Trehalose-6-phosphate Phosphatase Research Articles

Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

The opportunistic bacterium Pseudomonas aeruginosa has been recognized as an important pathogen of clinical relevance and is a leading cause of hospital-acquired infections. The presence of a glycolytic enzyme in Pseudomonas, which is known to be inhibited by trehalose 6-phosphate (T6P) in other organisms, suggests that these bacteria may be vulnerable to the detrimental effects of intracellular T6P accumulation. In the present study, we explored the structural and functional properties of trehalose 6-phosphate phosphatase (TPP) in P. aeruginosa in support of future target-based drug discovery. A survey of genomes revealed the existence of 2 TPP genes with either chromosomal or extrachromosomal location. Both TPPs were produced as recombinant proteins, and characterization of their enzymatic properties confirmed specific, magnesium-dependent catalytic hydrolysis of T6P. The 3-dimensional crystal structure of the chromosomal TPP revealed a protein dimer arising through β-sheet expansion of the individual monomers, which possess the overall fold of halo-acid dehydrogenases.-Cross, M., Biberacher, S., Park, S.-Y., Rajan, S., Korhonen, P., Gasser, R. B., Kim, J.-S., Coster, M. J., Hofmann, A. Trehalose 6-phosphate phosphatases of Pseudomonas aeruginosa.

Using M. marinum T6PP as a Model for M. tuberculosis Inhibitor Design

Trehalose-6-phosphate phosphatase (T6PP) is a member of the haloalkanoic acid dehalogenase superfamily (HADSF) acting upon trehalose 6-phosphate, an intermediate in the pathway by which glucose 6-phosphate is converted to trehalose (1). The gene encoding T6PP, otsB, is conserved among bacterial pathogens, such as Mycobacterium tuberculosis, but absent in mammalian species. Bacterial pathogens of the Mycolata taxon utilize trehalose to synthesize cord factor, a mycolic acid-derived membrane glycolipid enabling toxicity, pathogenicity, and phagocyte survival (2). The otsB-knockout of M. tuberculosis precludes pathogen growth in vitro and virulence in a mouse model (2); thus, T6PP inhibitors are desired as therapeutics. Due to the 76% sequence identity between M. marinum and M. tuberculosis T6PP, our study investigates the molecular structure of T6PP from the model organism M. marinum in order to understand the binding determinants of the M. tuberculosis ortholog. Toward this goal, we have determined the unliganded structure of M. marinum to 1.97 □ resolution with a magnesium ion cofactor. The structure consists of 3 domains: the N-terminal, Rossmann-like core and cap domains. Like many other unliganded T6PP structures, the active site, located at cap-core interface, is exposed to solvent and is not in a conformation competent to bind substrate. However, comparison of the individual domains with M. tuberculosis T6PP results in an RMSD score of 0.64, 0.60, and 0.85 for the N-terminal, core and cap domains, respectively. We have measured the steady-state kinetic constants of M. marinum T6PP as kcat of 13 s−1 and a Km of 150 μM, which are comparable to those from the M. tuberculosis enzyme (kcat of 10 s−1 and Km of 500 μM) (3). Together these data confirm that the M. marinum enzyme is a model for structural and kinetic studies. To date, collaboration with The Scripps Research Institute facilitated the discovery of two T6PP inhibitors: clinically employed compounds Closantel and Cephalosporin C. We have determined a Ki for M. marinum T6PP of 39 μM in the presence of Cephalosporin C. Based on clinical data reporting the usage of the drug for tuberculosis therapy (4), we postulate that Cephalosporin C inhibits T6PP by preventing cord factor synthesis and thereby bacterial pathogenicity. In the future, we seek to determine the liganded structure of M. marinum T6PP with potential inhibitors bound. Support or Funding Information This work was funded by the Arnold and Mabel Beckman Foundation and Boston University Undergraduate Research Opportunities Program. Newly determined structure of M. marinum T6PP overlaid onto known structure of M. tuberculosis T6PP. This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Invertebrate Trehalose-6-Phosphate Synthase Gene: Genetic Architecture, Biochemistry, Physiological Function, and Potential Applications.

