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Acid‐induced arginine decarboxylase AdiA is a typical homo‐oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole‐cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0–9.0), and its enzymatic activity is 35‐fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L−1 with a conversion of 0.94 mol·mol−1 in whole‐cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH‐adaptation engineering of other oligomeric decarboxylases. [ABSTRACT FROM AUTHOR]

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid statecultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao-ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti-Ao-ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production. [ABSTRACT FROM AUTHOR]

The role of protein modifications and amino acid metabolism in the dormancy breaking of pistachio (Pistacia vera L.) kernels during moist chilling (5 ºC) and warm stratification (25 ºC) were studied. Cold-stratified kernels showed germination up to 97%, while warm-stratified ones had low germination (40%). Increased protein solubility at neutral pH was accompanied by protein carbonylation in both cotyledons and embryonic axes during cold treatment, whereas these values decreased under warm incubation. Amino acid accumulation occurred in both tissues of cold- and warm-stratified kernels. Arginase activity increased in both tissues of cold-stratified kernels but significantly declined during warm treatment. While arginine decarboxylase activity of both organs increased under cold and warm stratification of pistachio kernels, ornithine aminotransferase activity declined during these periods. These results show that increased protein solubility and its carbonylation during cold stratification may induce the protein mobilization and accumulation of amino acids for their subsequent direction to the proper metabolic pathways. In this way, protein modification and arginine metabolism by arginase can be considered germination-specific events during cold stratification of kernels. [ABSTRACT FROM AUTHOR]

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min−1, and 63,218 min−1 mM−1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci. [ABSTRACT FROM AUTHOR]

Summary: Ralstonia solanacearum, a species complex of bacterial plant pathogens that causes bacterial wilt, comprises four phylotypes that evolved when a founder population was split during the continental drift ~180 million years ago. Each phylotype contains strains with RipTAL proteins structurally related to transcription activator‐like (TAL) effectors from the bacterial pathogen Xanthomonas. RipTALs have evolved in geographically separated phylotypes and therefore differ in sequence and potentially functionality. Earlier work has shown that phylotype I RipTAL Brg11 targets a 17‐nucleotide effector binding element (EBE) and transcriptionally activates the downstream arginine decarboxylase (ADC) gene. The predicted DNA binding preferences of Brg11 and RipTALs from other phylotypes are similar, suggesting that most, if not all, RipTALs target the Brg11‐EBE motif and activate downstream ADC genes. Here we show that not only phylotype I RipTAL Brg11 but also RipTALs from other phylotypes activate host genes when preceded by the Brg11‐EBE motif. Furthermore, we show that Brg11 and RipTALs from other phylotypes induce the same quantitative changes of ADC‐dependent plant metabolites, suggesting that most, if not all, RipTALs induce functionally equivalent changes in host cells. Finally, we report transgenic tobacco lines in which the RipTAL‐binding motif Brg11‐EBE mediates RipTAL‐dependent transcription of the executor‐type resistance (R) gene Bs4C from pepper, thereby conferring resistance to RipTAL‐delivering R. solanacearum strains. Our results suggest that cell death‐inducing executor‐type R genes, preceded by the RipTAL‐binding motif Brg11‐EBE, could be used to genetically engineer broad‐spectrum bacterial wilt resistance in crop plants without any apparent fitness penalty. [ABSTRACT FROM AUTHOR]

Abstract Acid‐induced arginine decarboxylase AdiA is a typical homo‐oligomeric protein biosynthesizing alkaline nylon monomer putrescine. However, upon loss of the AdiA decamer oligomeric state at neutral and alkaline conditions the activity also diminishes, obstructing the whole‐cell biosynthesis of alkaline putrescine. Here, a structure cohesion strategy is proposed to change the pH adaptation of AdiA to alkaline environments based on the rational engineering of meridional and latitudinal oligomerization interfaces. After integrating substitutions of E467K at the latitudinal interface and H736E at the meridional channel interface, the structural stability of AdiA decamer and its substrate transport efficiency at neutral and alkaline conditions are improved. Finally, E467K_H736E is well adapted to neutral and alkaline environments (pH 7.0–9.0), and its enzymatic activity is 35‐fold higher than that of wild AdiA at pH 8.0. Using E467K_H736E in the putrescine synthesis pathway, the titer of putrescine is up to 128.9 g·L−1 with a conversion of 0.94 mol·mol−1 in whole‐cell catalysis. Additionally, the neutral pH adaptation of lysine decarboxylase, with a decamer structure similar to AdiA, is also improved using this cohesion strategy, providing an option for pH‐adaptation engineering of other oligomeric decarboxylases.

Colorectal cancer (CRC) has been proven to be highly reliant on arginine availability. Limiting arginine-rich foods or treating patients with arginine-depleting enzymes arginine deiminase (ADI) or arginase can suppress colon cancer. However, arginase and ADI are not the best drug candidates for CRC. Ornithine, the product of arginase, can enhance the supply of polyamine, which favors CRC cell growth, while citrulline, the product of ADI, faces the problem of arginine recycling due to the overexpression of argininosuccinate synthetase (ASS). Biosynthetic arginine decarboxylase (ADC), an enzyme that catalyzes the conversion of arginine to agmatine and carbon dioxide, may be a better choice as it combines both arginine depletion and suppression of intracellular polyamine synthesis via its product agmatine. ADC has anti-tumor potential yet has received much less attention than the other two arginine-depleting enzymes. In order to gain a better understanding of ADC, the preparation and the anti-cancer properties of this enzyme were explored in this study. When tested in vitro, ADC inhibited the proliferation of three colorectal cancer cell lines regardless of their ASS cellular expression. In contrast, ADC had a lesser cytotoxic effect on the human foreskin fibroblasts and rat primary hepatocytes. Further in vitro studies revealed that ADC induced S and G2/M phase cell-cycle arrest and apoptosis in HCT116 and LoVo cells. ADC-induced apoptosis in HCT116 cells followed the mitochondrial apoptotic pathway and was caspase-3-dependent. With all results obtained, we suggest that arginine is a potential target for treating colorectal cancer with ADC, and the anti-cancer properties of ADC should be more deeply investigated in the future. [ABSTRACT FROM AUTHOR]

Polyamines are polycations derived from amino acids that play an important role in proliferation and growth in almost all living cells. In Streptococcus pneumoniae (the pneumococcus), modulation of polyamine metabolism not only plays an important regulatory role in central metabolism, but also impacts virulence factors such as the capsule and stress responses that affect survival in the host. However, functional annotation of enzymes from the polyamine biosynthesis pathways in the pneumococcus is based predominantly on computational prediction. In this study, we cloned SP_0166, predicted to be a pyridoxal-dependent decarboxylase, from the Orn/Lys/Arg family pathway in S. pneumoniae TIGR4 and expressed and purified the recombinant protein. We performed biochemical characterization of the recombinant SP_0166 and confirmed the substrate specificity. For polyamine analysis, we developed a simultaneous quantitative method using hydrophilic interaction liquid chromatography (HILIC)-based liquid chromatography–tandem mass spectrometry (LC–MS/MS) without derivatization. SP_0166 has apparent Km, kcat, and kcat/Km values of 11.3 mM, 715,053 min−1, and 63,218 min−1 mM−1, respectively, with arginine as a substrate at pH 7.5. We carried out inhibition studies of SP_0166 enzymatic activity with arginine as a substrate using chemical inhibitors DFMO and DFMA. DFMO is an irreversible inhibitor of ornithine decarboxylase activity, while DFMA inhibits arginine decarboxylase activity. Our findings confirm that SP_0166 is inhibited by DFMA and DFMO, impacting agmatine production. The use of arginine as a substrate revealed that the synthesis of putrescine by agmatinase and N-carbamoylputrescine by agmatine deiminase were both affected and inhibited by DFMA. This study provides experimental validation that SP_0166 is an arginine decarboxylase in pneumococci.

