Spermidine Synthase Research Articles

Polyamines and Their Biosynthesis/Catabolism Genes Are Differentially Modulated in Response to Heat Versus Cold Stress in Tomato Leaves (Solanum lycopersicum L.).

Polyamines (PAs) regulate growth in plants and modulate the whole plant life cycle. They have been associated with different abiotic and biotic stresses, but little is known about the molecular regulation involved. We quantified gene expression of PA anabolic and catabolic pathway enzymes in tomato (Solanum lycopersicum cv. Ailsa Craig) leaves under heat versus cold stress. These include arginase 1 and 2, arginine decarboxylase 1 and 2, agmatine iminohydrolase/deiminase 1, N-carbamoyl putrescine amidase, two ornithine decarboxylases, three S-adenosylmethionine decarboxylases, two spermidine synthases; spermine synthase; flavin-dependent polyamine oxidases (SlPAO4-like and SlPAO2) and copper dependent amine oxidases (SlCuAO and SlCuAO-like). The spatiotemporal transcript abundances using qRT-PCR revealed presence of their transcripts in all tissues examined, with higher transcript levels observed for SAMDC1, SAMDC2 and ADC2 in most tissues. Cellular levels of free and conjugated forms of putrescine and spermidine were found to decline during heat stress while they increased in response to cold stress, revealing their differential responses. Transcript levels of ARG2, SPDS2, and PAO4-like increased in response to both heat and cold stresses. However, transcript levels of ARG1/2, AIH1, CPA, SPDS1 and CuAO4 increased in response to heat while those of ARG2, ADC1,2, ODC1, SAMDC1,2,3, PAO2 and CuPAO4-like increased in response to cold stress, respectively. Transcripts of ADC1,2, ODC1,2, and SPMS declined in response to heat stress while ODC2 transcripts declined under cold stress. These results show differential expression of PA metabolism genes under heat and cold stresses with more impairment clearly seen under heat stress. We interpret these results to indicate a more pronounced role of PAs in cold stress acclimation compared to that under heat stress in tomato leaves.

The miR-1908/SRM regulatory axis contributes to extracellular vesicle secretion in prostate cancer.

Targeting extracellular vesicle (EV) secretion can have potential clinical implications for cancer therapy, however the precise regulatory mechanisms of EV secretion are not fully understood. Recently, we have shown a novel pathway of EV biogenesis in PCa cell lines, PC3 and PC3M. However, as the characteristics of EVs are divergent even among PCa cell lines, we hypothesized that other pathways or common regulatory pathways of EV biogenesis still exist. Here, we performed quantitative high‐throughput screening to determine the key regulatory genes involved in EV biogenesis in 22Rv1 cells, which secrete a different type of EVs. In total, 1728 miRNAs were screened and miR‐1908 was selected as the potential miRNA regulating EV biogenesis in 22Rv1 cells. Subsequently, we investigated target genes of miR‐1908 using siRNA screening and identified that spermidine synthase (SRM) was the key regulator of EV secretion in 22Rv1 cells. Attenuation of SRM expression significantly inhibited secretion of EVs in 22Rv1 cells, and overexpression of SRM was confirmed in PCa tissues. Furthermore, we found that the number of endosome compartments was increased in cellular cytoplasm after knockdown of the SRM gene. In conclusion, our results showed that miR‐1908‐mediated regulation of SRM can control secretion of EVs in PCa. In addition, these data suggested that the EV secretion pathway was dependent on cellular characteristics.

Regulation of stomatal aperture in response to drought stress mediating with polyamines, nitric oxide synthase and hydrogen peroxide in Rosa canina L.

