O was ingested, and [1-¹³C]acetate and [3-³H]glucose were infused in T2DM (n = 10) and healthy nondiabetic (ND; n = 8) subjects. TA was assessed from the ratio of ¹³C3 to ¹³C4 glucose enrichment (¹³C3/¹³C4) measured by ¹³C NMR. Glucose turnover was measured before (~16-h fast) and during hyperglycemic (~10 mM) moderate-dose insulin (~0.35 mU·kg⁻¹·min⁻¹) clamp. ¹³C3/¹³C4 in T2DM vs. ND was <1.0 and not different at baseline and clamp, indicating equivalent TA. To determine whether incomplete triose phosphate isomerase (TPI) exchange contributed to asymmetric ¹³C3/¹³C4, [U-¹³C]glycerol was infused in lieu of [1-¹³C]acetate during a separate visit in a subset of ND (n = 7) subjects. Ratio of ¹³C3/¹³C4 obtained following either tracer was <1.0 at baseline and during clamp, indicating that TPI exchange was essentially complete and did not contribute to asymmetric glucose enrichment. Uncorrected and corrected rates of gluconeogenesis were no different (P = not significant) in T2DM vs. ND both at baseline and during clamp. TA correction resulted in equivalent estimates of corrected gluconeogenesis in T2DM and ND that were ~25-35% lower than uncorrected gluconeogenesis both at baseline and during the clamp. The asymmetric enrichment of glucose from ¹³C-gluconeogenic tracers is attributable to TA exchange and can be utilized to correct for TA exchange. In conclusion, TA exchange does not differ between T2DM and ND under fasting or hyperglycemic clamp conditions, and the ²H₂O method continues to provide an accurate estimation of gluconeogenesis.

Protein expression patterns of an erythritol-producing yeast, Candida magnoliae, were analyzed to identify differentially expressed proteins in response to glucose perturbation. Specifically, wild type C. magnoliae was grown under high and low glucose conditions and the cells were harvested at both mid-exponential and erythritol production phases for proteomic studies. In order to analyze intracellular protein abundances from the harvested cells quantitatively, total intracellular proteins were extracted and applied to two-dimensional gel electrophoresis for separation and visualization of individual proteins. Among the proteins distributed in the range of pI 4-7 and molecular weight 29-97kDa, five osmo-responsive proteins were drastically changed in response to glucose perturbation. Hsp60 (Heat-shock protein 60), transaldolase and NADH:quinone oxidoreductase were down-regulated under the high glucose condition and Bro1 (BCK1-like Resistance to Osmotic shock) and Eno1 (enolase1) were up-regulated. These proteins are directly or indirectly related with cellular stress response. Importantly, protein expression patterns of Hsp60, Bro1 and Eno1 were strongly correlated with previous studies identifying the proteins perturbed by osmotic stress for other organisms including Saccharomyces cerevisiae., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Nucleotide degradation is a universal metabolic capability. Here we combine metabolomics, genetics and biochemistry to characterize the yeast pathway. Nutrient starvation, via PKA, AMPK/SNF1, and TOR, triggers autophagic breakdown of ribosomes into nucleotides. A protein not previously associated with nucleotide degradation, Phm8, converts nucleotide monophosphates into nucleosides. Downstream steps, which involve the purine nucleoside phosphorylase, Pnp1, and pyrimidine nucleoside hydrolase, Urh1, funnel ribose into the nonoxidative pentose phosphate pathway. During carbon starvation, the ribose-derived carbon accumulates as sedoheptulose-7-phosphate, whose consumption by transaldolase is impaired due to depletion of transaldolase's other substrate, glyceraldehyde-3-phosphate. Oxidative stress increases glyceraldehyde-3-phosphate, resulting in rapid consumption of sedoheptulose-7-phosphate to make NADPH for antioxidant defense. Ablation of Phm8 or double deletion of Pnp1 and Urh1 prevent effective nucleotide salvage, resulting in metabolite depletion and impaired survival of starving yeast. Thus, ribose salvage provides means of surviving nutrient starvation and oxidative stress.

