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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., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interestsKiyoshi Yasukawa reports financial support was provided by Japan Society for the Promotion of Science. If there are other authors, they declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

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., (© 2024 The Authors. Advanced Science published by Wiley‐VCH GmbH.)

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.

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.

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., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Masson SAS. All rights reserved.)

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., (© 2023 The Authors. Plant Biotechnology Journal published by Society for Experimental Biology and The Association of Applied Biologists and John Wiley & Sons Ltd.)

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.)

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., (Copyright © 2023 Elsevier Inc. All rights reserved.)

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.

The development of acid tolerance response (ATR) as a result of low pH in Escherichia coli O157:H7 (E. coli O157:H7) contaminating beef during processing is considered a major food safety concern. Thus, in order to explore the formation and molecular mechanisms of the tolerance response of E. coli O157:H7 in a simulated beef processing environment, the resistance of a wild-type (WT) strain and its corresponding ΔphoP mutant to acid, heat, and osmotic pressure was evaluated. Strains were pre-adapted under different conditions of pH (5.4 and 7.0), temperature (37 °C and 10 °C), and culture medium (meat extract and Luria-Bertani broth media). In addition, the expression of genes related to stress response and virulence was also investigated among WT and ΔphoP strains under the tested conditions. Pre-acid adaptation increased the resistance of E. coli O157:H7 to acid and heat treatment while resistance to osmotic pressure decreased. Moreover, acid adaptation in meat extract medium simulating slaughter environment increased ATR, whereas pre-adaptation at 10 °C reduced the ATR. Furthermore, it was shown that mildly acidic conditions (pH = 5.4) and the PhoP/PhoQ two-component system (TCS) acted synergistically to enhance acid and heat tolerance in E. coli O157:H7. Additionally, the expression of genes related to arginine and lysine metabolism, heat shock, and invasiveness was up-regulated, which revealed that the mechanism of acid resistance and cross-protection under mildly acidic conditions was mediated by the PhoP/PhoQ TCS. Both acid adaptation and phoP gene knockout reduced the relative expression of stx1 and stx2 genes which were considered as critical pathogenic factors. Collectively, the current findings indicated that ATR could occur in E. coli O157:H7 during beef processing. Thus, there is an increased food safety risk due to the persistence of tolerance response in the following processing conditions. The present study provides a more comprehensive basis for the effective application of hurdle technology in beef processing., Competing Interests: Declaration of competing interest No conflict of interest exits in the submission of this manuscript, and this manuscript has been approved by all authors for publication., (Copyright © 2023 Elsevier Ltd. All rights reserved.)

Chronic pain remains a significant burden worldwide, and treatments are often limited by safety or efficacy. The decarboxylated form of L-arginine, agmatine, antagonizes N-methyl-d-aspartate receptors, inhibits nitric oxide synthase, and reverses behavioral neuroplasticity. We hypothesized that expressing the proposed synthetic enzyme for agmatine in the sensory pathway could reduce chronic pain without motor deficits. Intrathecal delivery of an adeno-associated viral (AAV) vector carrying the gene for arginine decarboxylase (ADC) prevented the development of chronic neuropathic pain as induced by spared nerve injury in mice and rats and persistently reversed established hypersensitivity 266 days post-injury. Spinal long-term potentiation was inhibited by both exogenous agmatine and AAV-human ADC (hADC) vector pre-treatment but was enhanced in rats treated with anti-agmatine immunoneutralizing antibodies. These data suggest that endogenous agmatine modulates the neuroplasticity associated with chronic pain. Development of approaches to access this inhibitory control of neuroplasticity associated with chronic pain may yield important non-opioid pain-relieving options., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2023 The American Society of Gene and Cell Therapy. Published by Elsevier Inc. All rights reserved.)

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., (© 2022 The Authors. New Phytologist © 2022 New Phytologist Foundation.)

A plasmid was constructed to express the CapA-SpeA fusion protein from the capA gene, which encodes one of the subunits of capsular poly-γ-glutamate synthetase of Bacillus subtilis subsp. natto, and the speA gene, encoding biosynthetic arginine decarboxylase (EC 4.1.1.19) of Escherichia coli, under the control of the T5 promoter. The expression of SpeA on the extracellular surface of cells was confirmed by confocal microscopy with the anti-SpeA E. coli antibody and anti-rabbit IgG L & H conjugated with Alexa Fluor 488. The constructed strain SH2290 produced 200 mM agmatine from 200 mM arginine, 20 mM MgSO 4 , 0.9 % NaCl, and 0.02 mg/mL pyridoxal 5'-phosphate (initial pH 5.3) by adjusting pH of the reaction mixture to 6.8 with HCl after each sampling during the reaction. The addition of pyridoxal 5'-phosphate to the reaction mixture was required for the maximum agmatine production. The present results demonstrate that the expression of enzymes on the extracellular surface of cells is a very powerful method for enzymatic conversion., (Copyright © 2022 Elsevier Inc. All rights reserved.)

