The white lupin trehalase gene LaTRE1 regulates cluster root formation and function under phosphorus deficiency.

Under phosphorus (P) deficiency, white lupin (Lupinus albus L.) forms specialized root structure, called cluster root (CR), to improve soil exploration and nutrient acquisition. Sugar signaling is thought to play a vital role in the development of CR. Trehalose and its associated metabolites are the essential sugar signal molecules that link growth and development to carbon metabolism in plants, however, their roles in the control of CR are still unclear. Here, we investigated the function of the trehalose metabolism pathway by pharmacological and genetic manipulation of the activity of trehalase in white lupin, the only enzyme that degrades trehalose into glucose. Under P deficiency, validamycin A treatment, which inhibits trehalase, led to the accumulation of trehalose and promoted the formation of CR with enhanced organic acid production, whereas overexpression of the white lupin TREHALASE1 (LaTRE1) led to decreased trehalose levels, lateral rootlet density, and organic acid production. Transcriptomic and virus-induced gene silencing (VIGS) results revealed that LaTRE1 negatively regulates the formation of CRs, at least partially, by the suppression of LaLBD16, whose putative ortholog in Arabidopsis (Arabidopsis thaliana) acts downstream of ARF7- and ARF19-dependent auxin signaling in lateral root formation. Overall, our findings provide an association between the trehalose metabolism gene LaTRE1 and CR formation and function with respect to organic acid production in white lupin under P deficiency.

Elucidation of bacterial trehalose-degrading trehalase and trehalose phosphorylase: physiological significance and its potential applications.

Bacteria possess diverse metabolic and genetic processes, resulting in the inability of certain bacteria to degrade trehalose. However, some bacteria do have the capability to degrade trehalose, utilizing it as a carbon source, and for defense against environmental stress. Trehalose, a disaccharide, serves as a carbon source for many bacteria, including some that are vital for pathogens. The degradation of trehalose is carried out by enzymes like trehalase (EC 3.2.1.28) and trehalose phosphorylase (EC 2.4.1.64/2.4.1.231), which are classified under the glycoside hydrolase families GH37, GH15, and GH65. Numerous studies and reports have explored the physiological functions, recombinant expression, enzymatic characteristics, and potential applications of these enzymes. However, further research is still being conducted to understand their roles in bacteria. This review aims to provide a comprehensive summary of the current understanding of trehalose degradation pathways in various bacteria, focusing on three key areas: (i) identifying different trehalose-degrading enzymes in Gram-positive and Gram-negative bacteria, (ii) elucidating the mechanisms employed by trehalose-degrading enzymes belonging to the glycoside hydrolases GH37, GH15, and GH65, and (iii) discussing the potential applications of these enzymes in different sectors. Notably, this review emphasizes the bacterial trehalose-degrading enzymes, specifically trehalases (GH37, GH15, and GH65) and trehalose phosphorylases (GH65), in both Gram-positive and Gram-negative bacteria, an aspect that has not been highlighted before.

Toxicological, biochemical, and in silico investigations of three trehalase inhibitors for new ways to control aphids.

Insect trehalases have been identified as promising new targets for pest control. These key enzymes are involved in trehalose hydrolysis and plays an important role in insect growth and development. In this contribution, plant and microbial compounds, namely validamycin A, amygdalin, and phloridzin, were evaluated for their effect, through trehalase inhibition, on Acyrthosiphon pisum aphid. The latter is part of the Aphididae family, main pests as phytovirus vectors and being very harmful for crops. Validamycin A was confirmed as an excellent trehalase inhibitor with an half maximal inhibitory concentration and inhibitor constant of 2.2 × 10-7 and 5 × 10-8 M, respectively, with a mortality rate of ~80% on a A. pisum population. Unlike validamycin A, the insect lethal efficacy of amygdalin and phloridzin did not correspond to their trehalase inhibition, probably due to their hydrolysis by insect ß-glucosidases. Our docking studies showed that none of the three compounds can bind to the trehalase active site, unlike their hydrolyzed counterparts, that is, validoxylamine A, phloretin, and prunasin. Validoxylamine A would be by far the best trehalase binder, followed by phloretin and prunasin.

Synthesis and Antifungal Activities of Glucosamine Aromatic Derivativesagainst Four Phytopathogenic Fungi of Crops.