The relationship between hydrogen peroxide and the metabolism of polyamines and the role of NADPH oxidase (NOX) in that relationship under drought conditions remains unclear. To reveal the relationship, expression levels of the genes in polyamine metabolism, such as arginine decarboxylase, agmatine aminohydrolase, spermidine synthase, S-adenosyl methionine decarboxylase, diamine oxidase, and polyamine oxidase were determined by RT PCR under drought stress combined with exogenous hydrogen peroxide (H2O2) and diphenyleneiodonium chloride (DPI) treatments in maize seedlings. In addition, some basic stress parameters (leaf water potential, lipid peroxidation), levels of polyamines (putrescine, spermidine, and spermine), and gene expression of NOX were measured under drought stress. Exogenous H2O2 induced the polyamine content by up-regulating polyamine-synthesizing genes and downregulating polyamine oxidizing genes. When the NOX enzyme was inhibited by DPI, the polyamine pathway tended towards degradation instead of production. Exogenous H2O2 regulated the metabolism of polyamines to promote their synthesis, and NOX played a key role in that regulation.

In this study, the mechanism of cold stress tolerance in cucumber fruits pretreated with folic acid was investigated. Control group and the group treated with 5 mgL-1 folic acid were stored for 15 days at 4 °‌-C. The results showed that, in comparison with the control, treatment with folic acid resulted in reduced chilling injury and decreased electrolyte leakage. The cucumber fruits treated with folic acid showed higher chlorophyll contents in storage conditions with suppressed chlorophyllase enzyme activity. Exogenous folic acid treatment also increased the activity of arginine decarboxylase and ornithine decarboxylase resulting in the accumulation of polyamine contents. Also, higher levels of proline were observed in the fruits treated with folic acid, which is attributed to the increased activity of proline synthesizing enzymes △1-pyrroline-5-carboxylate syntheses and ornithine aminotransferase and also reduced activity of proline dehydrogenase enzyme that decompose proline. Generally, the results showed that folic acid treatment increased the resistance to the cold stress by regulating the activity of enzymes involved in the biosynthesis of proline, chlorophyll, and polyamines.

Summary: Arginine decarboxylase (ADC)‐mediated putrescine (Put) biosynthesis plays an important role in plant abiotic stress response. SNF1‐related protein kinases 2s (SnRK2s) and abscisic acid (ABA)‐response element (ABRE)‐binding factors (ABFs), are core components of the ABA signaling pathway involved in drought stress response. We previously reported that ADC of Poncirus trifoliata (PtrADC) functions in drought tolerance. However, whether and how SnRK2 and ABF regulate PtrADC to modulate putrescine accumulation under drought stress remains largely unclear.Herein, we employed a set of physiological, biochemical, and molecular approaches to reveal that a protein complex composed of PtrSnRK2.4 and PtrABF2 modulates putrescine biosynthesis and drought tolerance by directly regulating PtrADC.PtrABF2 was upregulated by dehydration in an ABA‐dependent manner. PtrABF2 activated PtrADC expression by directly and specifically binding to the ABRE core sequence within its promoter and positively regulated drought tolerance via modulating putrescine accumulation. PtrSnRK2.4 interacts with and phosphorylates PtrABF2 at Ser93. PtrSnRK2.4‐mediated PtrABF2 phosphorylation is essential for the transcriptional regulation of PtrADC. Besides, PtrSnRK2.4 was shown to play a positive role in drought tolerance by facilitating putrescine synthesis.Taken together, this study sheds new light on the regulatory module SnRK2.4‐ABF2‐ADC responsible for fine‐tuning putrescine accumulation under drought stress, which advances our understanding on transcriptional regulation of putrescine synthesis. [ABSTRACT FROM AUTHOR]

Current therapeutic strategies for spinal cord injury (SCI) cannot fully facilitate neural regeneration or improve function. Arginine decarboxylase (ADC) synthesizes agmatine, an endogenous primary amine with neuroprotective effects. Transfection of human ADC (hADC) gene exerts protective effects after injury in murine brain-derived neural precursor cells (mNPCs). Following from these findings, we investigated the effects of hADC-mNPC transplantation in SCI model mice. Mice with experimentally damaged spinal cords were divided into three groups, separately transplanted with fluorescently labeled (1) control mNPCs, (2) retroviral vector (pLXSN)-infected mNPCs (pLXSN-mNPCs), and (3) hADC-mNPCs. Behavioral comparisons between groups were conducted weekly up to 6 weeks after SCI, and urine volume was measured up to 2 weeks after SCI. A subset of animals was euthanized each week after cell transplantation for molecular and histological analyses. The transplantation groups experienced significantly improved behavioral function, with the best recovery occurring in hADC-mNPC mice. Transplanting hADC-mNPCs improved neurological outcomes, induced oligodendrocyte differentiation and remyelination, increased neural lineage differentiation, and decreased glial scar formation. Moreover, locomotor and bladder function were both rehabilitated. These beneficial effects are likely related to differential BMP-2/4/7 expression in neuronal cells, providing an empirical basis for gene therapy as a curative SCI treatment option. [ABSTRACT FROM AUTHOR]

Polyamines (PA) play an important role in the growth, development and stress resistance of plants, and arginine decarboxylase (ADC) is one of the key enzymes in the biosynthetic pathway of polyamines. Previously, the transcriptional regulation of the 'Manaohong' cherry under the shelter covering was carried out, and the PA synthase-related genes, particularly the ADC gene, were differentially expressed as exposure to drought stress. However, the mechanisms of how ADC is involved in the response of cherry to abiotic stress (especially drought stress) are still unknown. In the present work, the full-length coding sequence of this gene was isolated and named CpADC. Bioinformatics analysis indicated that the coding sequence of CpADC was 2529 bp in length. Cluster analysis showed that CpADC had the highest homologies with those of sweet cherry (Prunus avium, XP_021806331) and peach (Prunus persica, XP_007200307). Subcellular localization detected that the CpADC was localized in the plant nucleus. The qPCR quantification showed that CpADC was differentially expressed in roots, stems, leaves, flower buds, flowers, and fruits at different periods. Drought stress treatments were applied to both wild-type (WT) and transgenic Arabidopsis lines, and relevant physiological indicators were measured, and the results showed that the putrescine content of transgenic Arabidopsis was higher than that of WT under high-temperature treatment. The results showed that the MDA content of WT was consistently higher than that of transgenic plants and that the degree of stress in WT was more severe than in transgenic Arabidopsis, indicating that transgenic CpADC was able to enhance the stress resistance of the plants. Both the transgenic and WT plants had significantly higher levels of proline in their leaves after the stress treatment than before, but the WT plant had lower levels of proline than that of transgenic Arabidopsis in both cases. This shows that the accumulation of proline in the transgenic plants was higher than that in the wild type under drought and high and low-temperature stress, suggesting that the transgenic plants are more stress tolerant than the WT. Taken together, our results reveal that, under drought stress, the increase in both expressions of CpADC gene and Put (putrescine) accumulation regulates the activity of ADC, the content of MDA and Pro to enhance the drought resistance of Arabidopsis thaliana. [ABSTRACT FROM AUTHOR]