Studies of glycerol metabolism in the heart have largely emphasized its role in triglyceride synthesis. However, glycerol may also be oxidized in the citric acid cycle, and glycogen synthesis from glycerol has been reported in the nonmammalian myocardium. The intent of this study was to test the hypothesis that glycerol may be metabolized to glycogen in mammalian heart. Isolated rat hearts were supplied with a mixture of substrates including glucose, lactate, pyruvate, octanoate, [U-(13)C(3)]glycerol, and (2)H(2)O to probe various metabolic pathways including glycerol oxidation, glycolysis, the pentose phosphate pathway, and carbon sources of stored glycogen. NMR analysis confirmed that glycogen production from the level of the citric acid cycle did not occur and that the glycerol contribution to oxidation in the citric acid cycle was negligible in the presence of alternative substrates. Quite unexpectedly, (13)C from [U-(13)C(3)]glycerol appeared in glycogen in carbon positions 4-6 of glucosyl units but none in positions 1-3. The extent of [4,5,6-(13)C(3)]glucosyl unit enrichment in glycogen was enhanced by insulin but decreased by H(2)O(2). Given that triose phosphate isomerase is generally assumed to fully equilibrate carbon tracers in the triose pool, the marked (13)C asymmetry in glycogen can only be attributed to conversion of [U-(13)C(3)]glycerol to [U-(13)C(3)]dihydroxyacetone phosphate and [U-(13)C(3)]glyceraldehyde 3-phosphate followed by rearrangements in the nonoxidative branch of the pentose phosphate pathway involving transaldolase that places this (13)C-enriched 3-carbon unit only in the bottom half of hexose phosphate molecules contributing to glycogen.

The use of deuterated water as a method to measure gluconeogenesis has previously been well validated and is reflective of normal human physiology. However, there has been concern since the method was first introduced that transaldolase exchange may lead to the overestimation of gluconeogenesis. We examined the impact of transaldolase exchange on the estimation of gluconenogenesis using the deuterated water method under a variety of physiological conditions in humans by using the gluconeogenic tracer [U-(13)C]propionate, (2)H(2)O, and (2)H/(13)C nuclear magnetic resonance (NMR) spectroscopy. When [U-(13)C]propionate was used, (13)C labeling inequality occurred between the top and bottom halves of glucose in individuals fasted for 12-24 h who were weight stable (n = 18) or had lost weight via calorie restriction (n = 7), consistent with transaldolase exchange. Similar analysis of glucose standards revealed no significant difference in the total (13)C enrichment between the top and bottom halves of glucose, indicating that the differences detected were biological, not analytical, in origin. This labeling inequality was attenuated by extending the fasting period to 48 h (n = 12) as well as by dietary carbohydrate restriction (n = 7), both conditions associated with decreased glycogenolysis. These findings were consistent with a transaldolase effect; however, the resultant overestimation of gluconeogenesis in the overnight-fasted state was modest (7-12%), leading to an error of 14-24% that was easily correctable by using either a simultaneous (13)C gluconeogenic tracer or a correction nomogram generated from data in the present study.