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.

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 Arabidopsi s 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 Arabidopsi s 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.

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., (© 2022. The Author(s).)

C-repeat (CRT) binding factors (CBFs) are well known to act as crucial transcription factors that function in cold stress response. Arginine decarboxylase (ADC)- mediated putrescine (Put) biosynthesis has been reported to be activated in plants exposed to cold conditions, but it remains elusive whether CBFs can regulate ADC expression and Put accumulation. In this study, we show that cold upregulated ADC gene (Citrus sinensis ADC;CsADC) and elevated endogenous Put content in sweet orange (C.sinensis). The promoter of CsADC contains two CRT sequences that are canonical elements recognized by CBFs. Sweet orange genome contains four CBFs (CsCBF1-4), in which CsCBF1 was significantly induced by cold. CsCBF1, located in the nucleus, was demonstrated to bind directly and specifically to the promoter of CsADC and acted as a transcriptional activator. Overexpression of CsCBF1 led to notable elevation of CsADC and Put levels in sweet orange transgenic plants, along with remarkably enhanced cold tolerance, relative to the wild type. However, pretreatment with D-arginine, an ADC inhibitor, caused a prominent reduction of endogenous Put levels in the overexpressing lines, accompanied by greatly compromised cold tolerance. Taken together, these results demonstrate that the CBF1 of sweet orange directly regulates ADC expression and modulates Put synthesis for orchestrating the cold tolerance. Our findings shed light on the transcriptional regulation of Put accumulation through targeting the ADC gene in the presence of cold stress. Meanwhile, this study illustrates a new mechanism underlying the CBF-mediated cold stress response., (© The Author(s) 2021. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

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 K m , k cat , K i , and k cat /K m 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 K m (14.55 ± 1.45 mM) and k cat (12.62 ± 0.68 s -1 ) value obtained by assaying at 1-50 mM L-arginine without considering substrate inhibition. Both the k cat 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., (© 2021. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

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., (© 2021. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

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 (Δ speA ), 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.

Histamine or scombrotoxin fish poisoning is caused by ingestion of bacterially produced histamine in fish. Histamine-producing bacteria generally contain the histidine decarboxylase gene (hdc). However, some strains of Photobacterium phosphoreum are known to produce significant levels of histamine, although the hdc gene in these strains has not been recognized. The objective of this study was to investigate a previously unidentified mechanism of histamine production by P. phosphoreum. We identified a protein with histidine decarboxylase (HDC) activity comparable to activity of the pyridoxal-5-phosphate (PLP) dependent HDC from P. kishitanii and M. morganii. The newly identified protein (HDC2) in P. phosphoreum and P. kishitanii strains, was approximately 2× longer than the HDC protein from other Gram-negative bacteria and had 12% similarity to previously identified HDCs. In addition, the hdc2 gene cluster in P. phosphoreum was identical to the hdc gene cluster in P. kishitanii. HDC2 had optimal activity at 20-35 °C, at pH 4, and was not affected by 0-8% NaCl concentrations. Compared to the hdc gene from P. kishitanii, expression of the hdc2 gene was constitutive and not affected by pH or excess histidine. This newly identified protein explains possible mechanisms of histamine production in P. phosphoreum. Characterization of this protein will help in designing control measures to prevent or reduce histamine production in fish., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Published by Elsevier B.V.)

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., (Copyright © 2020 Elsevier Masson SAS. All rights reserved.)

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 ( IC 50 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., Competing Interests: The authors declare no conflict of interest.

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 H 2 O 2 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., (Copyright © 2019 Vuosku, Muilu-Mäkelä, Avia, Suokas, Kestilä, Läärä, Häggman, Savolainen and Sarjala.)

Nitric oxide (NO) is known to antagonize ethylene by various mechanisms; one of such mechanisms is reducing ethylene levels by competitive action on S-adenosyl-L-methionine (SAM)-a common precursor for both ethylene and polyamines (PAs) biosynthesis. In order to investigate whether this mechanism of SAM pool diversion by NO occur towards PAs biosynthesis in banana, we studied the effect of NO on alterations in the levels of PAs, which in turn modulate ethylene levels during ripening. In response to NO donor sodium nitroprusside (SNP) treatment, all three major PAs viz. putrescine, spermidine and spermine were induced in control as well as ethylene pre-treated banana fruits. However, the gene expression studies in two popular banana varieties of diverse genomes, Nanjanagudu rasabale (NR; AAB genome) and Cavendish (CAV; AAA genome) revealed the downregulation of SAM decarboxylase, an intermediate gene involved in ethylene and PA pathway after the fifth day of NO donor SNP treatment, suggesting that ethylene and PA pathways do not compete for SAM. Interestingly, arginine decarboxylase belonging to arginine-mediated route of PA biosynthesis was upregulated several folds in response to the SNP treatment. These observations revealed that NO induces PAs via l-arginine-mediated route and not via diversion of SAM pool.