The non-reducing disaccharide trehalose is widely distributed among various organisms. It plays a crucial role as an instant source of energy, being the major blood sugar in insects. In addition, it helps countering abiotic stresses. Trehalose synthesis in insects and other invertebrates is thought to occur via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. In many insects, the TPP gene has not been identified, whereas multiple TPS genes that encode proteins harboring TPS/OtsA and TPP/OtsB conserved domains have been found and cloned in the same species. The function of the TPS gene in insects and other invertebrates has not been reviewed in depth, and the available information is quite fragmented. The present review discusses the current understanding of the trehalose synthesis pathway, TPS genetic architecture, biochemistry, physiological function, and potential sensitivity to insecticides. We note the variability in the number of TPS genes in different invertebrate species, consider whether trehalose synthesis may rely only on the TPS gene, and discuss the results of in vitro TPS overexpression experiment. Tissue expression profile and developmental characteristics of the TPS gene indicate that it is important in energy production, growth and development, metamorphosis, stress recovery, chitin synthesis, insect flight, and other biological processes. We highlight the molecular and biochemical properties of insect TPS that make it a suitable target of potential pest control inhibitors. The application of trehalose synthesis inhibitors is a promising direction in insect pest control because vertebrates do not synthesize trehalose; therefore, TPS inhibitors would be relatively safe for humans and higher animals, making them ideal insecticidal agents without off-target effects.

Expression Profiles of the Trehalose-6-Phosphate Synthase Gene Associated With Thermal Stress in Ostrinia furnacalis (Lepidoptera: Crambidae)

Trehalose is the major blood sugar in insects. Physiological significance of this compound has been extensively reported. Trehalose-6-phosphate synthase (TPS) is an important enzyme in the trehalose biosynthesis pathway. Full-length cDNAs of TPS (Of tps) and its alternative splicing isoform (Of tps_isoformI) were cloned from the Asian corn borer (ACB), Ostrinia furnacalis (Guenée; Lepidoptera: Crambidae) larvae. The Of tps and Of tps_isoformI transcripts were 2913 and 1689 bp long, contained 2529 and 1293 bp open reading frames encoding proteins of 842 and 430 amino acids with a molecular mass of 94.4 and 48.6 kDa, respectively. Transcriptional profiling and response to thermal stress of Of tps gene were determined by quantitative real-time PCR showing that the Of tps was predominantly expressed in the larval fat body, significantly enhanced during molting and transformation; and thermal stress also induced Of tps expression. Gene structure analysis is indicating that one TPS domain and one trehalose-6-phosphate phosphatase (TPP) domain were located at the N- and C-termini of Of TPS, respectively, while only the TPS domain was detected in OfTPS_isoformI. Three-dimensional modeling and heterologous expression were developed to predict the putative functions of OfTPS and Of TPS_isoformI. We infer that the expression of Of tps gene is thermally induced and might be crucial for larvae survival.

Genome-wide analysis of the Solanum tuberosum (potato) trehalose-6-phosphate synthase (TPS) gene family: evolution and differential expression during development and stress

BackgroundTrehalose-6-phosphate synthase (TPS) serves important functions in plant desiccation tolerance and response to environmental stimuli. At present, a comprehensive analysis, i.e. functional classification, molecular evolution, and expression patterns of this gene family are still lacking in Solanum tuberosum (potato).ResultsIn this study, a comprehensive analysis of the TPS gene family was conducted in potato. A total of eight putative potato TPS genes (StTPSs) were identified by searching the latest potato genome sequence. The amino acid identity among eight StTPSs varied from 59.91 to 89.54%. Analysis of dN/dS ratios suggested that regions in the TPP (trehalose-6-phosphate phosphatase) domains evolved faster than the TPS domains. Although the sequence of the eight StTPSs showed high similarity (2571-2796 bp), their gene length is highly differentiated (3189-8406 bp). Many of the regulatory elements possibly related to phytohormones, abiotic stress and development were identified in different TPS genes. Based on the phylogenetic tree constructed using TPS genes of potato, and four other Solanaceae plants, TPS genes could be categorized into 6 distinct groups. Analysis revealed that purifying selection most likely played a major role during the evolution of this family. Amino acid changes detected in specific branches of the phylogenetic tree suggests relaxed constraints might have contributed to functional divergence among groups. Moreover, StTPSs were found to exhibit tissue and treatment specific expression patterns upon analysis of transcriptome data, and performing qRT-PCR.ConclusionsThis study provides a reference for genome-wide identification of the potato TPS gene family and sets a framework for further functional studies of this important gene family in development and stress response.