ABSTRACT To assess the role of genes involved in polyamines synthesis, nitric oxide synthase (NOS), copper amine oxidase activity (CuAO) and hydrogen peroxide (H2O2) in regulation of stomatal aperture to drought stress in Rosa canina L., a study was performed at three irrigating levels (25%, 50%, and 100% field capacity) with three replications at 1, 3, 6 and 12 days. The results showed that putrescine (Put) accumulation occurred under both 50% and 25% FC at 1 d. Furthermore, the role of the Put direct biosynthesis pathway ornithine decarboxylase (ODC) was more effective under 50% FC whereas in the 25% FC the Put indirect production pathway (agmatine iminohydrolase (AIH), N-carbamoyl putrescine amidohydrolase (CPA) and arginine decarboxylase (ADC)) was more effective. HPLC results showed that the accumulation of spermidine (Spd) and spermine (Spm) is consistent with the expression of S-adenosyl methionine decarboxylase (SAMDC), spermidine synthase (SPDS) and spermine synthase (SPMS) genes. Spd accumulation under both 50% and 25% FC occurred on the 3 d and then decreased in the other days. Spm content showed an increasing trend from 6 d under 50% FC and from 3 d under 25% FC. Our results suggest that among the measured polyamines, Put oxidation through CuAO activity increased resulted in an increase in H2O2 production. The H2O2 accumulation also as a secondary messenger led to enhance in NOS gene expression. Increase in NOS gene expression can act as a signal resulting in stomatal closure.

Shelter covering altered polyamines accumulation of cherry (Prunus pseudocerasus Lindl.) via elevating the expression of spermidine synthase (SPDS) gene

Polyamines (PAs), including putrescine (Put), spermidine (Spd), and spermine (Spm), play key roles in plant growth, development, and abiotic/biotic stress tolerance. Their functions appear to be intimately related to their synthesis, which occurs via arginine/ornithine decarboxylase (ADC/ODC), Spd synthase (SPDS) and Spm synthase (SPMS) etc. Previously, the transcriptional regulation of cherry under the shelter covering was carried out, and the PA synthase-related genes, particularly spermidine synthase (SPDS) gene, were differentially expressed in the fruits as exposure to the shelter covering. In the present work, the full-length cDNA sequence of this gene was cloned and named as CpSPDS (GenBank accession: MK045261). Bioinformatics analysis indicated that the cDNA sequence of CpSPDS was 1243 bp in length. Cluster analysis showed that CpSPDS had the highest homologies with those of sweet cherry (XP 021826297, 100 %) and peach (XP 020413638, 99 %). Subcellular localization detected that target gene was located in both nucleus and cytoplasm. The qPCR quantification showed that CpSPDS was differentially expressed in alabastrums, fruitlets, mature fruits and mature leaves of cherry. Interestingly, shelter covering significantly improved the fruitsetting in comparison with those from the shelter-free, gave a positive effect on the expression of CpSPDS gene. The endogenous free PAs were quantified in different tissues by high-performance liquid chromatography (HPLC). The PAs contents of the sheltered trees, i.e. Put, Spm and Spd were diversely higher in comparison to the shelter-free, especially in alabastrums, whose Spd titer of the sheltered was 3.29 folds higher than that of the unsheltered, which somehow collaborated result of mRNA quantification. Therefore, shelter covering for cherry might substantially enhance the expression of CpSPDS gene, leading to altering PAs accumulation, which at least partially contributed to the elevation in fruitsetting in rainy regions.

Novel chimeric spermidine synthase-saccharopine dehydrogenase gene with a possible role in postharvest development of the button mushroom (Agaricus bisporus)

ABSTRACT In plants, spermidine synthase (SPE) received detailed investigation due to its key roles in the biosynthesis of spermidine, a major form of polyamine. However, there is no report on the identification and characterisation of SPE in edible mushrooms. Here, a full-length cDNA of a gene encoding a potential SPE was isolated from fruiting bodies of Agaricus bisporus. Homology search, InterPro inquiry and multiple sequence alignment showed that it was a chimeric bifunctional gene (named as nSpe-Sdh) with a single transcript that could also encode saccharopine dehydrogenase (SDH). Furthermore, we found that the expression of nSPE-SDH mRNA and protein were both significantly up-regulated with the cap expansion of A. bisporus during storage. Once the protecting veil broken, both expression levels decreased to an initial storage level and remained relatively constant until the end of the storage, indicating the possible involvement of nSpe-Sdh in postharvest mushroom development. Besides, the up-regulation of the expression of nSPE-SDH mRNA and protein was both tissue-specially in cap tissues. To our knowledge, this is the first study focus on a specific gene for putative spermidine biosynthesis in edible mushrooms. Our present study provides valuable information for further functional characterisation of SPE-SDH proteins in postharvest mushroom physiology.