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. Key points 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.

Cell replacement therapy using neural progenitor cells (NPCs) following ischemic stroke is a promising potential therapeutic strategy, but lacks efficacy for human central nervous system (CNS) therapeutics. In a previous in vitro study, we reported that the overexpression of human arginine decarboxylase (ADC) genes by a retroviral plasmid vector promoted the neuronal differentiation of mouse NPCs. In the present study, we focused on the cellular mechanism underlying cell proliferation and differentiation following ischemic injury, and the therapeutic feasibility of NPCs overexpressing ADC genes (ADC-NPCs) following ischemic stroke. To mimic cerebral ischemia in vitro , we subjected the NPCs to oxygen-glucose deprivation (OGD). The overexpressing ADC-NPCs were differentiated by neural lineage, which was related to excessive intracellular calcium-mediated cell cycle arrest and phosphorylation in the ERK1/2, CREB, and STAT1 signaling cascade following ischemic injury. Moreover, the ADC-NPCs were able to resist mitochondrial membrane potential collapse in the increasingly excessive intracellular calcium environment. Subsequently, transplanted ADC-NPCs suppressed infarct volume, and promoted neural differentiation, synapse formation, and motor behavior performance in an in vivo tMCAO rat model. The results suggest that ADC-NPCs are potentially useful for cell replacement therapy following ischemic stroke.

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 Na v 1.8-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. NEW & NOTEWORTHY Our study is the first to report that agmatine preferentially antagonizes the GluN2B receptor subunit of the N-methyl-d-aspartate (NMDA) receptor in spinal cord. The preferential targeting of GluN2B receptor is consistent with the pharmacological profile of agmatine in that it reduces chronic pain without the motor side effects commonly seen with non-subunit-selective NMDA receptor antagonists.

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 Le SDC and Le ADC 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 Le SDC and Le ADC 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 Le SDC and Le ADC, exogenous spermidine significantly increased the arginase gene expression in Le SDC and Le ADC 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.

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., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: Ornithine (Orn) plays an essential role in the metabolism of plant cells through incorporation in polyamines biosynthesis, the urea cycle and nitrogen metabolism. Physiological response of the plant cells to its two enantiomers have not been widely investigated yet., Objectives: This study aimed to evaluate effect of ornithine enantiomers on expression of certain polyamine (PAs) biosynthetic genes in tobacco cells., Materials and Methods: Suspension-cultured tobacco cells were treated with different concentrations of L- and D- Orn for 24 h. Cell viability was assayed by Evans Blue and hydrogen peroxide content. The changes of gene expression were analyzed by semi-quantitative RT-PCR., Results: Exogenous D-Orn resulted in enhancement of expression of genes involved in Orn, arginine and S-adenosyl methionine metabolism. Additionally, exogenous D-Orn treatment resulted in sustained viability of cultured tobacco cells and normal levels of hydrogen peroxide were maintained. Supplied L-Orn increased the hydrogen peroxide level and lowered viability of cells. Treatment with L-Orn had a negative effect on the transcript levels for most analyzed PA-related genes. It was also illustrated that transcription of putrescine methyl transferase, key enzyme for nicotine production, was highly upregulated by L-Orn., Conclusions: Based on the results, D-Orn was shown to have a stereo-selective function in regulation of the PAs-related genes.