Modelling studies determing the mode of action of anthelmintics inhibiting in vitro trehalose-6-phosphate phosphatase (TPP) of Anisakis simplex s.l

The trehalose-6-phosphate phosphatase (TPP) enzyme is involved in the synthesis of trehalose, the main sugar in the energy metabolism of nematodes. TPP is a member of the HAD-like hydrolase superfamily and shows a robust and specific phosphatase activity for the substrate trehalose-6-phosphate. The presence of conserved active sites of TPP in closely related nematodes and its absence in humans makes it a promising target for antiparasitic drugs. In the present study, homology modeling, molecular docking and MD simulation techniques were used to explore the structure and dynamics of TPP. In the active site, a magnesium ion is stabilized by 3 coordinate bonds formed by D189, D191 and D400. The key amino acids involved in ligand binding by the enzyme are C198, Y201,T357, D191 and Y197. This study relied on docking to select potential inhibitors of TPP which were tested in vitro for sensitivity to anthelmintic drugs such as levamisole and ivermectin targeting Anisakis simplex. The higher toxicity of LEV than IVM was demonstrated after 96 h, 30% of larvae were motile in cultures with 100 μg/ml of LEV and 1000 μg/ml of IVM. We identified drug combination of LEV-IVM against in vitro A. simplex as agonistic effect (CI = 1.1). Levamisole appeared to be a more effective drug which inhibited enzyme activity after 48 h and expression of mRNA after 96 h at a concentration of 10 μg/ml. This preliminary study predicted the structure of TPP, and the results of an in vitro experiment involving A. simplex will contribute to the development of effective inhibitors with potential antiparasitic activity in the future.

An ERF transcription factor from Tamarix hispida, ThCRF1, can adjust osmotic potential and reactive oxygen species scavenging capability to improve salt tolerance

Ethylene-Responsive Factors (ERFs) are plant-specific transcription factors (TFs) involved in multiple biological processes, especially in abiotic stress tolerance. However, the ERFs from woody halophytes that are involved in salt stress have been little studied. In the present investigation, we characterized a subfamily member of ERF TFs from Tamarix hispida, ThCRF1, which responds to salt stress. ThCRF1 is a nuclear protein that binds to the motifs including TTG, DRE and GCC-box. Transient transformation was performed to generate T. hispida overexpressing ThCRF1 and RNA interference (RNAi)-silenced ThCRF1 to analyze its function using gain- and loss-of-function methods. Overexpression of ThCRF1 in T. hispida significantly improved tolerance to salt-shock-induced stress; by contrast, RNAi-silence of ThCRF1 significantly decreased tolerance to salt-shock-induced stress. Further experiments showed that ThCRF1 induces the expression of genes including those encoding pyrroline-5-carboxylate synthetase (P5CS), trehalose-6-phosphate synthase (TPS), trehalose-6-phosphate phosphatase (TPP), superoxide dismutase (SOD) and peroxidase (POD), which lead to enhanced proline and trehalose levels and increased SOD and POD activities. These results were further confirmed by studying transgenic Arabidopsis plants overexpressing ThCRF1. Therefore, the results suggested that ThCRF1 improves tolerance to salt-shock-induced stress by enhancing trehalose and proline biosynthesis to adjust the osmotic potential, and by improving SOD and POD activities to increase reactive oxygen species scavenging capability.

English

Trehalose-6-phosphate phosphatase (TPP) is one of the primary enzymes involved in the synthesis of trehalose, the main sugar found in insect hemolymph. In the present study, we report for the first time heterologous expression in Pichia pastoris, characterization and homology modeling of TPP from Anopheles gambiae mosquito. Purified TPP recombinant exhibited a molecular weight of approximately 36 kDa with optimum pH of 8.0, optimum temperature of 38°C, and the KM was 3.19 ± 0.10 mM. Inhibition tests revealed that CaCl2 and Ca(NO3)2 at 5 and 25 mM, respectively, were effective inhibitors of TPP activity. Homology studies and molecular modeling indicated high similarity of the TPP tridimensional structure with TPP enzymes deposited at a data bank and these homology studies confirmed that it is a trehalose phosphatase with conserved motifs of the superfamily haloacid dehydrogenase. These results may provide support for the discovering of TPP inhibitors to be used for development of insecticides to control mosquito vectors. Key words: Trehalose, trehalose-6-phosphate phosphatase, expression, enzyme characterization, homology modeling.