Understanding the Cellular Functions Polyamines Play in Proliferation, Replication, and Survival of Leishmania donovani

Leishmaniasis, a neglected tropical disease, affects an estimated billion people worldwide. A better understanding of parasite biology is vital for the development of much needed new therapeutic strategies. Our research addresses this critical need by focusing on the role of polyamines for growth and survival of Leishmania parasites. The main polyamine biosynthetic enzymes in Leishmania are ornithine decarboxylase (ODC) and spermidine synthase (SPDSYN), which sequentially convert ornithine to the polyamines, putrescine and spermidine. With generated gene deletion mutants, Δodc and Δspdsyn, we have demonstrated in vitro that the metabolite putrescine is essential beyond its function as the precursor for spermidine formation. Additionally, we observed that putrescine‐depleted Δodc parasites immediately ceased proliferation and died within two weeks. In contrast, putrescine‐rich Δspdsyn mutants showed an intermediate growth phenotype and entered a quiescent‐like state with cell death occurring after six weeks. Furthermore, putrescine‐depleted cells were unable to synthesize DNA and stopped replicating. We also observed two hallmarks of programmed cell death in the putrescine‐depleted cells: cell‐rounding and phosphatidylserine exposure. To reduce the variability associated with working with two cell lines and to further understand the functions polyamines play, we are in the process of generating a double gene deletion mutant, ΔodcΔspdsyn.Support or Funding InformationPacific University School of Pharmacy Research Incentive Grant

\u03b3-Aminobutyric acid confers cadmium tolerance in maize plants by concerted regulation of polyamine metabolism and antioxidant defense systems

Gamma-Aminobutyric acid (GABA) accumulates in plants following exposure to heavy metals. To investigate the role of GABA in cadmium (Cd) tolerance and elucidate the underlying mechanisms, GABA (0, 25 and 50 µM) was applied to Cd-treated maize plants. Vegetative growth parameters were improved in both Cd-treated and control plants due to GABA application. Cd uptake and translocation were considerably inhibited by GABA. Antioxidant enzyme activity was enhanced in plants subjected to Cd. Concurrently GABA caused further increases in catalase and superoxide dismutase activities, which led to a significant reduction in hydrogen peroxide, superoxide anion and malondealdehyde contents under stress conditions. Polyamine biosynthesis-responsive genes, namely ornithine decarboxylase and spermidine synthase, were induced by GABA in plants grown under Cd shock. GABA suppressed polyamine oxidase, a gene related to polyamine catabolism, when plants were exposed to Cd. Consequently, different forms of polyamines were elevated in Cd-exposed plants following GABA application. The maximum quantum efficiency of photosystem II (Fv/Fm) was decreased by Cd-exposed plants, but was completely restored by GABA to the same value in the control. These results suggest a multifaceted contribution of GABA, through regulation of Cd uptake, production of reactive oxygen species and polyamine metabolism, in response to Cd stress.

Differential regulation of genes co-involved in aroma production and stress amelioration during salt acclimation in indica rice cultivars

The present study investigated the effects of salt acclimation on the expression of genes, viz., proline dehydrogenase (PDH), Δ1-pyrroline-5-carboxylate-synthetase (P5CS), P5C dehydrogenase (P5CDH), ornithine aminotransferase (OAT), betaine aldehyde dehydrogenase 1 (BADH1), BADH2, triose phosphate isomerase (TPI), ornithine decarboxylase (ODC), arginine decarboxylase (ADC), S-adenosyl-methionine decarboxylase (SAMDC), spermidine synthase (SPDS), spermine synthase (SPMS), diamine oxidase (DAO) and polyamine oxidase (PAO) in four indigenous aromatic varieties, viz., Tulaipanji (TP), Radhunipagal (RP), Kalonunia (KN) and Gobindobhog (GB) along with a non-aromatic cultivar, IR-64. The PDH, P5CS, P5CDH and OAT expression showed the regulation of the P5C pathway of aroma synthesis, with OAT appearing to regulate the crosstalk between the P5C and the polyamine (PA) pathway for the production of aroma. The role of PA pathway was further validated by monitoring the expression of ODC, ADC, SAMDC, SPDS, SPMS, DAO and PAO. The BADH2 expression reflected the fact that the enzyme encoded by the gene was directly and negatively associated with aroma content in rice. The TPI expression established that the enzyme encoded by this gene leading to the synthesis of methylglyoxal (MG) is also a key regulator for aroma synthesis. The current study established the varietal differences in the expression of the aforementioned genes in the selected rice cultivars. The salt-stressed aromatic varieties followed the P5C pathway to synthesize aroma. TP and RP exhibited the capability to generate aroma via PA-dependent pathway. Salt-stressed KN, on the contrary, accumulated higher BADH2 and lower TPI transcripts, which correlated with our previous report of KN accumulating low aroma during salinization due to efficient metabolite channelization towards stress amelioration.