The phototropic bacterium Synechocystis sp. strain PCC 6803 is able to adapt its morphology in order to survive in a wide range of harsh environments. Under conditions of high salinity, planktonic cells formed cell aggregates in culture. Further observations using crystal violet staining, confocal laser scanning microscopy, and field emission-scanning electron microscopy confirmed that these aggregates were Synechocystis biofilms. Polyamines have been implicated in playing a role in biofilm formation, and during salt stress the content of spermidine, the major polyamine in Synechocystis , was reduced. Two putative arginine decarboxylases, Adc1 and Adc2, in Synechocystis were heterologously expressed in Escherichia coli and purified. Adc2 had high arginine decarboxylase activity, whereas Adc1 was much less active. Disruption of the adc genes in Synechocystis resulted in decreased spermidine content and formation of biofilms even under nonstress conditions. Based on the characterization of the adc mutants, Adc2 was the major arginine decarboxylase whose activity led to inhibition of biofilm formation, and Adc1 contributed only minimally to the process of polyamine synthesis. Taken together, in Synechocystis the shift from planktonic lifestyle to biofilm formation was correlated with a decrease in intracellular polyamine content, which is the inverse relationship of what was previously reported in heterotroph bacteria. IMPORTANCE There are many reports concerning biofilm formation in heterotrophic bacteria. In contrast, studies on biofilm formation in cyanobacteria are scarce. Here, we report on the induction of biofilm formation by salt stress in the model phototrophic bacterium Synechocystis sp. strain PCC 6803. Two arginine decarboxylases (Adc1 and Adc2) possess function in the polyamine synthesis pathway. Inactivation of the adc1 and adc2 genes leads to biofilm formation even in the absence of salt. The shift from planktonic culture to biofilm formation is regulated by a decrease in spermidine content in Synechocystis This negative correlation between biofilm formation and polyamine content, which is the opposite of the relationship reported in other bacteria, is important not only in autotrophic but also in heterotrophic bacteria., (Copyright © 2018 American Society for Microbiology.)

The extensive genetic resources of Chlamydomonas has led to its widespread use as a model system for understanding fundamental processes in plant cells, including rates of cell division potentially modulated through polyamines. Putrescine was the major polyamine in both free (88%) and membrane-bound fractions (93%) while norspermidine was the next most abundant in these fractions accounting for 11% and 6%, respectively. Low levels of diaminopropane, spermidine and spermine were also observed although no cadaverine or norspermine were detected. Ornithine decarboxylase (ODC, EC 4.1.1.17) activity was almost five times higher than arginine decarboxylase (ADC, EC 4.1.1.19) and is the major route of putrescine synthesis. The fluoride analogue of ornithine (α-DFMO) inhibited membrane associated ODC activity whilst simultaneously stimulating cell division in a dose dependent manner. Following exposure to α-DFMO the putrescine content in the cells declined while the norspermidine content increased over two fold. Addition of norspermidine to cultures stimulated cell division mimicking the effects observed using DFMO and also reversed the inhibitory effects of cyclohexylamine on growth. The results reveal that ODC is the major route to polyamine formation in the Chlamydomonas CC-406 cell-wall mutant, in contrast to the preferential ADC route reported for Chlorella vulgaris, suggesting that significant species differences exist in biosynthetic pathways which modulate endogenous polyamine levels in green algae., (Copyright © 2017 Elsevier Masson SAS. All rights reserved.)

Polyamine concentrations in the intestine are regulated by their biosynthesis by hundreds of gut microbial species and these polyamines are involved in host health and disease. However, polyamine biosynthesis has not been sufficiently analyzed in major members of the human gut microbiota, possibly owing to a lack of gene manipulation systems. In this study, we successfully performed markerless gene deletion in Bacteroides dorei, one of the major members of the human gut microbiota. The combination of a thymidine kinase gene (tdk) deletion mutant and a counter-selection marker tdk, which has been applied in other Bacteroides species, was used for the markerless gene deletion. Deletion of tdk in B. dorei caused 5-fluoro-2΄-deoxyuridine resistance, suggesting the utility of B. dorei Δtdk as the host for future markerless gene deletions. Compared to parental strains, an arginine decarboxylase gene (speA) deletion mutant generated in this system showed a severe growth defect and decreased concentration of spermidine in the cells and culture supernatant. Collectively, our results indicate the accessibility of gene deletion and the important role of speA in polyamine biosynthesis in B. dorei., (© FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

Immunolocalization for transmission electron microscopy is a powerful technique to identify subcellular localization of proteins. This can be combined with molecular and physiological data in order to have a complete overview of protein function. However, optimal sample preservation is required to avoid artefacts. When using chemically fixed samples, the progressive lowering of temperature (PLT) technique is a convenient procedure to dehydrate and embed samples at low temperature, thereby preserving the antigenicity of the proteins to be detected. Despite the advantages of immunogold labelling, it is a time-consuming cell biology technique. Therefore, the quality and specificity of the antibody should be previously checked by western blot. This approach also enables to identify changes in the amount of protein under study throughout development or in response to stress conditions.

Plants respond to pathogen attack by modifying defense gene expression and inducing the production of myriad proteins and metabolites. Among these responses, polyamine (PA) levels suffer remarkable modifications. Evidences demonstrate that plants make use of the polyamine biosynthetic pathway and the oxidative catabolism of these compounds in order to mount adequate defenses against pathogens. In Arabidopsis thaliana, putrescine is synthesized exclusively through the arginine decarboxylase (ADC) pathway, this enzyme exists as two isoforms named ADC1 and ADC2. Even though both isoforms participate in the response to pathogen attack, the mechanisms modulating ADC activity are not completely understood. Therefore, studies to clarify their roles are necessary. In this chapter, we describe the methods that can be applied for the study of plant-pathogen interactions using Arabidopsis adc mutant plants.