Streptomyces cattleya DSM 46488 is unusual in its ability to biosynthesise fluorine containing natural products, where it can produce fluoroacetate and 4-fluorothreonine. The individual enzymes involved in fluorometabolite biosynthesis have already been demonstrated in in vitro investigations. Candidate genes for the individual biosynthetic steps were located from recent genome sequences. In vivo inactivation of individual genes including those encoding the S-adenosyl-l-methionine:fluoride adenosyltransferase (fluorinase, SCATT&#95;41540), 5'-fluoro-5'-deoxyadenosine phosphorylase (SCATT&#95;41550), fluoroacetyl-CoA thioesterase (SCATT&#95;41470), 5-fluoro-5-deoxyribose-1-phosphate isomerase (SCATT&#95;20080) and a 4-fluorothreonine acetaldehyde transaldolase (SCATT&#95;p11780) confirm that they are essential for fluorometabolite production. Notably gene disruption of the transaldolase (SCATT&#95;p11780) resulted in a mutant which could produce fluoroacetate but was blocked in its ability to biosynthesise 4-fluorothreonine, revealing a branchpoint role for the PLP-transaldolase., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The activity of transaldolase and transketolase, key enzymes in the non-oxidative pentose phosphate pathway, is rate-limiting for xylose utilization in recombinant Saccharomyces cerevisiae. Overexpression of TAL1 and TKL1, the major transaldolase and transketolase genes, increases the flux from the pentose phosphate pathway into the glycolytic pathway. However, the functional roles of NQM1 and TKL2, the secondary transaldolase and transketolase genes, especially in xylose utilization, remain unclear. This study focused on characterization of NQM1 and TKL2, together with TAL1 and TKL1, regarding their roles in xylose utilization and fermentation. Knockout or overexpression of these four genes on the phenotype of xylose-utilizing S. cerevisiae strains was also examined. Transcriptional analysis indicated that the expression of TAL1, NQM1, and TKL1 was up-regulated in the presence of xylose. A significant decrease in both growth on xylose and xylose-fermenting ability in tal1Δ and tkl1Δ mutants confirmed that TAL1 and TKL1 are essential for xylose assimilation and fermentation. Gene disruption analysis using a tkl1Δ mutant revealed that TKL1 is also required for utilization of glucose. Growth on xylose and xylose-fermenting ability were slightly influenced by deletion of NQM1 or TKL2 when xylose was used as the sole carbon source. Moreover, the rate of xylose consumption and ethanol production was slightly impaired in TKL1- and TKL2-overexpressing strains. NQM1 and TKL2 may thus play a physiological role via an effect on the non-oxidative pentose phosphate pathway in the xylose metabolic pathway, although their roles in xylose utilization and fermentation are less important than those of TAL1 and TKL1., (Copyright © 2012 Elsevier Inc. All rights reserved.)

The ability of bifidobacteria to establish in the intestine of mammals is among the main factors considered to be important for achieving probiotic effects. The role of surface molecules from Bifidobacterium bifidum taxon in mucin adhesion capability and the aggregation phenotype of this bacterial species was analyzed. Adhesion to the human intestinal cell line HT29 was determined for a collection of 12 B. bifidum strains. In four of them-B. bifidum LMG13195, DSM20456, DSM20239, and A8-the involvement of surface-exposed macromolecules in the aggregation phenomenon was determined. The aggregation of B. bifidum A8 and DSM20456 was abolished after treatment with proteinase K, this effect being more pronounced for the strain A8. Furthermore, a mucin binding assay of B. bifidum A8 surface proteins showed a high adhesive capability for its transaldolase (Tal). The localization of this enzyme on the surface of B. bifidum A8 was unequivocally demonstrated by immunogold electron microscopy experiments. The gene encoding Tal from B. bifidum A8 was expressed in Lactococcus lactis, and the protein was purified to homogeneity. The pure protein was able to restore the autoaggregation phenotype of proteinase K-treated B. bifidum A8 cells. A recombinant L. lactis strain, engineered to secrete Tal, displayed a mucin- binding level more than three times higher than the strain not producing the transaldolase. These findings suggest that Tal, when exposed on the cell surface of B. bifidum, could act as an important colonization factor favoring its establishment in the gut.

Efficient conversion of xylose to ethanol is an essential factor for commercialization of lignocellulosic ethanol. To minimize production of xylitol, a major by-product in xylose metabolism and concomitantly improve ethanol production, Saccharomyces cerevisiae D452-2 was engineered to overexpress NADH-preferable xylose reductase mutant (XR(MUT)) and NAD⁺-dependent xylitol dehydrogenase (XDH) from Pichia stipitis and endogenous xylulokinase (XK). In vitro enzyme assay confirmed the functional expression of XR(MUT), XDH and XK in recombinant S. cerevisiae strains. The change of wild type XR to XR(MUT) along with XK overexpression led to reduction of xylitol accumulation in microaerobic culture. More modulation of the xylose metabolism including overexpression of XR(MUT) and transaldolase, and disruption of the chromosomal ALD6 gene encoding aldehyde dehydrogenase (SX6(MUT)) improved the performance of ethanol production from xylose remarkably. Finally, oxygen-limited fermentation of S. cerevisiae SX6(MUT) resulted in 0.64 g l⁻¹ h⁻¹ xylose consumption rate, 0.25 g l⁻¹ h⁻¹ ethanol productivity and 39% ethanol yield based on the xylose consumed, which were 1.8, 4.2 and 2.2 times higher than the corresponding values of recombinant S. cerevisiae expressing XR(MUT), XDH and XK only., (Copyright © 2011 Elsevier B.V. All rights reserved.)