Proteomic analysis revealed different responses to hypergravity of soleus and extensor digitorum longus muscles in mice

Investigating protein abundance profiles is important to understand the differences in the slow and fast skeletal muscle characteristics. The profiles in soleus (Sol) and extensor digitorum longus (EDL) muscles in mice exposed to 1 g or 3 g for 28 d were compared. The biological implications of the profiles revealed that hypergravity exposure activated a larger number of pathways involved in protein synthesis in Sol. In contrast, the inactivation of signalling pathways involved in oxidative phosphorylation were conspicuous in EDL. These results suggested that the reactivity of molecular pathways in Sol and EDL differed. Additionally, the levels of spermidine synthase and spermidine, an important polyamine for cell growth, increased in both muscles following hypergravity exposure, whereas the level of spermine oxidase (SMOX) increased in EDL alone. The SMOX level was negatively correlated with spermine content, which is involved in muscle atrophy, and was higher in EDL than Sol, even in the 1 g group. These results indicated that the contribution of SMOX to the regulation of spermidine and spermine contents in Sol and EDL differed. However, contrary to expectations, the difference in the SMOX level did not have a significant impact on the growth of these muscles following hypergravity exposure. SignificanceThe skeletal muscle-specific protein abundance profiles result in differences in the characteristics of slow and fast skeletal muscles. We investigated differences in the profiles in mouse slow-twitch Sol and fast-twitch EDL muscles following 28-d of 1 g and 3 g exposure by LC-MS/MS analysis and label-free quantitation. A two-step solubilisation of the skeletal muscle proteins increased the coverage of proteins identified by LC-MS/MS analysis. Additionally, this method reduced the complexity of samples more easily than protein or peptide fractionation by SDS-PAGE and offline HPLC while maintaining the high operability of samples and was reproducible. A larger number of hypergravity-responsive proteins as well as a prominent increase in the wet weights was observed in Sol than EDL muscles. The biological implications of the difference in the protein abundance profiles in 1 g and 3 g groups revealed that the reactivity of each molecular pathway in Sol and EDL muscles to hypergravity exposure differed significantly. In addition, we found that the biosynthetic and interconversion pathway of polyamines, essential factors for cell growth and survival in mammals, was responsive to hypergravity exposure; spermidine and spermine contents in Sol and EDL muscles were regulated by different mechanisms even in the 1 g group. However, our results indicated that the difference in the mechanism regulating polyamine contents is unlikely to have a significant effect on the differences in Sol and EDL muscle growth following hypergravity exposure.

Identification of a Spermidine Synthase Gene from Soybean by Recombinant Expression, Transcriptional Verification, and Sequence Analysis.

Spermidine possesses multiple healthy functions, and soybeans contain the most abundant spermidine. In this study, spermidine contents of soybeans from different varieties and production regions in China were evaluated, and a spermidine synthase gene (speE) was identified by recombinant expression, transcriptional verification, and sequence analysis. Spermidine contents of soybean samples from 18 varieties ranged 72.38-228.82 mg/kg, and those from 19 production regions ranged 134.64-242.32 mg/kg. The highest-spermidine sample GZ was used to clone four predicted speE genes. Expressing the gene speE5 improved the spermidine titer by 54% in Bacillus amyloliquefaciens, confirming that speE5 was involved in spermidine synthesis. Transcriptional verification was performed through a soybean germination model. Germination for 48 h led to a onefold increase of spermidine in samples SHX and HB, and corresponding speE5 transcriptional levels were improved by 26-fold and 18-fold, respectively, further verifying the function of speE5. Finally, the sequences of the speE5 gene and deduced amino acids were analyzed, and the conserved sites and catalysis mechanisms were presented. This study identified an active spermidine synthase gene from soybean for the first time, which provided an important gene resource for genetic breeding of spermidine-rich soybean or microbial cell factory.

