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Wheat (Triticum aestivum) is a major staple crop worldwide, and its yields are significantly threatened by wheat powdery mildew (Blumeria graminis f. sp. tritici). Enhancing disease resistance in wheat is crucial for meeting global food demand. This study investigated the disease response in wheat, focusing on the bioactive small molecules salicylic acid (SA), pipecolic acid (Pip), and N-hydroxypipecolic acid (NHP), to provide new insights for molecular breeding. We found that endogenous levels of SA, Pip, and NHP significantly increased in infected plants, with Pip and NHP levels rising earlier than those of SA. Notably, the rate of increase of NHP was substantially higher than that of SA. The gene expression levels of SARD1 and CBP60g, which are transcription factors for SA, Pip, and NHP biosynthesis, increased significantly during the early stages of infection. We also found that during the later stages of infection, the expression of ALD1, SARD4, and FMO1, which encode enzymes for Pip and NHP biosynthesis, dramatically increased. Additionally, ICS1, which encodes a key enzyme involved in SA biosynthesis, also showed increased expression during the later stages of infection. The temporal changes in ICS1 transcription closely mirrored the behavior of endogenous SA levels, suggesting that the ICS pathway is the primary route for SA biosynthesis in wheat. In conclusion, our results suggest that the early accumulation of Pip and NHP cooperates with SA in the disease response against wheat powdery mildew infection., 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 Inc. All rights reserved.)

The presented study aims to elucidate the regulatory role of Pipecolic acid (Pip) in modulating the antioxidant system activity of Mesembryanthemum crystallinum plants exposed to Pseudomonas syringae infestation. M. crystallinum, known for its semi-halophytic nature, can transition its metabolism from C 3  to CAM under salt stress conditions. The research encompasses the antioxidant system of the plants, covering both enzymatic and low molecular weight components. The findings indicate that Pip supplementation confers a beneficial effect on certain elements of the antioxidant system when the plants are subjected to stress induced by bacteria. Notably, during critical periods, particularly in the initial days post-bacterial treatment, M. crystallinum plants supplemented with Pip and exhibiting C 3 metabolism display heightened total antioxidant capacity. This enhancement includes increased superoxide dismutase activity and elevated levels of glutathione and proline. However, in plants with salinity-induced CAM, where these parameters are naturally higher, the supplementation of Pip does not yield significant effects. These results validate the hypothesis that the regulatory influence of Pip on defence mechanisms against biotic stress is contingent upon the metabolic state of the plant. Furthermore, this regulatory effect is more pronounced in C 3 plants of M. crystallinum than those undergoing CAM metabolism induced by salinity stress., (© 2024 Scandinavian Plant Physiology Society.)

l-Pipecolic acid (L-PA), an essential chiral cyclic nonprotein amino acid, is gaining prominence in the food and pharmaceutical sectors due to its wide-ranging biological and pharmacological properties. Historically, L-PA has been synthesized chemically for commercial purposes. This study introduces a novel and efficient microbial production method for L-PA using engineered strain Saccharomyces cerevisiae BY4743. Initially, an optimized biosynthetic pathway was constructed within S. cerevisiae , converting glucose to L-PA with a yield of 0.60 g/L in a 250 mL shake flask in vivo . Subsequently, a multifaceted engineering strategy was implemented to enhance L-PA production: substrate-enzyme affinity modification, global transcription machinery engineering modification, and Kozak sequence optimization for enhanced L-PA production. Approaches above led to an impressive 8.6-fold increase in L-PA yield, reaching 5.47 g/L in shake flask cultures. Further scaling up in a 5 L fed-batch fermenter achieved a remarkable L-PA concentration of 74.54 g/L. This research offers innovative insights into the industrial-scale production of L-PA.