Cloning of TaTPP-6AL1 associated with grain weight in bread wheat and development of functional marker

Trehalose 6-phosphate phosphatase (TPP) dephosphorylates trehalose 6-phosphate to trehalose, an important growth regulator, and is involved in starch accumulation and grain yield. In this study, wheat TPP homologs were isolated from chromosomes 6AL, 6BL, and 6DL, designated as TaTPP-6AL1, TaTPP-6BL1, and TaTPP-6DL1, respectively. Sequence alignment showed a single-nucleotide polymorphism (SNP) at TaTPP-6AL1 locus between cultivars with contrasting thousand grain weight (TGW), forming alleles TaTPP-6AL1a and TaTPP-6AL1b, respectively. A cleaved amplified polymorphic sequence (CAPS) marker, TaTPP-6AL1-CAPS, was developed to differentiate the two alleles. TaTPP-6AL1 was mapped within the interval of IWB65749 and IWB60449 in a recombinant inbred line (RIL) population derived from Zhou8425B/Chinese Spring using the wheat 90K SNP assay. A QTL for TGW identified in the interval explained 12.1–19.1% of the phenotypic variance across five environments. Association analysis on 141 Chinese wheat cultivars also indicated a significant correlation of TaTPP-6AL1 with TGW. In conclusion, TaTPP-6AL1 and its functional marker are valuable to improve grain yield in wheat breeding.

Enzyme characteristics of pathogen-specific trehalose-6-phosphate phosphatases

Owing to the key role of trehalose in pathogenic organisms, there has recently been growing interest in trehalose metabolism for therapeutic purposes. Trehalose-6-phosphate phosphatase (TPP) is a pivotal enzyme in the most prominent biosynthesis pathway (OtsAB). Here, we compare the enzyme characteristics of recombinant TPPs from five important nematode and bacterial pathogens, including three novel members of this protein family. Analysis of the kinetics of trehalose-6-phosphate hydrolysis reveals that all five enzymes display a burst-like kinetic behaviour which is characterised by a decrease of the enzymatic rate after the pre-steady state. The observed super-stoichiometric burst amplitudes can be explained by multiple global conformational changes in members of this enzyme family during substrate processing. In the search for specific TPP inhibitors, the trapping of the complex conformational transitions in TPPs during the catalytic cycle may present a worthwhile strategy to explore.

Trehalose‐6‐Phosphate Phosphatase Structure and Inhibitor Design

Trehalose is a sugar synthesized by many bacteria, fungi, plants and invertebrates. Trehalose is synthesized in a two‐step process involving trehalose‐6‐phosphate (T6P) synthase, which catalyzes the conversion of glucose‐6‐phosphate and uridine‐diphosphate‐glucose into T6P followed by T6P phosphatase (T6PP), which catalyzes the dephosphorylation of the phosphosugar to form trehalose. Without T6PP, the build‐up of T6P and absence of trehalose decreases the viability and virulence of fungi, bacteria and nematodes1,2,3. Therefore, T6PP has become a popular target for therapeutic intervention in diseases such as tuberculosis and lymphatic filariasis caused by infectious agents. Here, we present the 1.89 Å structure of Salmonella typhimurium (St) T6PP. The x‐ray crystallographic structure of apo St T6PP shows features seen in the haloalkanoic acid dehalogenase superfamily (HADSF) type II members4, with a core Rossmann‐like fold (residues 1 – 95, 175 – 246) and an α/β fold cap domain (residues 96 – 174). Overlaying a recently published structure of T6P‐bound fungal T6PP5 with St T6PP shows in addition to the four core motifs that bind the phosphoryl group in HADSF members, the cap residues Glu123, His132, and Glu167 are involved in binding the substrate. Unlike most unliganded HADSF structures, the St T6PP structure without cofactor or bound substrate has a closed conformation of the cap domain with respect to the core domain. Therefore, this crystal form may be amenable to soaking inhibitors into the active site. Inhibitor‐bound structures will help rationally design inhibitors to increase specificity and potency. In our laboratories, inhibitors of T6PP from several organisms have been uncovered by screening of an FDA approved drug library. These include Closantel and Cephalosporin C. Future efforts will explore incorporation of additional binding interactions from the Closantel and Cephalosporin C scaffolds to create a highly specific inhibitor for T6PP.Support or Funding InformationThis work was funded by R21‐AI103484 to KNA and DD‐M.