A series of diversified glucosamine derivatives (3a-3y) was synthesized and their antifungal activity was examined against four kinds of phytopathogens, Fusarium graminearum (F. graminearum), Fusarium moniliforme (F. moniliforme), Curvularia. lunata (C. lunata), and Rhizoctonia solani (R. solani)which cause seriously economic losses worldwide by affecting crops. The compound 3o showed remarkable antifungal activity against F. graminearum with EC50 values of 3.96 µg/mL, compared to the standard drug triadimefon (10.1µg/mL). 3D-QSAR model with the statistically recommended values (r2 = 0.915, q2=0.872) show that positive charge group and bulky group in the benzyl ring were favorable for the antifungal activity. Enzyme activity assays confirmed that 3o has amoderate inhibition of trehalase with inhibition rate of 51.4%at 5 µg/mL, which is comparable to those of commercial inhibitors validamycin A with inhibition rate of 83.3%.Molecular docking analysis revealed that 3o also had a hydrogen bond interaction with key amino acid residue compared to validoxylamine.

The vital hormone 20-hydroxyecdysone controls ATP production by upregulating binding of trehalase 1 with ATP synthase subunit α in Helicoverpa armigera.

Trehalose is the major "blood sugar" of insects and it plays a crucial role in energy supply and as a stress protectant. The hydrolysis of trehalose occurs only under the enzymatic control of trehalase (Treh), which plays important roles in growth and development, energy supply, chitin biosynthesis, and abiotic stress responses. Previous reports have revealed that the vital hormone 20-hydroxyecdysone (20E) regulates Treh, but the detailed mechanism underlying 20E regulating Treh remains unclear. In this study, we investigated the function of HaTreh1 in Helicoverpa armigera larvae. The results showed that the transcript levels and enzymatic activity of HaTreh1 were elevated during molting and metamorphosis stages in the epidermis, midgut, and fat body, and that 20E upregulated the transcript levels of HaTreh1 through the classical nuclear receptor complex EcR-B1/USP1. HaTreh1 is a mitochondria protein. We also found that knockdown of HaTreh1 in the fifth- or sixth-instar larvae resulted in weight loss and increased mortality. Yeast two-hybrid, coimmunoprecipitation, and glutathione-S-transferase (GST) pull-down experiments demonstrated that HaTreh1 bound with ATP synthase subunit alpha (HaATPs-α) and that this binding increased under 20E treatment. In addition, 20E enhanced the transcript level of HaATPs-α and ATP content. Finally, the knockdown of HaTreh1 or HaATPs-α decreased the induction effect of 20E on ATP content. Altogether, these findings demonstrate that 20E controls ATP production by up-regulating the binding of HaTreh1 to HaATPs-α in H. armigera.

Sulfur, sterol and trehalose metabolism in the deep-sea hydrocarbon seep tubeworm Lamellibrachia luymesi.

BACKGROUND: Lamellibrachia luymesi dominates cold sulfide-hydrocarbon seeps and is known for its ability to consume bacteria for energy. The symbiotic relationship between tubeworms and bacteria with particular adaptations to chemosynthetic environments has received attention. However, metabolic studies have primarily focused on the mechanisms and pathways of the bacterial symbionts, while studies on the animal hosts are limited. RESULTS: Here, we sequenced the transcriptome of L. luymesi and generated a transcriptomic database containing 79,464 transcript sequences. Based on GO and KEGG annotations, we identified transcripts related to sulfur metabolism, sterol biosynthesis, trehalose synthesis, and hydrolysis. Our in-depth analysis identified sulfation pathways in L. luymesi, and sulfate activation might be an important detoxification pathway for promoting sulfur cycling, reducing byproducts of sulfide metabolism, and converting sulfur compounds to sulfur-containing organics, which are essential for symbiotic survival. Moreover, sulfide can serve directly as a sulfur source for cysteine synthesis in L. luymesi. The existence of two pathways for cysteine synthesis might ensure its participation in the formation of proteins, heavy metal detoxification, and the sulfide-binding function of haemoglobin. Furthermore, our data suggested that cold-seep tubeworm is capable of de novo sterol biosynthesis, as well as incorporation and transformation of cycloartenol and lanosterol into unconventional sterols, and the critical enzyme involved in this process might have properties similar to those in the enzymes from plants or fungi. Finally, trehalose synthesis in L. luymesi occurs via the trehalose-6-phosphate synthase (TPS) and trehalose-6-phosphate phosphatase (TPP) pathways. The TPP gene has not been identified, whereas the TPS gene encodes a protein harbouring conserved TPS/OtsA and TPP/OtsB domains. The presence of multiple trehalases that catalyse trehalose hydrolysis could indicate the different roles of trehalase in cold-seep tubeworms. CONCLUSIONS: We elucidated several molecular pathways of sulfate activation, cysteine and cholesterol synthesis, and trehalose metabolism. Contrary to the previous analysis, two pathways for cysteine synthesis and the cycloartenol-C-24-methyltransferase gene were identified in animals for the first time. The present study provides new insights into particular adaptations to chemosynthetic environments in L. luymesi and can serve as the basis for future molecular studies on host-symbiont interactions and biological evolution.