Identification of Novel Prognosis and Prediction Markers in Advanced Prostate Cancer Tissues Based on Quantitative Proteomics.

Prostate cancer (PCa) is the most frequent cancer found in males worldwide, and its mortality rate is increasing every year. However, there are no known molecular markers for advanced or aggressive PCa, and there is an urgent clinical need for biomarkers that can be used for prognosis and prediction of PCa. Mass spectrometry-based proteomics was used to identify new biomarkers in tissues obtained from patients with PCa who were diagnosed with T2, T3, or metastatic PCa in regional lymph nodes. Among 1,904 proteins identified in the prostate tissues, 344 differentially expressed proteins were defined, of which 124 were up-regulated and 216 were down-regulated. Subsequently, based on the results of partial least squares discriminant analysis and Gene Ontology and Kyoto Encyclopedia of Genes and Genomes enrichment analyses, we proposed that spermidine synthase (SRM), nucleolar and coiled-body phosphoprotein 1 (NOLC1), and prostacyclin synthase (PTGIS) represent new protein biomarkers for diagnosis of advanced PCa. These proteomics results were verified by immunoblot assays in metastatic PCa cell lines and by indirect enzyme-linked immunosorbent assay in prostate specimens. SRM was significantly increased depending on the cancer stage, confirming the possibility of using SRM as a biomarker for prognosis and prediction of advanced PCa.

The eggplant transcription factor MYB44 enhances resistance to bacterial wilt by activating the expression of spermidine synthase.

Bacterial wilt (BW) caused by Ralstonia solanacearum is a serious disease affecting the production of Solanaceae species, including eggplant (Solanum melongena). However, few resistance genes have been identified in eggplant, and therefore the underlying mechanism of BW resistance remains unclear. Hence, we investigated a spermidine synthase (SPDS) gene from eggplant and created knock-down lines with virus-induced gene silencing. After eggplant was infected with R. solanacearum, the SmSPDS gene was induced, concurrent with increased spermidine (Spd) content, especially in the resistant line. We speculated that Spd plays a significant role in the defense response of eggplant to BW. Moreover, using the yeast one-hybrid approach and dual luciferase-based transactivation assay, an R2R3-MYB transcription factor, SmMYB44, was identified as directly binding to the SmSPDS promoter, activating its expression. Overexpression of SmMYB44 in eggplant induced the expression of SmSPDS and Spd content, increasing the resistance to BW. In contrast, the SmMYB44-RNAi transgenic plants showed more susceptibility to BW compared with the control plants. Our results provide insight into the SmMYB44-SmSPDS-Spd module involved in the regulation of resistance to R. solanacearum. This research also provides candidates to enhance resistance to BW in eggplant.

Fruit Architecture in Polyamine-Rich Tomato Germplasm Is Determined via a Medley of Cell Cycle, Cell Expansion, and Fruit Shape Genes.

Shape and size are important features of fruits. Studies using tomatoes expressing yeast Spermidine Synthase under either a constitutive or a fruit-ripening promoter showed obovoid fruit phenotype compared to spherical fruit in controls, suggesting that polyamines (PAs) have a role in fruit shape. The obovoid fruit pericarp exhibited decreased cell layers and pericarp thickness compared to wild-type fruit. Transgenic floral buds and ovaries accumulated higher levels of free PAs, with the bound form of PAs being predominant. Transcripts of the fruit shape genes, SUN1 and OVATE, and those of CDKB2, CYCB2, KRP1 and WEE1 genes increased significantly in the transgenic ovaries 2 and 5 days after pollination (DAP). The levels of cell expansion genes CCS52A/B increased at 10 and 20 DAP in the transgenic fruits and exhibited negative correlation with free or bound forms of PAs. In addition, the cell layers and pericarp thickness of the transgenic fruits were inversely associated with free or bound PAs in 10 and 20 DAP transgenic ovaries. Collectively, these results provide evidence for a linkage between PA homeostasis and expression patterns of fruit shape, cell division, and cell expansion genes during early fruit development, and suggest role(s) of PAs in tomato fruit architecture.