Enteric pathogens such as Salmonella typhimurium colonize the human gut in spite of the lethal acidic pH environment (pH < 2.5) due to the activation of inducible acid tolerance response (ATR) systems. The pyridoxal 5'-phosphate (PLP)-dependent enzyme, biodegradative arginine decarboxylase (ADC, encoded by AdiA), is a component of an ATR system. The enzyme consumes a cytoplasmic proton in the process of arginine degradation to agmatine. Arginine-agmatine antiporter (AdiC) exchanges the product agmatine for arginine. In this manuscript, we describe the structure of Salmonella typhimurium ADC (StADC). The decameric structure assembled from five dimers related by a non crystallographic 5-fold symmetry represents the first apo-form of the enzyme. The structure suggests that PLP-binding is not a prerequisite for oligomerization. Comparison with E. coli ADC reveals that PLP-binding is accompanied by the movement and ordering of two loops (residues 150-159 and 191-197) and a few active site residues such as His256 and Lys257. A number of residues important for substrate binding are disordered in the apo-StADC structure indicating that PLP binding is important for substrate binding. Unlike the interactions between 5-fold related protomers, interactions that stabilize the dimeric structure are not pH dependent., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Agmatine, the decarboxylation product of arginine, was largely neglected as an important player in mammalian metabolism until the mid-1990s, when it was re-discovered as an endogenous ligand of imidazoline and α 2 -adrenergic receptors. Since then, a wide variety of agmatine-mediated effects have been observed, and consequently agmatine has moved from a wallflower existence into the limelight of clinical neuroscience research. Despite this quantum jump in scientific interest, the understanding of the anabolism and catabolism of this amine is still vague. The purification and biochemical characterization of natural mammalian arginine decarboxylase and agmatinase still are open issues. Nevertheless, the agmatinergic system is currently one of the most promising candidates in order to pharmacologically interfere with some major diseases of the central nervous system, which are summarized in the present review. Particularly with respect to major depression, agmatine, its derivatives, and metabolizing enzymes show great promise for the development of an improved treatment of this common disease., (© 2017 The Author(s); published by Portland Press Limited on behalf of the Biochemical Society.)

Polyamines (PAs), such as spermidine and spermine, as well as amino acids that are substrates for their biosynthesis, are known to be essential for plant development. However, little is known about the gene expression and metabolic switches associated with the ornithine/arginine and PA biosynthetic pathway during seed development in conifers. To understand these metabolic switches, the enzyme activity of arginine decarboxylase and ornithine decarboxylase, as well as the contents of PAs and amino acids were evaluated in three Araucaria angustifolia (Bertol. Kuntze) seed developmental stages in combination with expression profile analyses of genes associated with the ornithine/arginine and PA biosynthetic pathway. Twelve genes were selected for further analysis and it was shown that the expression profiles of AaADC and AaSAMDC were up-regulated during zygotic embryo development. Polyamines and amino acids were found to accumulate differently in embryos and megagametophytes, and the transition from the globular to the cotyledonary stage was marked by an increase in free and conjugated spermidine and spermine contents. Putrescine is made from arginine, which was present at low content at the late embryogenesis stage, when high content of citrulline was observed. Differences in amino acids, PAs and gene expression profiles of biosynthetic genes at specific seed stages and at each seed transition stage were investigated, providing insights into molecular and physiological aspects of conifer embryogenesis for use in future both basic and applied studies.

Mercury compounds are well-known toxic environmental pollutants and potently induce severe neurotoxicological effects in human and experimental animals. Previous studies showed that one of the mechanisms of mercury compounds neurotoxicity arose from the over-activation of the N-methyl d-aspartate (NMDA)-type glutamate receptor induced by increased glutamate release. In this work, we aimed to investigate the molecular mechanisms of Hg compounds neurotoxicities by identifying their biological targets in cells. Firstly, the inhibitory effects of four Hg compounds, including three organic (methyl-, ethyl- and phenyl-mercury) and one inorganic (Hg 2+ ) Hg compounds, on the activity of arginine decarboxylase (ADC), a key enzyme in the central agmatinergic system, were evaluated. They were found to inhibit the ADC activity significantly with methylmercury (MeHg) being the strongest (IC 50 =7.96nM). Furthermore, they showed remarkable inhibitory effects on ADC activity in PC12 cells (MeHg>EtHg>PhHg>HgCl 2 ), and led to a marked loss in the level of agmatine, an endogenous neuromodulatory and neuroprotective agent that selectively blocks the activation of NMDA receptors. MeHg was detected in the immunoprecipitated ADC from the cells, providing unequivocal evidence for the direct binding of MeHg with ADC in the cell. Molecular dynamics simulation revealed that Hg compounds could form the coordination bond not only with cofactor PLP of ADC, but also with substrate arginine. Our finding indicated that MeHg could attenuate the neuroprotective effects of agmatine by the inhibition of ADC, a new cellular target of MeHg, which might be implicated in molecular mechanism of MeHg neurotoxicity., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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., (Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.)