Colorectal cancer (CRC) has been proven to be highly reliant on arginine availability. Limiting arginine-rich foods or treating patients with arginine-depleting enzymes arginine deiminase (ADI) or arginase can suppress colon cancer. However, arginase and ADI are not the best drug candidates for CRC. Ornithine, the product of arginase, can enhance the supply of polyamine, which favors CRC cell growth, while citrulline, the product of ADI, faces the problem of arginine recycling due to the overexpression of argininosuccinate synthetase (ASS). Biosynthetic arginine decarboxylase (ADC), an enzyme that catalyzes the conversion of arginine to agmatine and carbon dioxide, may be a better choice as it combines both arginine depletion and suppression of intracellular polyamine synthesis via its product agmatine. ADC has anti-tumor potential yet has received much less attention than the other two arginine-depleting enzymes. In order to gain a better understanding of ADC, the preparation and the anti-cancer properties of this enzyme were explored in this study. When tested in vitro, ADC inhibited the proliferation of three colorectal cancer cell lines regardless of their ASS cellular expression. In contrast, ADC had a lesser cytotoxic effect on the human foreskin fibroblasts and rat primary hepatocytes. Further in vitro studies revealed that ADC induced S and G2/M phase cell-cycle arrest and apoptosis in HCT116 and LoVo cells. ADC-induced apoptosis in HCT116 cells followed the mitochondrial apoptotic pathway and was caspase-3-dependent. With all results obtained, we suggest that arginine is a potential target for treating colorectal cancer with ADC, and the anti-cancer properties of ADC should be more deeply investigated in the future.

Abstract Background Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. Results In this study, we report the development in the Generally Regarded as Safe (GRAS) l-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speA I534D. Conclusions The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from l-arginine by Corynebacterium crenatum is feasible.

The relationship between hydrogen peroxide and the metabolism of polyamines and the role of NADPH oxidase (NOX) in that relationship under drought conditions remains unclear. To reveal the relationship, expression levels of the genes in polyamine metabolism, such as arginine decarboxylase, agmatine aminohydrolase, spermidine synthase, Sadenosyl methionine decarboxylase, diamine oxidase, and polyamine oxidase were determined by RT PCR under drought stress combined with exogenous hydrogen peroxide (H2O2) and diphenyleneiodonium chloride (DPI) treatments in maize seedlings. In addition, some basic stress parameters (leaf water potential, lipid peroxidation), levels of polyamines (putrescine, spermidine, and spermine), and gene expression of NOX were measured under drought stress. Exogenous H2O2 induced the polyamine content by up-regulating polyamine-synthesizing genes and downregulating polyamine oxidizing genes. When the NOX enzyme was inhibited by DPI, the polyamine pathway tended towards degradation instead of production. Exogenous H2O2 regulated the metabolism of polyamines to promote their synthesis, and NOX played a key role in that regulation. [ABSTRACT FROM AUTHOR]

Agmatine, a natural polyamine also known as 4-aminobutyl-guanidine, is biosynthesized from arginine by decarboxylation. Aspergillus oryzae contains high amounts of agmatine, suggesting highly active arginine decarboxylase (ADC) in this organism. However, genome analysis revealed no ADC homolog in A. oryzae. A. oryzae strain RIB40 has six homologs of phosphatidylserine decarboxylase (PSD), an enzyme that synthesizes phosphatidyl ethanolamine from phosphatidylserine. We previously discovered that one of these homologs, AO090102000327, encodes arginine decarboxylase, which we named ADC1. In the present study, we determined the crystal structures of ligand-free, arginine-treated, and agmatine-treated ADC1 each at 1.9–2.15 Å resolution. Each structure contained four ADC1 molecules (chains A–D) in the asymmetric unit of the cell. Each ADC1 molecule is a heterodimer consisting of the N-terminal region (Asn60–Gly441) and C-terminal region (Ser442–Thr482). In the ligand-free ADC1, the N-terminus of Ser442 was modified to form a pyruvoyl group. In the arginine-treated ADC1, arginine was converted to agmatine, with the pyruvoyl group covalently bound to agmatine by forming a Schiff base. The same structure was observed in agmatine-treated ADC1. These results indicate that ADC1 is a pyruvoyl-dependent decarboxylase and unveils the reaction mechanism of ADC from A. oryzae. [Display omitted] • Agmatine is biosynthesized from arginine by decarboxylation. Aspergillus oryzae. • The crystal structures of ligand-free, arginine-treated, and agmatine-treated ADC1 were determined. • In the ligand-free ADC1, the N-terminus of Ser442 was modified to form a pyruvoyl group. • In the arginine-treated ADC1, arginine was converted to agmatine. • These results indicate that ADC1 is a pyruvoyl-dependent decarboxylase. [ABSTRACT FROM AUTHOR]

Background: Agmatine is a member of biogenic amines and is an important medicine which is widely used to regulate body balance and neuroprotective effects. At present, the industrial production of agmatine mainly depends on the chemical method, but it is often accompanied by problems including cumbersome processes, harsh reaction conditions, toxic substances production and heavy environmental pollution. Therefore, to tackle the above issues, arginine decarboxylase was overexpressed heterologously and rationally designed in Corynebacterium crenatum to produce agmatine from glucose by one-step fermentation. Results: In this study, we report the development in the Generally Regarded as Safe (GRAS) l-arginine-overproducing C. crenatum for high-titer agmatine biosynthesis through overexpressing arginine decarboxylase based on metabolic engineering. Then, arginine decarboxylase was mutated to release feedback inhibition and improve catalytic activity. Subsequently, the specific enzyme activity and half-inhibitory concentration of I534D mutant were increased 35.7% and 48.1%, respectively. The agmatine production of the whole-cell bioconversion with AGM3 was increased by 19.3% than the AGM2. Finally, 45.26 g/L agmatine with the yield of 0.31 g/g glucose was achieved by one-step fermentation of the engineered C. crenatum with overexpression of speAI534D. Conclusions: The engineered C. crenatum strain AGM3 in this work was proved as an efficient microbial cell factory for the industrial fermentative production of agmatine. Based on the insights from this work, further producing other valuable biochemicals derived from l-arginine by Corynebacterium crenatum is feasible. [ABSTRACT FROM AUTHOR]

Arginine decarboxylase (ADC) catalyzes the decarboxylation of arginine to form agmatine, an important physiological and pharmacological amine, and attracts attention to the enzymatic production of agmatine. In this study, we for the first time overexpressed and characterized the marine Shewanella algae ADC (SaADC) in Escherichia coli. The recombinant SaADC showed the maximum activity at pH 7.5 and 40 °C. The SaADC displayed previously unreported substrate inhibition when the substrate concentration was higher than 50 mM, which was the upper limit of testing condition in other reports. In the range of 1–80 mM l-arginine, the SaADC showed the Km, kcat, Ki, and kcat/Km values of 72.99 ± 6.45 mM, 42.88 ± 2.63 s−1, 20.56 ± 2.18 mM, and 0.59 s/mM, respectively, which were much higher than the Km (14.55 ± 1.45 mM) and kcat (12.62 ± 0.68 s−1) value obtained by assaying at 1–50 mM l-arginine without considering substrate inhibition. Both the kcat values of SaADC with and without substrate inhibition are the highest ones to the best of our knowledge. This provides a reference for the study of substrate inhibition of ADCs. [ABSTRACT FROM AUTHOR]