Characterization of a Double Deletion Mutant of Fusarium verticillioides Lacking Two Putative Trehalose‐6‐Phosphate Phosphatase Genes

Fusarium verticillioides is a fungal pathogen that commonly infects the stalk, ear, and kernels of corn and can produce fumonisins, a family of mycotoxins linked to disease in livestock and humans. Our goal is to characterize the role of the disaccharide trehalose in growth and stress response in F. verticillioides. Trehalose synthesis in fungi occurs via two steps, the first catalyzed by trehalose‐6‐phosphate synthase (TPS), and the second by trehalose‐6‐phosphate phosphatase (TPP). Previously, we found that trehalose synthesis is completely abolished in a strain lacking TPS, encoded by the TPS1 gene. We report here that mutant strains lacking either one (ΔTPS2) or both (ΔTPS2/ΔTPS3) of the putative TPP genes in F. verticillioides retain the ability to produce trehalose, albeit at reduced levels. The effects of osmotic and thermal stress on these mutants are being investigated.

Development and Characterization of Molecular Markers Associated with Female Plants in Muscadine Grape

Muscadine (Vitis rotundifolia Michx.) vines may be male (M), female (F), or hermaphroditic (H). Male flowers have only filaments and anthers, whereas female and hermaphroditic flowers are morphologically perfect. Female flowers are distinguished from hermaphroditic flowers by their reflexed stamens (as opposed to upright) and nonfunctional pollen. Primers derived from previously identified candidate genes located at the sex locus of Vitis vinifera L. were used to generate amplicons from M, F, and H muscadine cultivars. Sequence analysis of the amplicons revealed insertion/deletion (indel) polymorphisms in a trehalose-6-phosphate phosphatase (TPP) gene (VR006) and a WRKY transcription factor 21 gene (VR009). Primers were designed to create diagnostic markers for each indel polymorphism. Associations between the marker alleles and the plant sex trait were examined in a wide range of muscadine germplasm and in segregating populations derived from F × H, F × M, and H × H crosses. VR006 produced a codominant marker that was able to differentiate the female-associated allele from the male- and hermaphroditic-associated alleles. The marker was able to detect female plants and will be suitable for screening breeding program progenies. VR009 was able to detect the presence of the female allele in most germplasm, but a crossover event appears to have separated the marker from the sex locus in some germplasm. As shown in previous muscadine genetic studies, H × H-derived populations produced male seedlings. Marker analysis of these populations indicates that male flowers only occur in seedlings which are heterozygous for F- and H-associated marker alleles and inheritance of flower type in muscadine remains unclear.

Rational design of reversible inhibitors for trehalose 6-phosphate phosphatases

In some organisms, environmental stress triggers trehalose biosynthesis that is catalyzed collectively by trehalose 6-phosphate synthase, and trehalose 6-phosphate phosphatase (T6PP). T6PP catalyzes the hydrolysis of trehalose 6-phosphate (T6P) to trehalose and inorganic phosphate and is a promising target for the development of antibacterial, antifungal and antihelminthic therapeutics. Herein, we report the design, synthesis and evaluation of a library of aryl d-glucopyranoside 6-sulfates to serve as prototypes for small molecule T6PP inhibitors. Steady-state kinetic techniques were used to measure inhibition constants (Ki) of a panel of structurally diverse T6PP orthologs derived from the pathogens Brugia malayi, Ascaris suum, Mycobacterium tuberculosis, Shigella boydii and Salmonella typhimurium. The binding affinities of the most active inhibitor of these T6PP orthologs, 4-n-octylphenyl α-d-glucopyranoside 6-sulfate (9a), were found to be in the low micromolar range. The Ki of 9a with the B. malayi T6PP ortholog is 5.3 ± 0.6 μM, 70-fold smaller than the substrate Michaelis constant. The binding specificity of 9a was demonstrated using several representative sugar phosphate phosphatases from the HAD enzyme superfamily, the T6PP protein fold family of origin. Lastly, correlations drawn between T6PP active site structure, inhibitor structure and inhibitor binding affinity suggest that the aryl d-glucopyranoside 6-sulfate prototypes will find future applications as a platform for development of tailored second-generation T6PP inhibitors.