The antifungal effect induced by itraconazole in Candida parapsilosis largely depends on the oxidative stress generated at the mitochondria.

In Candida parapsilosis, homozygous disruption of the two genes encoding trehalase activity increased the susceptibility to Itraconazole compared with the isogenic parental strain. The fungicidal effect of this azole can largely be counteracted by preincubating growing cells with rotenone and the protonophore 2,4-Dinitrophenol. In turn, measurement of endogenous reactive oxygen species formation by flow cytometry confirmed that Itraconazole clearly induced an internal oxidative stress, which can be significantly abolished in rotenone-exposed cells. Analysis of the antioxidant enzymatic activities of catalase and superoxide dismutase pointed to a moderate decrease of catalase in trehalase-deficient mutant cells compared to the wild type, with an additional increase upon addition of rotenone. These enzymatic changes were imperceptible in the case of superoxide dismutase. Alternative assays with Voriconazole led to a similar profile in the results regarding cell growth and antioxidant activities. Collectively, our data suggest that the antifungal action of Itraconazole on C. parapsilosis is dependent on a functional mitochondrial activity. They also suggest that the central metabolic pathways in pathogenic fungi should be considered as preferential antifungal targets in new research.

Functional characterization of Nosema bombycis (microsporidia) trehalase 3.

Nosema bombycis, an obligate intracellular parasite, is a single-celled eukaryote known to infect various tissues of silkworms, leading to the manifestation of pebrine. Trehalase, a glycosidase responsible for catalyzing the hydrolysis of trehalose into two glucose molecules, assumes a crucial role in thermal stress tolerance, dehydration, desiccation stress, and asexual development. Despite its recognized importance in these processes, the specific role of trehalase in N. bombycis remains uncertain. This investigation focused on exploring the functions of trehalase 3 in N. bombycis (NbTre3). Immunofluorescence analysis of mature (dormant) spores indicated that NbTre3 primarily localizes to the spore membrane or spore wall, suggesting a potential involvement in spore germination. Reverse transcription-quantitative polymerase chain reaction results indicated that the transcriptional level of NbTre3 peaked at 6 h post N. bombycis infection, potentially contributing to energy storage for proliferation. Throughout the life cycle of N. bombycis within the host cell, NbTre3 was detected in sporoplasm during the proliferative stage rather than the sporulation stage. RNA interference experiments revealed a substantial decrease in the relative transcriptional level of NbTre3, accompanied by a certain reduction in the relative transcriptional level of Nb16S rRNA. These outcomes suggest that NbTre3 may play a role in the proliferation of N. bombycis. The application of the His pull-down technique identified 28 proteins interacting with NbTre3, predominantly originating from the host silkworm. This finding implies that NbTre3 may participate in the metabolism of the host cell, potentially utilizing the host cell's energy resources.

Host trehalose metabolism disruption by validamycin A results in reduced fitness of parasitoid offspring.

The general cutworm, Spodoptera litura (Lepidoptera: Noctuidae) is a worldwide destructive omnivorous pest and the endoparasitoid wasp Meteorus pulchricornis (Hymenoptera: Braconidae) is the dominant endoparasitoid of S. litura larvae. Trehalase is a key enzyme in insect trehalose metabolism and plays an important role in the growth and development of insects. However, the specific function of trehalase in parasitoid and host associations has been less reported. In this study, we obtained two trehalase genes (SlTre1 and SlTre2) from our previously constructed S. litura transcriptome database; they were highly expressed in 3rd instar larvae. SlTre1 was mainly expressed in the midgut, and SlTre2 was expressed highest in the head. SlTre1 and SlTre2 were highly expressed 5 days after parasitization by M. pulchricornis. Treatment with the trehalase inhibitor validamycin A significantly inhibited the expression levels of SlTre1 and SlTre2, and the trehalase activity. Besides, the content of trehalose was increased but the content of glucose was decreased 24 h after validamycin A treatment in parasitized S. litura larvae. In addition, the immune-related genes in phenoloxidase (PO) pathway and fatty acid synthesis-related genes in lipid metabolism were upregulated in parasitized host larvae after validamycin A treatment. Importantly, the emergence rate, proportion of normal adults, and body size of parasitoid offspring was decreased in parasitized S. litura larvae after validamycin A treatment, indicating that validamycin A disrupts the trehalose metabolism of parasitized host and thus reduces the fitness of parasitoid offspring. The present study provides a novel perspective for coordinating the application of biocontrol and antibiotics in agroecosystem.