Polyamines (PA) play an important role in the growth, development and stress resistance of plants, and arginine decarboxylase (ADC) is one of the key enzymes in the biosynthetic pathway of polyamines. Previously, the transcriptional regulation of the ‘Manaohong’ cherry under the shelter covering was carried out, and the PA synthase-related genes, particularly the ADC gene, were differentially expressed as exposure to drought stress. However, the mechanisms of how ADC is involved in the response of cherry to abiotic stress (especially drought stress) are still unknown. In the present work, the full-length coding sequence of this gene was isolated and named CpADC. Bioinformatics analysis indicated that the coding sequence of CpADC was 2529 bp in length. Cluster analysis showed that CpADC had the highest homologies with those of sweet cherry (Prunus avium, XP_021806331) and peach (Prunus persica, XP_007200307). Subcellular localization detected that the CpADC was localized in the plant nucleus. The qPCR quantification showed that CpADC was differentially expressed in roots, stems, leaves, flower buds, flowers, and fruits at different periods. Drought stress treatments were applied to both wild-type (WT) and transgenic Arabidopsis lines, and relevant physiological indicators were measured, and the results showed that the putrescine content of transgenic Arabidopsis was higher than that of WT under high-temperature treatment. The results showed that the MDA content of WT was consistently higher than that of transgenic plants and that the degree of stress in WT was more severe than in transgenic Arabidopsis, indicating that transgenic CpADC was able to enhance the stress resistance of the plants. Both the transgenic and WT plants had significantly higher levels of proline in their leaves after the stress treatment than before, but the WT plant had lower levels of proline than that of transgenic Arabidopsis in both cases. This shows that the accumulation of proline in the transgenic plants was higher than that in the wild type under drought and high and low-temperature stress, suggesting that the transgenic plants are more stress tolerant than the WT. Taken together, our results reveal that, under drought stress, the increase in both expressions of CpADC gene and Put (putrescine) accumulation regulates the activity of ADC, the content of MDA and Pro to enhance the drought resistance of Arabidopsis thaliana.

Aspergillus oryzae spores, when sprinkled onto steamed rice and allowed to propagate, are referred to as rice "koji ." Agmatine, a natural polyamine derived from arginine through the action of arginine decarboxylase (ADC), is abundantly produced by solid state-cultivated rice koji of A. oryzae RIB40 under low pH conditions, despite the apparent absence of ADC orthologs in its genome. Mass spectrometry imaging revealed that agmatine was accumulated inside rice koji at low pH conditions, where arginine was distributed. ADC activity was predominantly observed in substrate mycelia and minimally in aerial mycelia. Natural ADC was isolated from solid state-cultivated A. oryzae rice koji containing substrate mycelia, using ammonium sulfate fractionation, ion exchange, and gel-filtration chromatography. The purified protein was subjected to sodium dodecyl sulfate poly-acrylamide gel electrophoresis (SDS-PAGE), and the detected peptide band was digested for identification by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The gene AO090102000327 of strain RIB40 was identified, previously annotated as phosphatidylserine decarboxylase (PSD), and encoded a 483-amino acid peptide. Recombinant protein encoded by AO090102000327 was expressed in Escherichia coli cells cultivated at 20°C, resulting in the detection of 49 kDa and 5 kDa peptides. The protein exhibited pyruvoyl-dependent decarboxylase activity, favoring arginine over ornithine and showing no activity with phosphatidylserine. The gene was designated Ao-adc1. Ao -ADC1 expression in rice koji at pH 4-6 was confirmed through western blotting using the anti- Ao -ADC1 serum. These findings indicate that Ao-adc1 encodes arginine decarboxylase involved in agmatine production.IMPORTANCEGene AO090102000327 in A. oryzae RIB40, previously annotated as a PSD, falls into a distinct clade when examining the phylogenetic distribution of PSDs. Contrary to the initial PSD annotation, our analysis indicates that the protein encoded by AO090102000327 is expressed in the substrate mycelia area of solid state-cultivated A. oryzae rice koji and functions as an arginine decarboxylase (ADC). The clade to which Ao- ADC1 belongs includes three other Ao- ADC1 paralogs (AO090103000445, AO090701000800, and AO090701000802) that presumably encode ADC rather than PSDs. Regarding PSD, AO090012000733 and AO090005001124 were speculated to be nonmitochondrial and mitochondrial PSDs in A. oryzae RIB40, respectively., Competing Interests: The authors declare no conflict of interest.

Cancer is the second leading cause of death globally. Chemotherapy and radiation therapy and other medications are employed to treat various types of cancer. However, each treatment has its own set of side effects, owing to its low specificity. As a result, there is an urgent need for newer therapeutics that do not disrupt healthy cells' normal functioning. Depriving nutrient or non/semi-essential amino acids to which cancerous cells are auxotrophic remains one such promising anticancer strategy. L-Arginine (Arg) is a semi-essential vital amino acid involved in versatile metabolic processes, signaling pathways, and cancer cell proliferation. Hence, the administration of Arg depriving enzymes (ADE) such as arginase, arginine decarboxylase (ADC), and arginine deiminase (ADI) could be effective in cancer therapy. The Arg auxotrophic cancerous cells like hepatocellular carcinoma, human colon cancer, leukemia, and breast cancer cells are sensitive to ADE treatment due to low expression of crucial enzymes argininosuccinate synthetase (ASS), argininosuccinate lyase (ASL), and ornithine transcarbamylase (OCT). These therapeutic enzyme treatments induce cell death through inducing autophagy, apoptosis, generation of oxidative species, i.e., oxidative stress, and arresting the progression and expansion of cancerous cells at certain cell cycle checkpoints. The enzymes are undergoing clinical trials and could be successfully exploited as potential anticancer agents in the future. [ABSTRACT FROM AUTHOR]

Polyamines, such as spermidine (Spd) spermine (Spm) and their direct precursor, the diamine putrescine (Put) are vital and essential aliphatic amines which are also present in plants. Although ethylene and polyamines are also involved in fruit ripening, the genes coding them must also take part in other biosynthetic pathways. In the ethylene and polyamines play an important role in development of salt stress tolerance, and in responses for biotic and abiotic stresses. Exogenous application of all three main polyamines (Put, Spd, Spm) increase salt tolerance of plants, but, accordingly to previous experiments, spermidine has the main effect on the enhancement of salt tolerance. Nicotiana tabacum L. plants were grown in vitro on MS medium, the treatments were as follows: arginine (150 mg l -1), putrescine (10 mg l -1), spermidine (10 mg l -1). Proline, chlorophyll a, b and polyamine contents were measured. The obtained results show that the arginine decarboxylase and the spermidine synthase genes involved in polyamine metabolism, cannot be enhanced by exogenous addition of their precursor molecules. On the contrary, the spermine synthase gene has a positive effect to the lower-class forms of polyamines. [ABSTRACT FROM AUTHOR]