Transaldolase (Tal) is involved in the central carbon metabolism, i.e. the non-oxidative pentose phosphate pathway, and is therefore a ubiquitous enzyme. However, Tals show a low degree in sequence identity and vary in length within the enzyme family which previously led to the definition of five subfamilies. We wondered how this variation is conserved in structure and function. To answer this question we characterised and compared the Tals from Bacillus subtilis, Corynebacterium glutamicum and Escherichia coli, each belonging to a different subfamily, with respect to their biochemical properties and structures. The overall structure of the Tal domain, a (β/α)(8) -barrel fold, is well conserved between the different subfamilies but the enzymes show different degrees of oligomerisation (monomer, dimer and decamer). The substrate specificity of the three enzymes investigated is quite similar which is reflected in the conservation of the active site, the phosphate binding site as well as the position of a catalytically important water molecule. All decameric enzymes characterised so far appear to be heat stable no matter whether they originate from a mesophilic or thermophilic organism. Hence, the thermostability might be due to the structural properties, i.e. tight packing, of these enzymes. Database The crystal structures have been deposited in the Protein Data Bank with accession code 3R8R for BsTal and 3R5E for CgTal., (© 2011 The Authors Journal compilation © 2011 FEBS.)

Thermophilic enzymes are in high demand for various applications due to their prolonged lifetimes and high reaction rates at elevated temperatures. In this work, an open reading frame TM0295, which encodes a putative transaldolase (TAL) from a hyper-thermophilic microorganism, Thermotoga maritima, was cloned and expressed in Escherichia coli. The enzyme activity of transaldolase at high temperatures (e.g., at 80 °C) was reported here for the first time. The recombinant T. maritima transaldolase was extremely thermostable, with a half-life time of 198 and 13.0 h at 60 °C and 80 °C, respectively. The estimated total turn-over number was 1.5 × 10(6) mol of product per mol of enzyme at 80 °C. This enzyme also exhibited high activities within a broad pH range of 6.0-9.0. This ultra-thermostable TAL with high activity shows great potential for use in such applications as the production of enzymatic biofuels production and the synthesis of high-value carbohydrates by cell-free synthetic pathway biotransformation.

Bacillus coagulans XZL4 is an efficient pentose-utilizing producer of important platform compounds, such as l-lactic acid, 2,3-butanediol, and acetoin. Here we present a 2.8-Mb assembly of its genome. Simple and efficient carbohydrate metabolism systems, especially the transketolase/transaldolase pathway, make it possible to convert pentose sugars to products at high levels.

Cyanophages infecting the marine cyanobacteria Prochlorococcus and Synechococcus encode and express genes for the photosynthetic light reactions. Sequenced cyanophage genomes lack Calvin cycle genes, however, suggesting that photosynthetic energy harvested via phage proteins is not used for carbon fixation. We report here that cyanophages carry and express a Calvin cycle inhibitor, CP12, whose host homologue directs carbon flux from the Calvin cycle to the pentose phosphate pathway (PPP). Phage CP12 was coexpressed with phage genes involved in the light reactions, deoxynucleotide biosynthesis, and the PPP, including a transaldolase gene that is the most prevalent PPP gene in cyanophages. Phage transaldolase was purified to homogeneity from several strains and shown to be functional in vitro, suggesting that it might facilitate increased flux through this key reaction in the host PPP, augmenting production of NADPH and ribose 5-phosphate. Kinetic measurements of phage and host transaldolases revealed that the phage enzymes have k(cat)/K(m) values only approximately one third of the corresponding host enzymes. The lower efficiency of phage transaldolase may be a tradeoff for other selective advantages such as reduced gene size: we show that more than half of host-like cyanophage genes are significantly shorter than their host homologues. Consistent with decreased Calvin cycle activity and increased PPP and light reaction activity under infection, the host NADPH/NADP ratio increased two-fold in infected cells. We propose that phage-augmented NADPH production fuels deoxynucleotide biosynthesis for phage replication, and that the selection pressures molding phage genomes involve fitness advantages conferred through mobilization of host energy stores.