Background and Aims: Although silicon (Si) has been widely reported to alleviate plant nutrient deficiency, the alleviating effect of Si on potassium (K) deficiency and its underlying mechanism are poorly understood. Here, we examined whether Si-regulated putrescine (Put) metabolisms are involved in Si-alleviated K deficiency., Methods: Sorghum seedlings were grown in K deficiency solution with and without Si for 15 d. The influence of K deficiency and Si on leaf chlorosis symptoms, K(+) concentration, polyamine (PA) levels, amine oxidase activities, the transcription of Put synthesis genes, antioxidant enzyme activities and H2O2 accumulation were measured., Key Results: Under K-sufficient conditions, plant growth was not affected by Si application. Si application significantly alleviated the growth inhibition induced by K-deficient stress, however. K deficiency induced leaf chlorosis and reduction in several leaf chlorosis-related metrics, including photosynthesis, efficiency of photosystem II photochemistry, chlorophyll content and chlorophyll a/b ratio; all of these changes were moderated by Si application. Si application did not influence the K(+) concentration in leaves under K-sufficient or K-deficient conditions. It did, however, decrease the excessive accumulation of Put that was otherwise induced by K deficiency. Simultaneously, Put synthesis gene transcription and activation of amine oxidases were down-regulated by Si application under K-deficient conditions. In addition, Si reduced K-deficiency-enhanced antioxidant enzyme activities and decreased K-deficiency-induced H2O2 accumulation., Conclusions: These results indicate that Si application could reduce K-deficiency-induced Put accumulation by inhibiting Put synthesis and could decrease H2O2 production via PA oxidation. Decreased H2O2 accumulation contributes to the alleviation of cell death, thereby also alleviating K-deficiency-induced leaf chlorosis and necrosis., (© The Author 2016. Published by Oxford University Press on behalf of the Annals of Botany Company. All rights reserved. For Permissions, please email: journals.permissions@oup.com.)

Polyamines (PAs) are small aliphatic polycations that are found ubiquitously in all organisms. In plants, PAs are involved in diverse biological processes such as growth, development, and stress responses. In Arabidopsis thaliana, the arginine decarboxylase enzymes (ADC1 and 2) catalyze the first step of PA biosynthesis. For a better understanding of PA biological functions, mutants in PA biosynthesis have been generated; however, the double adc1/adc2 mutant is not viable in A. thaliana. In this study, we generated non-lethal A. thaliana lines through an artificial microRNA that simultaneously silenced the two ADC genes (amiR:ADC). The generated transgenic lines (amiR:ADC-L1 and -L2) showed reduced AtADC1 and AtADC2 transcript levels. For further analyses the amiR:ADC-L2 line was selected. We found that the amiR:ADC-L2 line showed a significant decrease of their PA levels. The co-silencing revealed a stunted growth in A. thaliana seedlings, plantlets and delay in its flowering rate; these phenotypes were reverted with PA treatment. In addition, amiR:ADC-L2 plants displayed two seed phenotypes, such as yellow and brownish seeds. The yellow mutant seeds were smaller than adc1, adc2 mutants and wild type seeds; however, the brownish were the smallest seeds with arrested embryos at the torpedo stage. These data reinforce the importance of PA homeostasis in the plant development processes.

Polyamines are organic compounds involved in various biological roles in plants, including cell growth and organ development. In the present study, the expression profile, the accumulation of free polyamines and the transcript localisation of the genes involved in Put metabolism, such as Ornithine decarboxylase (ODC), Arginine decarboxylase (ADC) and copper containing Amine oxidase (CuAO), were examined during Solanum lycopersicum cv. Chiou fruit development and maturation. Moreover, the expression of genes coding for enzymes involved in higher polyamine metabolism, including Spermidine synthase (SPDS), Spermine synthase (SPMS), S-adenosylmethionine decarboxylase (SAMDC) and Polyamine oxidase (PAO), were studied. Most genes participating in PAs biosynthesis and metabolism exhibited an increased accumulation of transcripts at the early stages of fruit development. In contrast, CuAO and SPMS were mostly expressed later, during the development stages of the fruits where a massive increase in fruit volume occurs, while the SPDS1 gene exhibited a rather constant expression with a peak at the red ripe stage. Although Put, Spd and Spm were all exhibited decreasing levels in developing immature fruits, Put levels maxed late during fruit ripening. In contrast to Put both Spd and Spm levels continue to decrease gradually until full ripening. It is worth noticing that in situ RNA-RNA hybridisation is reported for the first time in tomato fruits. The localisation of ADC2, ODC1 and CuAO gene transcripts at tissues such as the locular parenchyma and the vascular bundles fruits, supports the theory that all genes involved in Put biosynthesis and catabolism are mostly expressed in fast growing tissues. The relatively high expression levels of CuAO at the ImG4 stage of fruit development (fruits with a diameter of 3 cm), mature green and breaker stages could possibly be attributed to the implication of polyamines in physiological processes taking place during fruit ripening., (Copyright © 2016 Elsevier Masson SAS. All rights reserved.)