Putrescine, produced via the arginine decarboxylase (ADC)/ornithine decarboxylase (ODC)-mediated pathway, is an initial precursor for polyamines metabolism and the root-specific biosynthesis of medicinal tropane alkaloids (TAs). These alkaloids are widely used as muscarinic acetylcholine antagonists in clinics. Although the functions of ODC in biosynthesis of polyamines and TAs have been well investigated, the role of ADC is still poorly understood. In this study, enzyme inhibitor treatment showed that ADC was involved in the biosynthesis of putrescine-derived metabolites and root growth in Atropa belladonna. Further analysis found that there were six ADC unigenes in the A. belladonna transcriptome, with two of them, AbADC1 and AbADC2 , exhibiting high expression in the roots. To investigate their roles in TAs/polyamines metabolism and root growth, RNA interference (RNAi) was used to suppress either AbADC1 or AbADC2 expression in A. belladonna hairy roots. Suppression of the AbADC1 expression resulted in a significant reduction in the putrescine content and hairy root biomass. However, it had no noticeable effect on the levels of N -methylputrescine and the TAs hyoscyamine, anisodamine, and scopolamine. On the other hand, suppression of AbADC2 expression markedly reduced the levels of putrescine, N -methylputrescine, and TAs, but had no significant effect on hairy root biomass. According to β -glucuronidase (GUS) staining assays, AbADC1 was mainly expressed in the root elongation and division region while AbADC2 was mainly expressed in the cylinder of the root maturation region. These differences in expression led to functional divergence, with AbADC1 primarily regulating root growth and AbADC2 contributing to TA biosynthesis. • RNAi reduced putrescine biosynthesis by suppressing AbADC1 or AbADC2 in A. belladonna hairy roots. • Knocking down AbADC1 reduced hairy root biomass, while knocking down AbADC2 did not. • Knocking down AbADC2 suppressed the biosynthesis of N -methylputrescine and TAs. • AbADC1 exhibited high expression in the root elongation and division region. • AbADC2 was highly expressed in the cylinder of the root maturation region. [ABSTRACT FROM AUTHOR]

Sucrose, proline, and polyamines are compatible solutes accumulating in plant tissues and increasing cellular osmolarity under environmental stresses. These compatible solutes and hydrogen peroxide can function as signaling molecules in plants. There has been very little evidence how the supply of sucrose changes the biosynthesis of compatible solutes. This study aimed to assess the cross-talk among sucrose, H2O2, and compatible solutes on the expression of genes encoding key enzymes in the pathways of proline and polyamine metabolism in drought stressed maize seedlings. Drought stress (induced by polyethylene glycol solution) increased the expressions of genes encoding pyrroline-5-carboxylate synthetase (P5CS), arginine decarboxylase (ADC), and S-adenosylmethionine decarboxylase (SAMDC), while decreased proline dehydrogenase (ProDH), diamine oxidase (DAO), and polyamine oxidase (PAO) expressions. Addition of sucrose to the stressed seedlings increased the P5CS, ADC and SAMDC expressions more than drought stress alone and reduced more the ProDH, DAO, and PAO expressions. Moreover, exogenous sucrose increased leaf water potential and the content of proline, polyamines, and total soluble sugars, whereas decreased H2O2 content and membrane damages under the drought stress conditions. Consequently, exogenous sucrose contributed to the preservation of water status and the amelioration of damage in maize seedlings under the drought stress.

Copyright of Modern Food Science & Technology is the property of Editorial Office of Modern Food Science & Technology and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

During plant-microbe interactions, polyamines participate in the plant defense response. Previously, we reported that silencing of ADC genes in Arabidopsis thaliana causes a drastic reduction of polyamine levels as well as increments in reactive oxygen species content. In this study, we examined the response of the adc -silenced line to Botrytis cinerea and Pseudomonas syringae infection. The adc -silenced line was more susceptible to Botrytis cinerea , showing larger lesion length and a higher incidence of fungal infection. Pre-treatments with putrescine reestablished the response of the adc -silenced line to Botrytis cinerea , resulting in a similar phenotype to the parental plant. Expression levels of defense-related genes were analyzed during fungal infection showing that the salicylic acid-induced gene PR1 was up-regulated, while the jasmonic acid-related genes LOX3 and PDF1.2 , as well as, the camalexin biosynthetic gene PAD3 were down-regulated in the adc -silenced line. Furthermore, methyl jasmonate pre-treatments reduced Botrytis cinerea infection in the adc -silenced line. On the other hand, the adc -silenced line showed an increased resistance to Pseudomonas syringae infection. SA-related genes such as PR1 , ZAT1.2 , WRKY54 and WRKY70 were highly expressed in the adc -silenced line upon bacterial interaction. Our data show that the adc -silenced line has altered the defense-response against Botrytis cinerea and Pseudomonas syringae , that is consistent with deregulation of SA- and JA-mediated response pathways. • The adc -silenced line with low polyamine content is more susceptible to Botrytis cinerea. • MeJA reduces the severity of the disease caused by Botrytis cinerea in adc -silenced line. • The adc -silenced line showed an increased resistance to Pseudomonas syringae infection. • Arabidopsis adc -silenced line shows a deregulation in the SA and JA response genes. [ABSTRACT FROM AUTHOR]

Unlike in flowering plants, the detailed roles of the enzymes in the polyamine (PA) pathway in conifers are poorly known. We explored the sequence conservation of the PA biosynthetic genes and diamine oxidase (DAO) in conifers and flowering plants to reveal the potential functional diversification of the enzymes between the plant lineages. The expression of the genes showing different selective constraints was studied in Scots pine zygotic embryogenesis and early seedling development. We found that the arginine decarboxylase pathway is strongly preferred in putrescine production in the Scots pine as well as generally in conifers and that the reduced use of ornithine decarboxylase (ODC) has led to relaxed purifying selection in ODC genes. Thermospermine synthase (ACL5) genes evolve under strong purifying selection in conifers and the DAO gene is also highly conserved in pines. In developing Scots pine seeds, the expression of both ACL5 and DAO increased as embryogenesis proceeded. Strong ACL5 expression was present in the procambial cells of the embryo and in the megagametophyte cells destined to die via morphologically necrotic cell death. Thus, the high sequence conservation of ACL5 genes in conifers may indicate the necessity of ACL5 for both embryogenesis and vascular development. Moreover, the result suggests the involvement of ACL5 in morphologically necrotic cell death and supports the view of the genetic regulation of necrosis in Scots pine embryogenesis and in plant development. DAO transcripts were located close to the cell walls and between the walls of adjacent cells in Scots pine zygotic embryos and in the roots of young seedlings. We propose that DAO, in addition to the role in Put oxidation for providing H2O2 during the cell-wall structural processes, may also participate in cell-to-cell communication at the mRNA level. To conclude, our findings indicate that the PA pathway of Scots pines possesses several special functional characteristics which differ from those of flowering plants.