VdPT1 Encoding a Neutral Trehalase of Verticillium dahliae Is Required for Growth and Virulence of the Pathogen.

Verticillum dahliae is a soil-borne phytopathogenic fungus causing destructive Verticillium wilt disease. We previously found a trehalase-encoding gene (VdPT1) in V. dahliae being significantly up-regulated after sensing root exudates from a susceptible cotton variety. In this study, we characterized the function of VdPT1 in the growth and virulence of V. dahliae using its deletion-mutant strains. The VdPT1 deletion mutants (ΔVdPT1) displayed slow colony expansion and mycelial growth, reduced conidial production and germination rate, and decreased mycelial penetration ability and virulence on cotton, but exhibited enhanced stress resistance, suggesting that VdPT1 is involved in the growth, pathogenesis, and stress resistance of V. dahliae. Host-induced silencing of VdPT1 in cotton reduced fungal biomass and enhanced cotton resistance against V. dahliae. Comparative transcriptome analysis between wild-type and mutant identified 1480 up-regulated and 1650 down-regulated genes in the ΔVdPT1 strain. Several down-regulated genes encode plant cell wall-degrading enzymes required for full virulence of V. dahliae to cotton, and down-regulated genes related to carbon metabolism, DNA replication, and amino acid biosynthesis seemed to be responsible for the decreased growth of the ΔVdPT1 strain. In contrast, up-regulation of several genes related to glycerophospholipid metabolism in the ΔVdPT1 strain enhanced the stress resistance of the mutated strain.

[Prevalence of trehalase enzymopathy genetic determinants in Siberian and Russian Far East populations].

To date, it has been established that the patient's genotype plays a significant role in the formation of trehalase enzymopathy: the level of enzyme activity decreases when the G→A allele replacement occurs in the rs2276064 locus of the TREH gene. To assess the prevalence of trehalase deficiency, extensive population-based studies are needed. Clinical observations show that the reduced activity of bowel trehalase is more common in the Arctic than in European populations. The aim of this research was to analyze the frequency of the alleles and variants of trehalase gene (rs2276064 TREH) in the indigenous small-numbered populations of Siberia and the Russian Far East. Material and methods. Using the Infinium iSelect HD Custom BeadChip biochip on the iScan platform and real-time polymerase chain reaction on a Bio-Rad CFX96 Touch amplifier, genotyping of 1068 DNA samples was carried out, of which 711 represent 10 ethnic groups of the indigenous people of the North of Siberia and the Far East of the Russian Federation. Two reference groups of Russians (n=311) and Yakuts (n=46) represented the "Caucasoid" and "Mongoloid" poles of the Russian population. Results. The reduced trehalase activity that the heterozygous GA*TREH genotype determines can manifest itself in 19.8-53.7% of indigenous northerners. An additional 1.0 to 19.7% of the population are carriers of the AA*TREH genotype, which is associated with apparent trehalose malabsorption. The carriers may experience nausea, abdominal pain, and other dyspeptic symptoms after eating trehalose containing foods. The total risk of trehalase enzymopathy among the indigenous northerners in the Asian part of the Russian Federation is very high and can reach 60-70%. There is a gradient in the A*TREH allele frequencies in the small-numbered indigenous northern groups of Russia from the west (Khanty, Mansi, Nenets) to the east (peoples of the Far East). Conclusion. The results are consistent with previously reported data on the higher carriage of the A*TREH mutant allele in Mongoloid populations compared to Caucasoid groups. It was hypothesized that, while the initial A*TREH allele prevalence in Mongoloid groups was moderately high, an adaptation to a low-sugar protein-lipid "high-latitude" diet led to a weaker control over the maintenance of the carriage of the ancestral G allele. Trehalose malabsorption requires special attention of specialists in the field of nutrition, gastroenterology, public health, and medical genetics working in high-latitude regions.