Nexus Between Spermidine and Floral Organ Identity and Fruit/Seed Set in Tomato.

Polyamines (PAs) constituting putrescine (Put), spermidine (Spd), and spermine (Spm) are ubiquitous in all organisms and play essential roles in the growth and developmental processes in living organisms, including plants. Evidences obtained through genetic, biochemical, and transgenic approaches suggest a tight homeostasis for cellular PA levels. Altered cellular PA homeostasis is associated with abnormal phenotypes. However, the mechanisms involved for these abnormalities are not yet fully understood, nor is it known whether cellular ratios of different polyamines play any role(s) in specific plant processes. We expressed a yeast spermidine synthase gene (ySpdSyn) under a constitutive promoter CaMV35S in tomato and studied the different phenotypes that developed. The constitutive expression of ySpdSyn resulted in variable flower phenotypes in independent transgenic lines, some of which lacked fruit and seed set. Quantification of PA levels in the developing flowers showed that the transgenic plants without fruit and seed set had significantly reduced Spd levels as well as low Spd/Put ratio compared to the transgenic lines with normal fruit and seed set. Transcript levels of SlDELLA, GA-20oxidase-1, and GA-3oxidase-2, which impact gibberellin (GA) metabolism and signaling, were significantly reduced in bud tissue of transgenic lines that lacked fruit and seed set. These findings indicate that PAs, particularly Spd, impact floral organ identity and fruit set in tomato involving GA metabolism and signaling. Furthermore, we suggest that a nexus exists between PA ratios and developmental programs in plants.

A polyamine-independent role for S-adenosylmethionine decarboxylase.

The only known function of S-adenosylmethionine decarboxylase (AdoMetDC) is to supply, with its partner aminopropyltransferase enzymes such as spermidine synthase (SpdSyn), the aminopropyl donor for polyamine biosynthesis. Polyamine spermidine is probably essential for the growth of all eukaryotes, most archaea and many bacteria. Two classes of AdoMetDC exist, the prokaryotic class 1a and 1b forms, and the eukaryotic class 2 enzyme, which is derived from an ancient fusion of two prokaryotic class 1b genes. Herein, we show that 'eukaryotic' class 2 AdoMetDCs are found in bacteria and are enzymatically functional. However, the bacterial AdoMetDC class 2 genes are phylogenetically limited and were likely acquired from a eukaryotic source via transdomain horizontal gene transfer, consistent with the class 2 form of AdoMetDC being a eukaryotic invention. We found that some class 2 and thousands of class 1b AdoMetDC homologues are present in bacterial genomes that also encode a gene fusion of an N-terminal membrane protein of the Major Facilitator Superfamily (MFS) class of transporters and a C-terminal SpdSyn-like domain. Although these AdoMetDCs are enzymatically functional, spermidine is absent, and an entire fusion protein or its SpdSyn-like domain only, does not biochemically complement a SpdSyn deletion strain of E. coli This suggests that the fusion protein aminopropylates a substrate other than putrescine, and has a role outside of polyamine biosynthesis. Another integral membrane protein found clustered with these genes is DUF350, which is also found in other gene clusters containing a homologue of the glutathionylspermidine synthetase family and occasionally other polyamine biosynthetic enzymes.

Integrated Systems Pharmacology, Urinary Metabonomics, and Quantitative Real-Time PCR Analysis to Uncover Targets and Metabolic Pathways of the You-Gui Pill in Treating Kidney-Yang Deficiency Syndrome.