Urea is a product of arginine catabolism in plants and its Nitrogen is recycled into the plant metabolism as ammonium after hydrolysis by urease. The eu3-a soybean mutant is null for the Ni insertion protein (UreG) necessary for urease activity. No UreG protein nor any activity of the urease enzymes is detectable in these eu3-a mutants. In order to understand the mechanisms of nitrogen cycling in soybean and the possible physiological benefits to N metabolism, eu3-a (urease-null) and control soybean near-isogenic Eu3 plants were studied. They were grown to two different developmental stages (vegetative-V5 and reproductive-R5) with 15 mM nitrate as the sole source of nitrogen. Growth and biochemical parameters (such as amino acid, nitrate, and polyamine pools) were evaluated in leaves. Gene transcript levels were determined for some enzymes related to Arg catabolism, together with those of the DUR3 active urea transporter and the UreG Ni-insertion accessory protein, whose transcript was confirmed to be absent in eu3-a. The absence of urease activity in the eu3-a null plants did not affect growth or yield although there was a substantial and progressive accumulation of urea in the leaves. Metabolic changes occurred mainly in the pool of amino acids and in the expression of genes related to the pathway of Arg degradation. There are indications that the pathway may be diverted to form polyamines, but to a limited extent. Thus, considering both developmental stages, the degradation of Arg to urea and Orn remains the main path for nitrogen recycling from Arg, despite the progressive accumulation of urea and consequently immobilization of N. [ABSTRACT FROM AUTHOR]

Unlike in flowering plants, the detailed roles of the enzymes in the polyamine (PA) pathway in conifers are poorly known. We explored the sequence conservation of the PA biosynthetic genes and diamine oxidase (DAO) in conifers and flowering plants to reveal the potential functional diversification of the enzymes between the plant lineages. The expression of the genes showing different selective constraints was studied in Scots pine zygotic embryogenesis and early seedling development. We found that the arginine decarboxylase pathway is strongly preferred in putrescine production in the Scots pine as well as generally in conifers and that the reduced use of ornithine decarboxylase (ODC) has led to relaxed purifying selection in ODC genes. Thermospermine synthase (ACL5) genes evolve under strong purifying selection in conifers and the DAO gene is also highly conserved in pines. In developing Scots pine seeds, the expression of both ACL5 and DAO increased as embryogenesis proceeded. Strong ACL5 expression was present in the procambial cells of the embryo and in the megagametophyte cells destined to die via morphologically necrotic cell death. Thus, the high sequence conservation of ACL5 genes in conifers may indicate the necessity of ACL5 for both embryogenesis and vascular development. Moreover, the result suggests the involvement of ACL5 in morphologically necrotic cell death and supports the view of the genetic regulation of necrosis in Scots pine embryogenesis and in plant development. DAO transcripts were located close to the cell walls and between the walls of adjacent cells in Scots pine zygotic embryos and in the roots of young seedlings. We propose that DAO, in addition to the role in Put oxidation for providing H2O2 during the cell-wall structural processes, may also participate in cell-to-cell communication at the mRNA level. To conclude, our findings indicate that the PA pathway of Scots pines possesses several special functional characteristics which differ from those of flowering plants. [ABSTRACT FROM AUTHOR]

Copyright of Mycosystema is the property of Mycosystema Editorial Board and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

The effect of polyamine biosynthesis inhibitors on the salt stress response of rice seedlings was investigated. For this, DL-α-difluoromethylarginine (DFMA) and DL-α-difluoromethylornithine (DFMO), two competitive inhibitors of arginine decarboxylase (ADC) and ornithine decarboxylase (ODC), were used. The ADC and ODC are rate-limiting enzymes involved in synthesis of putrescine. The effective quantum yield of photosynthetic energy conversion (ΦPSII) decreased with the salt stress, and this decrease was highly significant in the treatments with DFMA and DFMO. Interestingly, addition of exogenous putrescine reduced the decline of ΦPSII. Putrescine content strongly decreased after one day of the inhibitor treatment. Although the content of spermidine (converted from putrescine) also showed an initial decrease in response to the inhibitors, it recovered to a similar level to that in the control after 3 d of treatment. Under the salt stress, the effect of the inhibitors on the different compounds was similar. Moreover, the addition of exogenous putrescine partially suppressed the decrease in spermidine and spermine content. A positive correlation between the spermidine and spermine content and the ΦPSII was observed. The results suggest that, under salt stress, a decrease in polyamine biosynthesis and/or polyamine content has a strong negative effect on leaves and increases salt stress sensitivity.

The pressing challenge of cancer's high mortality and invasiveness demands improved therapeutic approaches. Targeting the nutrient dependencies within cancer cells has emerged as a promising approach. This study is dedicated to demonstrating the potential of arginine depletion for cancer treatment. Notably, the focus centers on arginine decarboxylase (RDC), a pH-dependent enzyme expecting enhanced activity within the slightly acidic microenvironments of tumors. To investigate the effect of a single-site mutation on the catalytic efficacy of RDC, diverse amino acids, including glycine, alanine, phenylalanine, tyrosine, tryptophan, p-azido-phenylalanine, and a phenylalanine analog with a hydrogen-substituted tetrazine, were introduced at the crucial threonine site (position 39) in the multimer-forming interface. Remarkably, the introduction of either a natural or a non-natural aromatic amino acid at position 39 substantially boosted enzymatic activity, while amino acids with smaller side chains did not show the same effect. This enhanced enzymatic activity is likely attributed to the reinforced formation of multimer structures through favorable interactions between the introduced aromatic amino acid and the neighboring subunit. Noteworthy, at slightly acidic pH, the RDC variant featuring tryptophan at position 39 demonstrated augmented cytotoxicity against tumor cells compared to the wild-type RDC. This attribute aligns with the tumor microenvironment and positions these variants as potential candidates for targeted cancer therapy., (© 2023 Wiley-VCH GmbH.)

Heat-Stable Antifungal Factor (HSAF) and its analogs are antifungal natural products produced by the biocontrol agent Lysobacter enzymogenes. The production of HSAF is greatly influenced by environmental stimuli and nutrients, but the underlying molecular mechanism is mostly unclear. Here, we found that HSAF production in L. enzymogenes OH11 is strictly controlled by spermidine, which is the most prevalent triamine in bacteria. When added into OH11 cultures, spermidine regulated the production of HSAF and analogs in a concentration-dependent manner. To verify the role of spermidine, we deleted LeSDC and LeADC genes, encoding S-adenosylmethionine decarboxylase and arginine decarboxylase, respectively, that are the key enzymes for spermidine biosynthesis. Both deletion mutants produced barely detectable spermidine and HSAF including its analogs, whereas the antifungals production was restored by exogenous spermidine. The results showed that the OH11 cells must maintain a proper spermidine homeostasis for the antifungals production. Indeed, the expression level of the key HSAF biosynthetic genes was significantly impaired in LeSDC and LeADC mutants, and exogenous spermidine restored the gene expression level in the mutants. Ornithine is a key substrate for HSAF biosynthesis, and OH11 genome contains arg1 and arg2 genes, encoding arginases that convert arginine to ornithine. While the expression of arg1 and arg2 was affected slightly upon mutation of LeSDC and LeADC, exogenous spermidine significantly increased the arginase gene expression in LeSDC and LeADC mutants. Together, the data revealed a previously unrecognized mechanism, in which spermidine controls antibiotic production through controlling both the biosynthetic genes and the substrate-production genes.