The formation of signalling boundaries is one of the strategies employed by the Notch (N) pathway to give rise to two distinct signalling populations of cells. Unravelling the mechanisms involved in the regulation of these signalling boundaries is essential to understanding the role of N during development and diseases. The function of N in the segmentation of the Drosophila leg provides a good system to pursue these mechanisms at the molecular level. Transcriptional and post-transcriptional regulation of the N ligands, Serrate (Ser) and Delta (Dl) generates a signalling boundary that allows the directional activation of N in the distalmost part of the segment, the presumptive joint. A negative feedback loop between odd-skipped-related genes and the N pathway maintains this signalling boundary throughout development in the true joints. However, the mechanisms controlling N signalling boundaries in the tarsal joints are unknown. Here we show that the non-canonical tarsal-less (tal) gene (also known as pri), which encodes for four small related peptides, is expressed in the N-activated region and required for joint development in the tarsi during pupal development. This function of tal is both temporally and functionally separate from the tal-mediated tarsal intercalation during mid-third instar that we reported previously. In the pupal function described here, N signalling activates tal expression and reciprocally Tal peptides feedback on N by repressing the transcription of Dl in the tarsal joints. This Tal-induced repression of Dl is mediated by the post-transcriptional activation of the Shavenbaby transcription factor, in a similar manner as it has been recently described in the embryo. Thus, a negative feedback loop involving Tal regulates the formation and maintenance of a Dl+/Dl- boundary in the tarsal segments highlighting an ancient mechanism for the regulation of N signalling based on the action of small cell signalling peptides., (Copyright © 2011. Published by Elsevier Inc.)

Background: The conserved Notch signaling pathway regulates cell fate decisions and maintains stem cells in multicellular organisms. Up-regulation of Notch signaling is observed in several types of cancer and is causally involved in proliferation and survival of cancer cells. Thus, it is of great interest to look for anti-Notch reagents for therapeutic purposes. In model animal Drosophila, Notch signaling restricts selection of sensory organ precursors (SOPs) during external sensory (ES) organ development. To look for novel genes that can suppress Notch signaling, we performed a gain-of-function modifier screen to look for genes that enhance the phenotype of ectopic ES organs induced by overexpression of phyllopod, a gene required for SOP specification., Results: From the gain-of-function screen, we discovered that overexpression of polished rice/tarsal-less (pri/tal) increases the numbers of ES organs as well as SOPs. pri/tal is a polycistronic gene that contains four short open reading frames encoding three 11-amino acid and one 32-amino acid peptides. Ectopic expression of the 11 amino-acid peptides recapitulates the pri/tal misexpression phenotype in ectopic ES organ formation. In situ hybridization experiment reveals that pri/tal mRNA is expressed in the SOPs of the chemosensory organs and the stretch-sensing chordotonal organs.In Drosophila wing development, the Notch signaling pathway mediates the formation of the dorsal-ventral (DV) compartmental boundary and the restriction of the vein width from the primordial veins, the proveins. We also found that pri/tal mRNA is expressed in the DV boundary and the longitudinal proveins, and overexpression of Pri/Tal peptides disrupts the DV boundary formation and helps to expand the width of the wing vein. Genetic analyses further show that a Notch loss-of-function allele strongly enhances these two phenotypes. Cut and E(spl)mβ are target genes of the Notch pathway in DV boundary formation and vein specification, respectively. We also found that overexpression of Pri/Tal peptides abolishes Cut expression and co-expression of Pri/Tal peptides with phyl strongly reduces E(spl)mβ expression., Conclusions: We show for the first time that the overexpression of Pri/Tal 11-amino acid peptides disrupts multiple Notch-mediated processes and reduces Notch target gene expression in Drosophila, suggesting that these peptides have novel antagonistic activity to the Notch pathway. Thus, our discovery might provide insights into designing new therapeutic reagents for Notch-related diseases.