Two trehalase isoforms, produced from a single transcript, regulate drought stress tolerance in Arabidopsis thaliana.

KEY MESSAGE: Alternative translation initiation of the unique Arabidopsis trehalase gene allows for the production of two isoforms with different subcellular localization, providing enzyme access to both intra- and extra-cellular trehalose. The trehalose-hydrolyzing enzyme trehalase mediates drought stress tolerance in Arabidopsis thaliana by controlling ABA-induced stomatal closure. We now report the existence of two trehalase isoforms, produced from a single transcript by alternative translation initiation. The longer full-length N-glycosylated isoform (AtTRE1L) localizes in the plasma membrane with the catalytic domain in the apoplast. The shorter isoform (AtTRE1S) lacks the transmembrane domain and localizes in the cytoplasm and nucleus. The two isoforms can physically interact and this interaction affects localization of AtTRE1S. Consistent with their role in plant drought stress tolerance, both isoforms are activated by AtCPK10, a stress-induced calcium-dependent guard cell protein kinase. Transgenic plants expressing either isoform indicate that both can mediate ABA-induced stomatal closure in response to drought stress but that the short (cytoplasmic/nuclear) isoform, enriched in those conditions, is significantly more effective.

Trehalase is required for sex pheromone biosynthesis in Helicoverpa armigera.

Trehalase (Treh) hydrolyzes trehalose to generate glucose and it plays important role in many physiological processes. Acetyl-CoA, the precursor of sex pheromone biosynthesis in the pheromone gland (PG) of Helicoverpa armigera, originates from glucose during glycolysis. However, the function of Treh in sex pheromone biosynthesis remains elusive. In the present study, H. armigera was used as a model to investigate the function of two Trehs (Treh1 and Treh2) in sex pheromone biosynthesis. Results demonstrated that knockdown of HaTreh1 or HaTreh2 in female PGs led to significant decreases in Z11-16:Ald production, female ability to attract males, and successful mating proportions. Pheromone biosynthesis activating neuropeptide (PBAN) treatment triggered HaTreh1 and HaTreh2 activities in the isolated PGs and Sf9 cells. However, the activities of HaTreh1 and HaTreh2 triggered by PBAN were offset by H-89, the specific inhibitor of protein kinase A (PKA). Furthermore, the H-89 treatment significantly decreased the phosphorylation level of Trhe2, which was induced by PBAN. In addition, sugar feeding (5% sugar) increased the enzyme activities of Treh1 and Treh2. In summary, our findings confirmed that PBAN activates Treh1/2 activities by recruiting cAMP/PKA signalling, promotes glycolysis to ensure the supply of acetyl-CoA, and ultimately facilitates sex pheromone biosynthesis and mating behaviour.

Validamycin A Enhances the Interaction Between Neutral Trehalase and 14-3-3 Protein Bmh1 in Fusarium graminearum.

In agriculture, Trehalase is considered the main target of the biological fungicide validamycin A, and the toxicology mechanism of validamycin A is unknown. 14-3-3 proteins, highly conserved proteins, participate in diverse cellular processes, including enzyme activation, protein localization, and acting as a molecular chaperone. In Saccharomyces cerevisiae, the 14-3-3 protein Bmh1could interact with Nth1 to respond to specific external stimuli. Here, we characterized FgNth, FgBmh1, and FgBmh2 in Fusarium graminearum. ΔFgNth, ΔFgBmh1, and ΔFgBmh2 displayed great growth defects and their peripheral tips hyphae generated more branches when compared with wild-type (WT) PH-1. When exposed to validamycin A as well as high osmotic and high temperature stresses, ΔFgNth, ΔFgBmh1, and ΔFgBmh2 showed more tolerance than WT. Both ΔFgNth and ΔFgBmh1 displayed reduced deoxynivalenol production but opposite for ΔFgBmh2, and all three deletion mutants showed reduced virulence on wheat coleoptiles. In addition, coimmunoprecipitation (Co-IP) experiments suggested that FgBmh1 and FgBmh2 both interact with FgNth, but no interaction was detected between FgBmh1 and FgBmh2 in our experiments. Further, validamycin A enhances the interaction between FgBmh1 and FgNth in a positive correlation under concentrations of 1 to 100 µg/ml. In addition, both high osmotic and high temperature stresses promote the interaction between FgBmh1 and FgNth. Co-IP assay also showed that neither FgBmh1 nor FgBmh2 could interact with FgPbs2, a MAPKK kinase in the high-osmolarity glycerol pathway. However, FgBmh2 but not FgBmh1 binds to the heat shock protein FgHsp70 in F. graminearum. Taken together, our results demonstrate that FgNth and FgBmh proteins are involved in growth and responses to external stresses and virulence; and validamycin enhanced the interaction between FgNth and FgBmh1in F. graminearum.