Polyamines such as putrescine, cadaverine, and spermidine are small cationic molecules that play significant roles in cellular processes, including bacterial stress responses and host–pathogen interactions. Streptococcus pneumoniae is an opportunistic human pathogen, which causes several diseases that account for significant morbidity and mortality worldwide. As it transits through different host niches, S. pneumoniae is exposed to and must adapt to different types of stress in the host microenvironment. We earlier reported that S. pneumoniae TIGR4, which harbors an isogenic deletion of an arginine decarboxylase (ΔspeA), an enzyme that catalyzes the synthesis of agmatine in the polyamine synthesis pathway, has a reduced capsule. Here, we report the impact of arginine decarboxylase deletion on pneumococcal stress responses. Our results show that ΔspeA is more susceptible to oxidative, nitrosative, and acid stress compared to the wild-type strain. Gene expression analysis by qRT-PCR indicates that thiol peroxidase, a scavenger of reactive oxygen species and aguA from the arginine deiminase system, could be important for peroxide stress responses in a polyamine-dependent manner. Our results also show that speA is essential for endogenous hydrogen peroxide and glutathione production in S. pneumoniae. Taken together, our findings demonstrate the critical role of arginine decarboxylase in pneumococcal stress responses that could impact adaptation and survival in the host.

Objectives: Agmatine is a cationic amine resulting from the decarboxylation of l-arginine. Agmatine has neuroprotective, anti-inflammatory, anti-stress, and anti-depressant properties. In this study, plasma agmatine, arginine decarboxylase, and agmatinase levels were measured during manic episode and remission period in patients with bipolar disorder. Methods: Thirty healthy volunteers and 30 patients who meet Bipolar Disorder Manic Episode diagnostic criteria were included in the study. Additionally, the changes in the patient group between manic episode and remission period were examined. We evaluated the relationship between levels of l-arginine and arginine decarboxylase in the agmatine synthesis pathway, and level of agmatinase that degrades agmatine. Results: Levels of agmatine and l-arginine were significantly increased than control group during manic episode (p <.01). All parameters were increased during manic episode compared to remission period (p <.05). Agmatinase was significantly decreased both during manic episode (p <.01) and remission period (p <.05) in comparison to the control group. Arginine decarboxylase levels did not show a significant difference between the groups (p >.05). Conclusions: This study indicate that there may be a relationship between bipolar disorder and agmatine and its metabolic pathway. Nonetheless, we believe more comprehensive studies are needed in order to reveal the role of agmatine in etiology of bipolar disorder. Agmantine, agmatinase, l-arginine and arginine decarboxylase levels in BD have not been explored before. Various neuro-chemical mechanisms act to increase agmatine in BD; however, agmatine could have elevated to compensate agmatine deficit prior to the manifestation of the disease as in schizophrenia. Elevated agmatine degradation resulting from excess expression of agmatinase which is suggested to be effective in pathogenesis of mood disorders was compensated by this way. Elevated agmatine may be one of the causes which play a role in mania development. Elevated agmatine levels are also suggested to trigger psychosis and be related with the etiology of manic episode and lead to BD. [ABSTRACT FROM AUTHOR]

The Gram-negative bacterium Proteus mirabilis is a common cause of catheter-associated urinary tract infections (CAUTI), which can progress to secondary bacteremia. While numerous studies have investigated experimental infection with P. mirabilis in the urinary tract, little is known about pathogenesis in the bloodstream. This study identifies the genes that are important for survival in the bloodstream using a whole-genome transposon insertion-site sequencing (Tn-Seq) approach. A library of 50,000 transposon mutants was utilized to assess the relative contribution of each non-essential gene in the P. mirabilis HI4320 genome to fitness in the livers and spleens of mice at 24 hours following tail vein inoculation compared to growth in RPMI, heat-inactivated (HI) naïve serum, and HI acute phase serum. 138 genes were identified as ex vivo fitness factors in serum, which were primarily involved in amino acid transport and metabolism, and 143 genes were identified as infection-specific in vivo fitness factors for both spleen and liver colonization. Infection-specific fitness factors included genes involved in twin arginine translocation, ammonia incorporation, and polyamine biosynthesis. Mutants in sixteen genes were constructed to validate both the ex vivo and in vivo results of the transposon screen, and 12/16 (75%) exhibited the predicted phenotype. Our studies indicate a role for the twin arginine translocation (tatAC) system in motility, translocation of potential virulence factors, and fitness within the bloodstream. We also demonstrate the interplay between two nitrogen assimilation pathways in the bloodstream, providing evidence that the GS-GOGAT system may be preferentially utilized. Furthermore, we show that a dual-function arginine decarboxylase (speA) is important for fitness within the bloodstream due to its role in putrescine biosynthesis rather than its contribution to maintenance of membrane potential. This study therefore provides insight into pathways needed for fitness within the bloodstream, which may guide strategies to reduce bacteremia-associated mortality. [ABSTRACT FROM AUTHOR]

The role of the N-methyl-d-aspartate receptor (NMDAr) as a contributor to maladaptive neuroplasticity underlying the maintenance of chronic pain is well established. Agmatine, an NMDAr antagonist, has been shown to reverse tactile hypersensitivity in rodent models of neuropathic pain while lacking the side effects characteristic of global NMDAr antagonism, including sedation and motor impairment, indicating a likely subunit specificity of agmatine's NMDAr inhibition. The present study assessed whether agmatine inhibits subunit-specific NMDAr-mediated current in the dorsal horn of mouse spinal cord slices. We isolated NMDAr-mediated excitatory postsynaptic currents (EPSCs) in small lamina II dorsal horn neurons evoked by optogenetic stimulation of Nav1.8-containing nociceptive afferents. We determined that agmatine abbreviated the amplitude, duration, and decay constant of NMDAr-mediated EPSCs similarly to the application of the GluN2B antagonist ifenprodil. In addition, we developed a site-specific knockdown of the GluN2B subunit of the NMDAr. We assessed whether agmatine and ifenprodil were able to inhibit NMDAr-mediated current in the spinal cord dorsal horn of mice lacking the GluN2B subunit of the NMDAr by analysis of electrically evoked EPSCs. In control mouse spinal cord, agmatine and ifenprodil both inhibited amplitude and accelerated the decay kinetics. However, agmatine and ifenprodil failed to attenuate the decay kinetics of NMDAr-mediated EPSCs in the GluN2B-knockdown mouse spinal cord. The present study indicates that agmatine preferentially antagonizes GluN2B-containing NMDArs in mouse dorsal horn neurons. [ABSTRACT FROM AUTHOR]

Summary: Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying the freezing tolerance of the potato is largely unknown. In the present research, we studied the transcriptome and metabolome of the freezing‐tolerant wild species Solanum acaule (Aca) and freezing‐sensitive cultivated S. tuberosum (Tub) to identify the main pathways and important factors related to freezing tolerance. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway annotation indicated that polyamine and amino acid metabolic pathways were specifically upregulated in Aca under cold treatment. The transcriptome changes detected in Aca were accompanied by the specific accumulation of putrescine, saccharides, amino acids and other metabolites. The combination of transcriptome and metabolome analyses revealed that putrescine exhibited an accumulative pattern in accordance with the expression of the arginine decarboxylase gene ADC1. The primary role of putrescine was further confirmed by analyzing all three polyamines (putrescine, spermidine, and spermine) and the genes encoding the corresponding enzymes in two sets of potato genotypes with distinct freezing tolerance, implying that only putrescine and ADC1 were uniquely enhanced by cold in the freezing‐tolerant genotypes. The function of putrescine was further analyzed by its exogenous application and the overexpression of SaADC1 in S. tuberosum cv. E3, indicating its important role(s) in cold‐acclimated freezing tolerance, which was accompanied with the activation of C‐repeat binding factor genes (CBFs). The present research has identified that the ADC1‐associated putrescine pathway plays an important role in cold‐acclimated freezing tolerance of potato, probably by enhancing the expression of CBF genes. Significance Statement: Low temperature severely influences potato production as the cultivated potato (Solanum tuberosum) is frost sensitive, however the mechanism underlying freezing tolerance of potato is largely unknown. The cold‐responsive genes and metabolites of freezing‐tolerant wild species Solanum acaule were identified and the arginine decarboxylase gene ADC1‐associated putrescine pathway was demonstrated to contribute functionally to potato cold‐acclimated freezing tolerance probably through the upregulation of C‐repeat binding factor gene expression. [ABSTRACT FROM AUTHOR]