Kidney-yang deficiency syndrome (KYDS) is a metabolic disease caused by a neuro-endocrine disorder. The You-gui pill (YGP) is a classic traditional Chinese medicine (TCM) formula for the treatment of KYDS and has been widely used to warm and recuperate KYDS clinically for hundreds of years in China. However, it is unknown whetherthe corresponding targets and metabolic pathways can also be found via using metabonomics based on one platform (e.g., 1H NMR) to study different biological samples of KYDS. At the same time, relevant reports on further molecular verification (e.g., RT-qPCR analysis) of these targets associated with biomarkers and metabolic pathways have not yet, to our knowledge, been seen in KYDS’s research. In the present study, a comprehensive strategy integrating systems pharmacology and 1H NMR-based urinary metabonomics analysis was proposed to identify the target proteins and metabolic pathways that YGP acts on KYDS. Thereafter, further validation of target proteins in kidney tissue was performed through quantitative real-time PCR analysis (RT-qPCR). Furthermore, biochemical parameters and histopathological analysis were studied. As a result, seven target proteins (L-serine dehydratase; phosphoenolpyruvate carboxykinase; spermidine synthase; tyrosyl-tRNA synthetase, glutamine synthetase; 3-hydroxyacyl-CoA dehydrogenase; glycine amidinotransferase) in YGP were discovered to play a therapeutic role in KYDS via affecting eight metabolic pathways (glycine, serine and threonine metabolism; butanoate metabolism; TCA cycle, etc.). Importantly, three target proteins (i.e., 3-hydroxyacyl-CoA dehydrogenase; glutamine synthetase; and glycine amidinotransferase) and two metabolic pathways (butanoate metabolism and dicarboxylate metabolism) related to KYDS, to our knowledge, had been newly discovered in our study. The mechanism of action mainly involved energy metabolism, oxidative stress, ammonia metabolism, amino acid metabolism, and fatty acid metabolism. In short, our study demonstrated that targets and metabolic pathways for the treatment of KYDS by YGP can be effectively found via combining with systems pharmacology and urinary metabonomics. In addition to this, common and specific targets and metabolic pathways of KYDS treated by YGP can be found effectively by integration with the analysis of different biological samples (e.g., serum, urine, feces, and tissue). It is; therefore, important that this laid the foundation for deeper mechanism research and drug-targeted therapy of KYDS in future.

Biological conversion of methane to putrescine using genome-scale model-guided metabolic engineering of a methanotrophic bacterium Methylomicrobium alcaliphilum 20Z

BackgroundMethane is the primary component of natural gas and biogas. The huge abundance of methane makes it a promising alternative carbon source for industrial biotechnology. Herein, we report diamine compound, putrescine, production from methane by an industrially promising methanotroph Methylomicrobium alcaliphilum 20Z.ResultsWe conducted adaptive evolution to improve putrescine tolerance of M. alcaliphilum 20Z because putrescine highly inhibits the cell growth. The evolved strain 20ZE was able to grow in the presence of 400 mM of putrescine dihydrochloride. The expression of linear pathway ornithine decarboxylase genes from Escherichia coli and Methylosinus trichosporium OB3b allowed the engineered strain to produce putrescine. A higher putrescine titer of 12.44 mg/L was obtained in the strain 20ZE-pACO with ornithine decarboxylase from M. trichosporium OB3b. For elimination of the putrescine utilization pathway, spermidine synthase (MEALZ_3408) was knocked out, resulting in no spermidine formation in the strain 20ZES1-pACO with a putrescine titer of 18.43 mg/L. Next, a genome-scale metabolic model was applied to identify gene knockout strategies. Acetate kinase (MEALZ_2853) and subsequently lactate dehydrogenase (MEALZ_0534) were selected as knockout targets, and the deletion of these genes resulted in an improvement of the putrescine titer to 26.69 mg/L. Furthermore, the putrescine titer was improved to 39.04 mg/L by overexpression of key genes in the ornithine biosynthesis pathway under control of the pTac promoter. Finally, suitable nitrogen sources for growth of M. alcaliphilum 20Z and putrescine production were optimized with the supplement of 2 mM ammonium chloride to nitrate mineral salt medium, and this led to the production of 98.08 mg/L putrescine, almost eightfold higher than that from the initial strain. Transcriptome analysis of the engineered strains showed upregulation of most genes involved in methane assimilation, citric acid cycle, and ammonia assimilation in ammonia nitrate mineral salt medium, compared to nitrate mineral salt medium.ConclusionsThe engineered M. alcaliphilum 20ZE4-pACO strain was able to produce putrescine up to 98.08 mg/L, almost eightfold higher than the initial strain. This study represents the bioconversion of methane to putrescine—a high value-added diamine compound.