Chemical Genetics of AGC-kinases Reveals Shared Targets of Ypk1, Protein Kinase A and Sch9.

Protein phosphorylation cascades play a central role in the regulation of cell growth and protein kinases PKA, Sch9 and Ypk1 take center stage in regulating this process in S. cerevisiae To understand how these kinases co-ordinately regulate cellular functions we compared the phospho-proteome of exponentially growing cells without and with acute chemical inhibition of PKA, Sch9 and Ypk1. Sites hypo-phosphorylated upon PKA and Sch9 inhibition were preferentially located in RRxS/T-motifs suggesting that many are directly phosphorylated by these enzymes. Interestingly, when inhibiting Ypk1 we not only detected several hypo-phosphorylated sites in the previously reported RxRxxS/T-, but also in an RRxS/T-motif. Validation experiments revealed that neutral trehalase Nth1, a known PKA target, is additionally phosphorylated and activated downstream of Ypk1. Signaling through Ypk1 is therefore more closely related to PKA- and Sch9-signaling than previously appreciated and may perform functions previously only attributed to the latter kinases.

Evaluation of trehalase as an enhancer for a green biocide in the mitigation of Desulfovibrio vulgaris biocorrosion of carbon steel.

Trehalase can biocatalyze the conversion of trehalose to glucose. It is an enzyme that plays an important role in biofilm formation. Thus, trehalase has been patented as a chemical for preventing and treating biofilms. Sulfate-reducing bacteria (SRB) biofilms are often found responsible for biocorrosion, also known as microbiologically infuenced corrosion (MIC), especially in the oil and gas industries and in water utilities. The MIC treatment process typically requires biocide treatment of biofilms, sometimes together with scrubbing. Owing to environmental concerns, a lower biocide dosage is desired in the treatment process. In this work, trehalase was tested as a green biocide enhancer to enhance tetrakis hydroxymethyl phosphonium sulfate (THPS) in the prevention of Desulfovibrio vulgaris MIC of C1018 carbon steel in ATCC 1249 culture medium at 37 °C. THPS is one of the most popular industrial biocides owing to its broad-spectrum efficacy and green chemical status. After 7 days of incubation in 50 mL anaerobic vials containing 40 mL culture medium at pH 7.0, the sessile cell counts indicated that 50 ppm (w/w) THPS + 30 ppm (w/w) trehalase led to an extra 5.7-fold sessile cell reduction when compared with the 50 ppm THPS alone treatment. As a consequence, the combination treatment also resulted in an extra 54% in pit depth reduction and 30% in weight loss reduction when compared with the 50 ppm THPS alone treatment (with 9.0 µm and 1.0 mg/cm2). The biofilm images corroborated the decreased sessile cell count and pitting corrosion.

Expression and characterization of a novel trehalase from Microvirga sp. strain MC18.

Trehalase catalyzes the hydrolysis of trehalose into two glucose molecules and is present in nearly all tissues in various forms. In this study, a putative bacterial trehalase gene, encoding a glycoside hydrolase family 15 (GH15) protein was identified in Microvirga sp. strain MC18 and heterologously expressed in E. coli. The specific activity of the purified recombinant trehalase MtreH was 24 U/mg, with Km and Vmax values of 23.45 mg/mL and 184.23 µmol/mg/min, respectively. The enzyme exhibited optimal activity at 40 °C and pH 7.0, whereby Ca2+ had a considerable positive effects on the catalytic activity and thermostability. The optimized enzymatic reaction conditions for the bioconversion of trehalose using rMtreH were determined as 40 °C, pH 7.0, 10 h and 1% trehalose concentration. The characterization of this bacterial trehalase improves our understanding of the metabolism and biological role of trehalose in prokaryotic organism.

Evaluation of the roles of hydrophobic residues in the N-terminal region of archaeal trehalase in its folding.