Aluminum (Al) stress induces putrescine (Put) accumulation in several plants and this response is proposed to alleviate Al toxicity. However, the mechanisms underlying this alleviation remain largely unknown. Here, we show that exposure to Al clearly increases Put accumulation in the roots of wheat plants (Triticum aestivum L. 'Xi Aimai-1') and that this was accompanied by significant increase in the activity of arginine decarboxylase (ADC), a Put producing enzyme. Application of an ADC inhibitor (d-arginine) terminated the Al-induced Put accumulation, indicating that increased ADC activity may be responsible for the increase in Put accumulation in response to Al. The d-arginine treatment also increased the Al-induced accumulation of cell wall polysaccharides and the degree of pectin demethylation in wheat roots. Thus, it elevated Al retention in cell walls and exacerbated Al accumulation in roots, both of which aggravate Al toxicity in wheat plants. The opposite effects were true for exogenous Put application. These results suggest that ADC-dependent Put accumulation plays important roles in providing protection against Al toxicity in wheat plants through decreasing cell wall polysaccharides and increasing the degree of pectin methylation, thus decreasing Al retention in the cell walls., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Abscisic acid-responsive element (ABRE)-binding factors (ABFs) play important roles in abiotic stress responses; however, the underlying mechanisms are poorly understood. In this study, it is reported that overexpression of Poncirus trifoliata PtrABF significantly enhanced dehydration tolerance. The transgenic lines displayed smaller stomatal apertures, reduced stomatal density/index, and lower expression levels of genes associated with stomatal development. PtrABF was found to interact with PtrICE1, a homologue of ICE1 (Inducer of CBF Expression 1) that has been shown to be critical for stomatal development. Microarray analysis revealed that a total of 70 genes were differentially expressed in the transgenic line, 42 induced and 28 repressed. At least two units of ABREs and coupling elements were present in the promoters of most of the induced genes, among which peroxidase and arginine decarboxylase were verified as bona fide targets of PtrABF. Transgenic plants exhibited higher antioxidant enzyme activities and free polyamine levels, but lower levels of reactive oxygen species (ROS) and malondialdehyde. Polyamines were revealed to be associated with ROS scavenging in the transgenic plants due to a modulation of antioxidant enzymes triggered by signalling mediated by H2O2 derived from polyamine oxidase (PAO)-mediated catabolism. Taken together, the results indicate that PtrABF functions positively in dehydration tolerance by limiting water loss through its influence on stomatal movement or formation and maintaining ROS homeostasis via modulation of antioxidant enzymes and polyamines through transcriptional regulation of relevant target genes., (© The Author 2015. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

Polyamine biosynthesis starts with putrescine production through the decarboxylation of arginine or ornithine. In Arabidopsis thaliana, putrescine is synthesised exclusively by arginine decarboxylase (ADC), which exists as two isoforms (ADC1 and 2) that are differentially regulated by abiotic stimuli, but their role in defence against pathogens has not been studied in depth. This work analysed the participation of ADC in Arabidopsis defence against Pseudomonas viridiflava. ADC activity and expression, polyamine levels and bacterial resistance were analysed in null mutants of each ADC isoform. In non-infected wild-type (WT) plants, ADC2 expression was much higher than ADC1. Analysis of adc mutants demonstrated that ADC2 contributes to a much higher extent than ADC1 to basal ADC activity and putrescine biosynthesis. In addition, adc2 mutants showed increased basal expression of salicylic acid- and jasmonic acid-dependent PR genes. Bacterial infection induced putrescine accumulation and ADC1 expression in WT plants, but pathogen-induced putrescine accumulation was blocked in adc1 mutants. Results suggest a specific participation of ADC1 in defence, although basal resistance was not decreased by dysfunction of either of the two ADC genes. In addition, and as opposed to WT plants, bacterial infection increased ADC2 expression and ADC activity in adc1 mutants, which could counterbalance the lack of ADC1. Results demonstrate a major contribution of ADC2 to total ADC activity and the specific induction of ADC1 in response to infection. A certain degree of functional redundancy between the two isoforms in relation to their contribution to basal resistance is also evident., (© 2015 German Botanical Society and The Royal Botanical Society of the Netherlands.)