Characterization of a highly conserved Antheraea pernyi spermidine synthase gene.

In the present study, we isolated a spermidine synthase gene from Antheraea pernyi (ApSpds) using expressed sequence tag method. The obtained cDNA sequence of 1483bp contains an open-reading frame of 864bp encoding a polypeptide of 287 amino acids. Sequence analysis revealed that ApSpds belonged to class I of AdoMet-MTase family, and exhibited 30% identity to those from bacteria, 45-48% identity to fungi, 36-47% identity to plants, 52-54% identity to vertebrates and 53-80% identity to invertebrates. Phylogenetic analysis found that the used Spds protein sequences were well divided into five groups corresponding to bacteria, fungi, plants, invertebrates and vertebrates, respectively. These results further confirmed that Spds is highly conserved through evolution of life organisms. The ApSpds mRNA is expressed during all four developmental stages and is present in all examined tissues with the highest abundance in the muscle, in which the relative mRNA expression level was 1.6 times higher than in the fat body. Although not significant, the mRNA level decreased after high-temperature exposure suggesting that the Spds gene may not be involved in temperature stress tolerance in A. pernyi. Taken together, our results suggested that ApSpds play an important role in development of silkworm.

Spermidine Synthase (SPDS) Undergoes Concerted Structural Rearrangements Upon Ligand Binding - A Case Study of the Two SPDS Isoforms From Arabidopsis thaliana.

Spermidine synthases (SPDSs) catalyze the production of the linear triamine, spermidine, from putrescine. They utilize decarboxylated S-adenosylmethionine (dc-SAM), a universal cofactor of aminopropyltransferases, as a donor of the aminopropyl moiety. In this work, we describe crystal structures of two SPDS isoforms from Arabidopsis thaliana (AtSPDS1 and AtSPDS2). AtSPDS1 and AtSPDS2 are dimeric enzymes that share the fold of the polyamine biosynthesis proteins. Subunits of both isoforms present the characteristic two-domain structure. Smaller, N-terminal domain is built of the two β-sheets, while the C-terminal domain has a Rossmann fold-like topology. The catalytic cleft composed of two main compartments, the dc-SAM binding site and the polyamine groove, is created independently in each AtSPDS subunits at the domain interface. We also provide the structural details about the dc-SAM binding mode and the inhibition of SPDS by a potent competitive inhibitor, cyclohexylamine (CHA). CHA occupies the polyamine binding site of AtSPDS where it is bound at the bottom of the active site with the amine group placed analogously to the substrate. The crystallographic snapshots show in detail the structural rearrangements of AtSPDS1 and AtSPDS2 that are required to stabilize ligands within the active site. The concerted movements are observed in both compartments of the catalytic cleft, where three major parts significantly change their conformation. These are (i) the neighborhood of the glycine-rich region where aminopropyl moiety of dc-SAM is bound, (ii) the very flexible gate region with helix η6, which interacts with both, the adenine moiety of dc-SAM and the bound polyamine or inhibitor, and (iii) the N-terminal β-hairpin, that limits the putrescine binding grove at the bottom of the catalytic site.

Critical functions of the polyamine putrescine for proliferation and viability of Leishmania donovani parasites

Polyamines are metabolites that play important roles in rapidly proliferating cells, and recent studies have highlighted their critical nature in Leishmania parasites. However, little is known about the function of polyamines in parasites. To address this question, we assessed the effect of polyamine depletion in Leishmania donovani mutants lacking ornithine decarboxylase (Δodc) or spermidine synthase (Δspdsyn). Intracellular putrescine levels depleted rapidly in Δodc mutants and accumulated in Δspdsyn mutants, while spermidine levels were maintained at low but stable levels in both cell lines. Putrescine depletion in the Δodc mutants led to cell rounding, immediate cessation of proliferation, and loss of viability, while putrescine-rich Δspdsyn mutants displayed an intermediate proliferation phenotype and were able to arrest in a quiescent-like state for 6 weeks. Supplementation of Δodc mutants with spermidine had little effect on cell proliferation and morphology but enabled parasites to persist for 14 weeks. Thus, putrescine is not only essential as precursor for spermidine formation but also critical for parasite proliferation, morphology, and viability.