Thermoplasma trehalase Tvn1315 is predicted to be composed of a ß-sandwich domain (BD) and a catalytic domain (CD) based on the structure of the bacterial GH15 family glucoamylase (GA). Tvn1315 as well as Tvn1315 (Δ5), in which the 5 N-terminal amino acids are deleted, could be expressed in Escherichia coli as active enzymes, but deletion of 10 residues (Δ10) led to inclusion body formation. To further investigate the role of the N-terminal region of BD, we constructed five mutants of Δ5, in which each of the 5th to 10th residues of the N-terminus of Tvn1315 was mutated to Ala. Every mutant protein could be recovered in soluble form, but only a small fraction of the Y9A mutant was recovered in the soluble fraction. The Y9A mutant recovered in soluble form had similar specific activity to the other proteins. Subsequent mutation analysis at the 9th position of Tvn1315 in Δ5 revealed that aromatic as well as bulky hydrophobic residues could function properly, but residues with hydroxy groups impaired the solubility. Similar results were obtained with mutants based on untruncated Tvn1315. When the predicted BD, Δ5BD, Δ10BD, and BD mutants were expressed, the Δ10BD protein formed inclusion bodies, and the BD mutants behaved similarly to the Δ5 and full-length enzyme mutants. These results suggest that the hydrophobic region is involved in the solubilization of BD during the folding process. Taken together, these results indicate that the solubility of CD depends on BD folding. KEY POINTS: • N-terminal hydrophobic region of the BD is involved in the protein folding. • The N-terminal hydrophobic region of the BD is also involved in the BD folding. • BD is able to weakly interact with the insoluble ß-glucan.

Estrogen-Related Receptor Influences the Hemolymph Glucose Content by Regulating Midgut Trehalase Gene Expression in the Last Instar Larvae of Bombyx mori.

The expression of trehalase in the midgut of insects plays an important role in glucose supply to the hemolymph. Energy metabolism is usually regulated by the estrogen-related receptor (ERR). A decrease in ATP levels is caused by the ERR hindering glycolysis. However, the relationship between trehalose accumulation and ERR expression is still unclear. Here, we found that silkworm ERR (BmERR) is concentrated and BmERR expression is strongly correlated with trehalase in the midgut during the last instar silkworm larval stage. We cloned the promoter of the trehalase from Bombyx mori (BmTreh) and found that the ERR bound directly to the core response elements of the promoter. Cell level interference and the overexpression of ERR can reduce or enhance BmTreh transcription and promoter activity. Overexpressed transgenic BmERR can significantly increase the expression of BmTreh in the midgut of the last instar silkworm larvae, thereby hydrolyzing trehalose into glucose and releasing it into the hemolymph. Additionally, increased hemolymph glucose content reduces silkworm pupa weight but does not affect silk protein production from the silk gland. Our results suggest a novel function for BmERR through its involvement in BmTreh regulation and expand the understanding of ERR functions in insect trehalose metabolism.

Trehalase Gene as a Molecular Signature of Dietary Diversification in Mammals.

Diet is a key factor in determining and structuring animal diversity and adaptive radiations. The mammalian fossil record preserves phenotypic evidence of many dietary shifts, whereas genetic changes followed by dietary diversification in mammals remain largely unknown. To test whether living mammals preserve molecular evidence of dietary shifts, we examined the trehalase gene (Treh), which encodes an enzyme capable of digesting trehalose from insect blood, in bats and other mammals with diverse diets. Bats represent the largest dietary radiation among all mammalian orders, with independent origins of frugivory, nectarivory, carnivory, omnivory, and even sanguivory in an otherwise insectivorous clade. We found that Treh has been inactivated in unrelated bat lineages that independently radiated into noninsectivorous niches. Consistently, purifying selection has been markedly relaxed in noninsectivorous bats compared with their insectivorous relatives. Enzymatic assays of intestinal trehalase in bats suggest that trehalase activity tends to be lost or markedly reduced in noninsectivorous bats compared with their insectivorous relatives. Furthermore, our survey of Treh in 119 mammal species, which represent a deeper evolutionary timeframe, additionally identified a number of other independent losses of Treh in noninsectivorous species, recapitulating the evolutionary pattern that we found in bats. These results document a molecular record of dietary diversification in mammals, and suggest that such molecular signatures of dietary shifts would help us understand both historical and modern changes of animal diets.