Tuber wound-healing processes are complex, and the associated regulation and modulation of these processes are poorly understood. Polyamines (PA) are involved in modulating a variety of responses to biotic and abiotic plant stresses and have been suggested to be involved in tuber wound responses. However, the time course of wound-induced changes in tuber PA content, activity of key biosynthetic enzymes and associated gene expression has not been determined and coordinated with major wound-healing processes. The objective of this study was to determine these wound-induced changes and their coordination with wound-healing processes. Wounding induced increases in putrescine (Put) and spermidine (Spd), but had only minor effects on spermine (Spm) content during the 168 h time course which encompassed the initiation and completion of the closing layer formation, and the initiation of cell division and wound periderm formation. As determinants of the first committed step in PA biosynthesis, arginine and ornithine decarboxylase (ADC and ODC, respectively) activities were below levels of detectability in resting tubers and expression of genes encoding these two enzymes was low. Within 6h of wounding, increases in the in vitro activities of ADC and ODC and expression of their cognate genes were observed. Expression of a gene encoding S-adenosylmethionine decarboxylase, required for Spd and Spm biosynthesis, was also increased 6h after wounding and remained elevated throughout the time course. Expression of a polyamine catabolic gene, encoding polyamine oxidase, was down-regulated after wounding. Results indicated a rapid wound-induced increase in PA biosynthesis during closing layer formation and the time of nuclei entry and exit from S-phase. PA content remained elevated as wound-induced cells became meristematic and initiated formation of the wound periderm suggesting sustained involvement in wound-healing., (Published by Elsevier GmbH.)

Background Aims: Mesenchymal stromal cells (MSCs) promote functional recovery in central nervous system (CNS) injury. Neuroprotective effects of MSCs are being tested in clinical trials for the treatment of CNS injury; however, the underlying mechanisms remain unclear. Arginine decarboxylase (ADC) is a rate-limiting enzyme of agmatine synthesis and is known to exist in the CNS of mammals. The present study investigated whether transplantation of ADC-overexpressing human MSCs (ADC-hMSCs) after spinal cord injury (SCI) could increase the production of neurotrophic factors and promote cell survival, differentiation, axonal regeneration and the restoration of functional recovery., Methods: Retroviral human ADC was constructed with the use of an LXSN vector. After compression injury in thoracic level 9, PKH26-labeled ADC-hMSCs were transplanted into the dorsolateral funiculus 1 mm rostral and caudal to the lesion site. The tissues were sampled at 2, 4 and 10 weeks after SCI., Results: Behavioral analysis revealed that locomotor functions of the ADC-hMSC group were significantly restored. Histological analysis showed that the fibrotic scar volume was smaller in the ADC-hMSC-injected group than in any other group. Brain-derived neurotrophic factor level was significantly higher in the ADC-hMSC-injected group than in any other group throughout 10 weeks. Terminal deoxynucleotidyl transferase-mediated nick-end labeling assay showed decreased cell death, and co-localization analysis showed significant increase in the number of neurons and oligodendrocytes originating from transplanted hMSCs when they had been transduced with the ADC gene., Conclusions: The results suggested that ADC-hMSCs are a more suitable candidate than hMSCs for stem cell therapy after SCI., (Copyright © 2015 International Society for Cellular Therapy. Published by Elsevier Inc. All rights reserved.)

Agmatine, decarboxylated arginine, is an important intermediary in polyamine production for many prokaryotes, but serves higher functions in eukaryotes such as nitric oxide inhibition and roles in neurotransmission. Pseudomonas aeruginosa relies on the arginine decarboxylase and agmatine deiminase pathways to convert arginine into putrescine. One of the two known agmatine deiminase operons, aguBA, contains an agmatine sensitive TetR promoter controlled by AguR. We have discovered that this promoter element can produce a titratable induction of its gene products in response to agmatine, and utilized this discovery to make a luminescent agmatine biosensor in P. aeruginosa. The genome of the P. aeruginosa lab strain UCBPP-PA14 was altered to remove both its ability to synthesize or destroy agmatine, and insertion of the luminescent reporter construct allows it to produce light in proportion to the amount of exogenous agmatine applied from ~100 nM to 1mM. Furthermore it does not respond to related compounds including arginine or putrescine. To demonstrate potential applications the biosensor was used to detect agmatine in spent supernatants, to monitor the development of arginine decarboxylase over time, and to detect agmatine in the spinal cords of live mice.