Abstract Arabidopsis amiR: ADC -L2 is a non-lethal line with several developmental defects, it is characterized by a drastic reduction in free polyamine content. Herein, we found that catalase application had growth-promoting effects in amiR: ADC -L2 and parental Ws seedlings. Differences in ROS content between amiR: ADC -L2 and Ws seedlings were detected. Increased H 2 O 2 levels were found in the amiR: ADC -L2, as well as low AtCAT2 gene expression and reduced catalase activity. Estimation of polyamine oxidase activity in amiR: ADC -L2 line indicated that the over-accumulation of H 2 O 2 is independent of polyamine catabolism. However, increments in NADPH oxidase activity and O 2 •− content could be associated to the higher H 2 O 2 levels in the amiR: ADC -L2 line. Our data suggest that low polyamine levels in Arabidopsis seedlings are responsible for the accumulation of ROS, by altering the activities of enzymes involved in ROS production and detoxification. Highlights • ROS content and CAT activity is deregulated in a non-lethal line (amiR: ADC -L2) with low polyamine levels. • Catalase exogenous treatment induces a promoting growth in A. thaliana. • Accumulation of ROS in amiR: ADC -L2 line is independent of PA catabolism. [ABSTRACT FROM AUTHOR]

Polyamines play essential roles in plant growth and survival. Arginine decarboxylase (ADC), which converts arginine to agmatine, catalyzes the first step in polyamine biosynthesis from arginine. However, few biochemical studies with purified plant ADCs have been conducted to evaluate their catalytic efficiency. Tomato genome encodes two arginine decarboxylases: SlADC1 and SlADC2 , which are critical for growth, development, and immune responses against bacterial pathogens. We expressed and purified soluble SlADC1 as a recombinant protein fused with maltose-binding protein tag from E. coli Rosetta 2(DE3) cells. Using the purified fusion protein, we characterized the biochemical properties of SlADC1 in vitro and explored it as a target of the bacterial small molecule phevamine A. We confirmed that the activity of SlADC1 depends on the cofactor pyridoxal 5′-phosphate. SlADC1 is specific toward l -arginine and its kinetic parameters were measured using a liquid chromatography-mass spectrometry method. Phevamine A is a competitive inhibitor of SlADC1 and reduces the activity of SlADC1 at high micromolar concentrations. Our purification and biochemical characterization of SlADC1 sets the stage for inhibition studies of this enzyme. • SlADC1 was purified as a maltose binding protein fusion from E. coli Rosetta 2(DE3) cells. • SlADC1 is pyridoxal 5′-phosphate-dependent. • SlADC1 is specific toward l -arginine. • SlADC1 has a K M of 0.6 ± 0.1 mM and a k cat of 16.3 ± 0.5 s−1. • The bacterial small molecule phevamine A is a competitive inhibitor of SlADC1. [ABSTRACT FROM AUTHOR]

Growing evidence suggests that the clinical use of neural progenitor cells (NPCs) is hampered by heterogeneity, poor neuronal yield and low survival rate. Recently, we reported that retrovirus-delivered human arginine decarboxylase (hADC) genes improve cell survival against oxidative insult in murine NPCs in vitro. This study investigates whether the induced expression of hADC gene in mNPCs induces any significant change in the cell fate commitment. The evaluation of induced hADC gene function was assessed by knockdown of hADC gene using specific siRNA. The hADC gene delivery triggered higher expression of N-CAM, cell adhesion molecule and MAP-2, neuronal marker. However, the hADC gene knockdown showed downregulation of N-CAM and MAP-2 expression suggesting that hADC gene delivery favors cell fate commitment of mNPCs towards neuronal lineage. Neurite outgrowth was significantly longer in the hADC infected cells. The neurotrophic signal, BDNF aided in the neuronal commitment, differentiation, and maturation of hADC-mNPCs through PI3K and ERK1/2 activation. The induction of neuron-like differentiation is believed to be regulated by the expression of GSK-3β and Wnt/β-catenin signaling pathways. Our findings suggest that hADC gene delivery favors cell fate commitment of mNPCs towards neuronal lineage, bring new advances in the field of neurogenesis and cell therapy.

Background: The identification of C-repeat binding factor (CBF), and the characterization as an inducer of CBF Expression 1 (ICE1), and a major activator for C-repeat binding factor, were important breakthroughs in the cold signaling network. Methods: In the present study, the full length cDNA of ICE1 was isolated from the tea tree (Camellia sinensis). CsICE1 protein was located in the cell nucleus as revealed by subcellular localization analysis. To investigate the biological functions of CsICE1, a transgenic line fused with the CsICE1 gene in Arabidopsis thaliana (arabidopsis) was generated by the floral dip method. Results: The CsICE1 was expressed differentially in various tea tree tissues, mostly in buds and leaves, and the transcript level of CsICE1 was increased after 1 h and peaked at 2 h under cold treatment. Transcription activity assay indicated that the spermine synthase (SPMS) and arginine decarboxylase (ADC) genes were possible targets of CsICE1. In addition, the values of net photosynthetic rate, transpiration rate, stomatal conductance in transgenic lines declined by less extent than wild-type plants under low temperatures. Furthermore, transcript levels of ADC genes in the transgenic lines had no apparent alteration under normal growth conditions but substantially increased under cold conditions, consistent of changes in free polyamine levels. Taken together, these results demonstrated that CsICE1 plays a positive role in cold tolerance, which may be due to the modulation of polyamine levels through interacting with CsADC.

The biogenic polyamines, spermine, spermidine (Spd) and putrescine (Put) are present at micro-millimolar concentrations in eukaryotic and prokaryotic cells (many prokaryotes have no spermine), participating in the regulation of cellular proliferation and differentiation. In mammalian cells Put is formed exclusively from L-ornithine by ornithine decarboxylase (ODC) and many potent ODC inhibitors are known. In bacteria, plants, and fungi Put is synthesized also from agmatine, which is formed from L-arginine by arginine decarboxylase (ADC). Here we demonstrate that the isosteric hydroxylamine analogue of agmatine (AO-Agm) is a new and very potent (IC50 3•10-8 M) inhibitor of E. coli ADC. It was almost two orders of magnitude less potent towards E. coli ODC. AO-Agm decreased polyamine pools and inhibited the growth of DU145 prostate cancer cells only at high concentration (1 mM). Growth inhibitory analysis of the Acremonium chrysogenum demonstrated that the wild type (WT) strain synthesized Put only from L-ornithine, while the cephalosporin C high-yielding strain, in which the polyamine pool is increased, could use both ODC and ADC to produce Put. Thus, AO-Agm is an important addition to the set of existing inhibitors of the enzymes of polyamine biosynthesis, and an important instrument for investigating polyamine biochemistry.