The deuterated water method is used extensively to measure gluconeogenesis in humans. This method assumes negligible exchange of the lower three carbons of fructose 6-phsophate via transaldolase exchange since this exchange will result in enrichment of carbon 5 of glucose in the absence of net gluconeogenesis. The present studies tested this assumption. ²H₂O and acetaminophen were ingested and [1-¹³C]acetate infused in 11 nondiabetic subjects after a 16-h fast. Plasma and urinary glucuronide enrichments were measured using nuclear magnetic resonance spectroscopy before and during a 0.35 mU·kg FFM⁻¹·min⁻¹ insulin infusion. Rates of endogenous glucose production measured with [3-³H]- and [6,6-²H₂]glucose did not differ either before (14.0 ± 0.7 vs. 13.8 ± 0.7 μmol·kg⁻¹·min⁻¹) or during the clamp (10.4 ± 0.9 vs. 10.9 ± 0.7 μmol·kg⁻¹·min⁻¹), consistent with equilibration and quantitative removal of tritium during triose isomerase exchange. Plasma [3-¹³C] glucose-to-[4-¹³C]glucose and urinary [3-¹³C] glucuronide-to-[4-¹³C]glucuronide ratios were <1.0 (P < 0.001) in all subjects both before (0.66 ± 0.04 and 0.60 ± 0.04) and during (059 ± 0.05 and 0.56 ± 0.06) the insulin infusion, respectively, indicating that ∼35-45% of the labeling of the 5th carbon of glucose by deuterium was due to transaldolase exchange rather than gluconeogenesis. When corrected for transaldolase exchange, rates of gluconeogenesis were lower (P < 0.001) and glycogenolysis higher (P < 0.001) than uncorrected rates both before and during the insulin infusion. In conclusion, assuming negligible dilution by glycerol and near-complete triose isomerase equilibration, these data provide strong experimental evidence that transaldolase exchange occurs in humans, resulting in an overestimate of gluconeogenesis and an underestimate of glycogenolysis when measured with the ²H₂O method. Use of appropriate ¹³C tracers provides a means of correcting for transaldolase exchange.

Background: The development of novel yeast strains with increased tolerance toward inhibitors in lignocellulosic hydrolysates is highly desirable for the production of bio-ethanol. Weak organic acids such as acetic and formic acids are necessarily released during the pretreatment (i.e. solubilization and hydrolysis) of lignocelluloses, which negatively affect microbial growth and ethanol production. However, since the mode of toxicity is complicated, genetic engineering strategies addressing yeast tolerance to weak organic acids have been rare. Thus, enhanced basic research is expected to identify target genes for improved weak acid tolerance., Results: In this study, the effect of acetic acid on xylose fermentation was analyzed by examining metabolite profiles in a recombinant xylose-fermenting strain of Saccharomyces cerevisiae. Metabolome analysis revealed that metabolites involved in the non-oxidative pentose phosphate pathway (PPP) [e.g. sedoheptulose-7-phosphate, ribulose-5-phosphate, ribose-5-phosphate and erythrose-4-phosphate] were significantly accumulated by the addition of acetate, indicating the possibility that acetic acid slows down the flux of the pathway. Accordingly, a gene encoding a PPP-related enzyme, transaldolase or transketolase, was overexpressed in the xylose-fermenting yeast, which successfully conferred increased ethanol productivity in the presence of acetic and formic acid., Conclusions: Our metabolomic approach revealed one of the molecular events underlying the response to acetic acid and focuses attention on the non-oxidative PPP as a target for metabolic engineering. An important challenge for metabolic engineering is identification of gene targets that have material importance. This study has demonstrated that metabolomics is a powerful tool to develop rational strategies to confer tolerance to stress through genetic engineering.