Rational design of first generation inhibitors for trehalose 6-phosphate phosphatases

In this study, trehalose 6-phosphate phosphatase (T6PP) was targeted for inhibitor development. T6PP catalyzes the hydrolysis of trehalose-6-phosphate to form trehalose and inorganic phosphate, a reaction essential to important fungal, bacterial, and nematodal pathogens. At the current time, there are no specific inhibitors of T6PP available to serve as tools for interrogating its structure and function nor as leads for pharmaceutical applications. Herein, we describe the synthesis of non-hydrolysable mimics of trehalose-6-phosphate, which incorporate 6-sulfate (1), -phosphonate (2), -fluorophosphonate (3) and –boronate (4) groups in place of the 6-phosphate moiety of the substrate. The inhibitory efficacies of these adducts were evaluated against trehalose 6-phosphate phosphatases selected from evolutionarily distant pathogenic bacteria and nematodes. Phosphonates 2 and 3 were found to display good inhibitory activities against the T6PPs, while the sulfate analog, trehalose-6-sulfate, proved to be a particularly effective broad-spectrum inhibitor of these phosphatases and an ideal prototype for optimization.

Two Subclasses of Differentially Expressed TPS1 Genes and Biochemically Active TPS1 Proteins May Contribute to Sugar Signalling in Kiwifruit Actinidia chinensis.

Trehalose metabolism and its intermediate trehalose-6-phosphate (T6P) are implicated in sensing and signalling sucrose availability. Four class I TREHALOSE-6-PHOSPHATE SYNTHASE (TPS1) genes were identified in kiwifruit, three of which have both the TPS and trehalose-6-phosphate phosphatase (TPP) domain, while the fourth gene gives rise to a truncated transcript. The transcript with highest sequence homology to Arabidopsis TPS1, designated TPS1.1a was the most highly abundant TPS1 transcript in all examined kiwifruit tissues. An additional exon giving rise to a small N-terminal extension was found for two of the TPS1 transcripts, designated TPS1.2a and TPS1.2b. Homology in sequence and gene structure with TPS1 genes from Solanaceae suggests they belong to a separate, asterid-specific class I TPS subclade. Expression of full-length and potential splice variants of these two kiwifruit TPS1.2 transcripts was sufficient to substitute for the lack of functional TPS1 in the yeast tps1Δ tps2Δ mutant, but only weak complementation was detected in the yeast tps1Δ mutant, and no or very weak complementation was obtained with the TPS1.1a construct. Transgenic Arabidopsis lines expressing kiwifruit TPS1.2 under the control of 35S promoter exhibited growth and morphological defects. We investigated the responses of plants to elevated kiwifruit TPS1 activity at the transcriptional level, using transient expression of TPS1.2a in Nicotiana benthamiana leaves, followed by RNA-seq. Differentially expressed genes were identified as candidates for future functional analyses.

Trehalose-6-Phosphate-Mediated Toxicity Determines Essentiality of OtsB2 in Mycobacterium tuberculosis In Vitro and in Mice.

Trehalose biosynthesis is considered an attractive target for the development of antimicrobials against fungal, helminthic and bacterial pathogens including Mycobacterium tuberculosis. The most common biosynthetic route involves trehalose-6-phosphate (T6P) synthase OtsA and T6P phosphatase OtsB that generate trehalose from ADP/UDP-glucose and glucose-6-phosphate. In order to assess the drug target potential of T6P phosphatase, we generated a conditional mutant of M. tuberculosis allowing the regulated gene silencing of the T6P phosphatase gene otsB2. We found that otsB2 is essential for growth of M. tuberculosis in vitro as well as for the acute infection phase in mice following aerosol infection. By contrast, otsB2 is not essential for the chronic infection phase in mice, highlighting the substantial remodelling of trehalose metabolism during infection by M. tuberculosis. Blocking OtsB2 resulted in the accumulation of its substrate T6P, which appears to be toxic, leading to the self-poisoning of cells. Accordingly, blocking T6P production in a ΔotsA mutant abrogated otsB2 essentiality. T6P accumulation elicited a global upregulation of more than 800 genes, which might result from an increase in RNA stability implied by the enhanced neutralization of toxins exhibiting ribonuclease activity. Surprisingly, overlap with the stress response caused by the accumulation of another toxic sugar phosphate molecule, maltose-1-phosphate, was minimal. A genome-wide screen for synthetic lethal interactions with otsA identified numerous genes, revealing additional potential drug targets synergistic with OtsB2 suitable for combination therapies that would minimize the emergence of resistance to OtsB2 inhibitors.

Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

The trehalose biosynthetic pathway is of great interest for the development of novel therapeutics because trehalose is an essential disaccharide in many pathogens but is neither required nor synthesized in mammalian hosts. As such, trehalose-6-phosphate phosphatase (TPP), a key enzyme in trehalose biosynthesis, is likely an attractive target for novel chemotherapeutics. Based on a survey of genomes from a panel of parasitic nematodes and bacterial organisms and by way of a structure-based amino acid sequence alignment, we derive the topological structure of monoenzyme TPPs and classify them into 3 groups. Comparison of the functional roles of amino acid residues located in the active site for TPPs belonging to different groups reveal nuanced variations. Because current literature on this enzyme family shows a tendency to infer functional roles for individual amino acid residues, we investigated the roles of the strictly conserved aspartate tetrad in TPPs of the nematode Brugia malayi by using a conservative mutation approach. In contrast to aspartate-213, the residue inferred to carry out the nucleophilic attack on the substrate, we found that aspartate-215 and aspartate-428 of BmTPP are involved in the chemistry steps of enzymatic hydrolysis of the substrate. Therefore, we suggest that homology-based inference of functionally important amino acids by sequence comparison for monoenzyme TPPs should only be carried out for each of the 3 groups.-Cross, M., Lepage, R., Rajan, S., Biberacher, S., Young, N. D., Kim, B.-N., Coster, M. J., Gasser, R. B., Kim, J.-S., Hofmann, A. Probing function and structure of trehalose-6-phosphate phosphatases from pathogenic organisms suggests distinct molecular groupings.

Structural insight into dephosphorylation by trehalose 6-phosphate phosphatase (OtsB2) from Mycobacterium tuberculosis.

Trehalose serves as a key structural component in the cell wall of Mycobacterium tuberculosis. M. tuberculosis trehalose-6-phosphate phosphatase (MtbTPP), an essential enzyme in the trehalose biosynthesis OtsAB pathway, catalyzes the dephosphorylation of trehalose-6-phosphate (trehalose-6-P) to generate trehalose, and plays a critical role in M. tuberculosis survival-associated cell wall formation and permeability. Therefore, MtbTPP (OtsB2) is considered a promising potential target for discovery of antimicrobial drugs. However, the absence of structural information of MtbTPP restrains our understanding of its underlying catalytic mechanism. Here, we report the high-resolution crystal structures of apo active MtbTPP and its trehalose-6-P bound complex. The apo structure presents a canonical haloacid dehalogenase superfamily structural fold plus an extra N-terminal domain. The catalytic center is located in a positively charged cleft between the hydrolase domain and the cap domain, demonstrating a highly conserved substrate binding pocket. The role of residues interacting with the substrate in catalysis were probed by site-directed mutagenesis. Asp147, Asp149, Asp330, and Asp331 were found to be pivotal for the enzymatic activity of MtbTPP. The MtbTPP structures reported here provide insight into a key step in the biosynthesis of trehalose, which would facilitate future development of anti-TB therapeutics.-Shan, S., Min, H., Liu, T., Jiang, D., Rao, Z. Structural insight into dephosphorylation by trehalose 6-phosphate phosphatase (OtsB2) from Mycobacterium tuberculosis.

The gene expression and the activity of enzyme synthesis of trehalose during development of Ascaris suum (Nematoda) eggs

The activity of trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) and their mRNA expression was determined in Ascaris suum eggs developing from a zygote to invasive larvae in an in vitro culture. The stages of nematode embryonic development, i.e. cleavage, gastrulation and formation of larvae, were always accompanied by an increase in TPS activity. An increase in TPP activity, the second enzyme of the analysed pathway, followed in subsequent developmental stages. Key changes in trehalose metabolism were observed in the tadpole stage. The development of moving larvae was accompanied by the highest mRNA expression and activity level of TPP, which converts metabolic intermediate T6P to trehalose. The trehalose synthesis enzymes activity was positively correlated with their mRNA expression. For three genes: tps1, tps2 (TPS coding) and tpp (TPP coding) there was very strong and statistically significant correlation with the activity of a specific enzyme. The Pearson’s coefficient was r = 0.903714 (p < 0.001) for tps1 gene, r = 0.803012 ( < 0.01) for tps2 and r = 0.856244 (p < 0.01) for tpp over development time.