N-hydroxy pipecolic acid (NHP) plays an important role in plant immunity. In contrast to its biosynthesis, our current knowledge with respect to the transcriptional regulation of the NHP pathway is limited. This study commences with the engineering of Arabidopsis plants that constitutively produce high NHP levels and display enhanced immunity. Label-free proteomics reveals a NAC-type transcription factor (NAC90) that is strongly induced in these plants. We find that NAC90 is a target gene of SAR DEFICIENT 1 (SARD1) and induced by pathogen, salicylic acid (SA), and NHP. NAC90 knockout mutants exhibit constitutive immune activation, earlier senescence, higher levels of NHP and SA, as well as increased expression of NHP and SA biosynthetic genes. In contrast, NAC90 overexpression lines are compromised in disease resistance and accumulated reduced levels of NHP and SA. NAC90 could interact with NAC61 and NAC36 which are also induced by pathogen, SA, and NHP. We next discover that this protein triad directly represses expression of the NHP and SA biosynthetic genes AGD2-LIKE DEFENSE RESPONSE PROTEIN 1 (ALD1), FLAVIN MONOOXYGENASE 1 (FMO1), and ISOCHORISMATE SYNTHASE 1 (ICS1). Constitutive immune response in nac90 is abolished once blocking NHP biosynthesis in the fmo1 background, signifying that NAC90 negative regulation of immunity is mediated via NHP biosynthesis. Our findings expand the currently documented NHP regulatory network suggesting a model that together with NHP glycosylation, NAC repressors take part in a 'gas-and-brake' transcriptional mechanism to control NHP production and the plant growth and defense trade-off., (© 2024. The Author(s).)

The disturbed gut microbiota is a key factor in activating the aflatoxin B 1 (AFB 1 )-induced liver pyroptosis by promoting inflammatory hepatic injury; however, the pathogen associated molecular pattern (PAMP) from disturbed gut microbiota and its mechanism in activating liver pyroptosis remain undefined. By transplanting AFB 1 -originated fecal microbiota and sterile fecal microbial metabolites filtrate, we determined the association of PAMP in AFB 1 -induced liver pyroptosis. Notably, AFB 1 -originated sterile fecal microbial metabolites filtrate were more active in triggering liver pyroptosis in mice, as compared to parental fecal microbiota. This result supported a critical role of the metabolic homeostasis of gut microbiota in AFB 1 -induced liver pyroptosis, rather than an injurious response to direct exposure of AFB 1 in liver. Among the gut-microbial metabolites, pipecolic acid and norepinephrine were proposed to bind TLR4 and NLRP3, the upstream proteins of pyroptosis signaling pathway. Besides, the activations of TLR4 and NLRP3 were linearly correlated with the concentrations of pipecolic acid and norepinephrine in the serum of mice. In silenced expression of TLR4 and NLRP3 in HepG2 cells, pipecolic acid or norepinephrine did not able to activate hepatocyte pyroptosis. These results demonstrated the necessity of gut microbial metabolism in sustaining liver homeostasis, as well as the potential to provide new insights into targeted intervention for AFB 1 hepatotoxicity., 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 B.V. All rights reserved.)

N-Hydroxypipecolic acid (NHP) is a signaling molecule crucial for systemic acquired resistance (SAR), a systemic immune response in plants that provides long-lasting and broad-spectrum protection against secondary pathogen infections. To identify negative regulators of NHP biosynthesis, we performed a forward genetic screen to search for mutants with elevated expression of the NHP biosynthesis gene FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1). Analysis of two constitutive expression of FMO1 (cef) and one induced expression of FMO1 (ief) mutants revealed that the AIPP3-PHD2-CPL2 protein complex, which is involved in the recognition of the histone modification H3K27me3 and transcriptional repression, contributes to the negative regulation of FMO1 expression and NHP biosynthesis. Our study suggests that epigenetic regulation plays a crucial role in controlling FMO1 expression and NHP levels in plants., (© 2023 Institute of Botany, Chinese Academy of Sciences.)

Systemic acquired resistance (SAR) is a plant immune response established in uninfected leaves after colonization of local leaves with biotrophic or hemibiotrophic pathogens. The amino acid-derived metabolite N-hydroxypipecolic acid (NHP) travels from infected to systemic leaves, where it activates salicylic acid (SA) biosynthesis through the isochorismate pathway. The resulting increased SA levels are essential for induction of a large set of SAR marker genes and full SAR establishment. In this study, we show that pharmacological treatment of Arabidopsis thaliana with NHP induces a subset of SAR-related genes even in the SA induction-deficient2 (sid2/isochorismate synthase1) mutant, which is devoid of NHP-induced SA. NHP-mediated induction is abolished in sid2-1 NahG plants, in which basal SA levels are degraded. The SA receptor NON-EXPRESSOR OF PATHOGENESIS-RELATED GENES1 (NPR1) and its interacting TGACG SEQUENCE-SPECIFIC BINDING PROTEIN (TGA) transcription factors are required for the NHP-mediated induction of SAR genes at resting SA levels. Isothermal titration analysis determined a KD of 7.9 ± 0.5 µM for the SA/NPR1 complex, suggesting that basal levels of SA would not bind to NPR1 unless yet unknown potentially NHP-induced processes increase the affinity. Moreover, the nucleocytoplasmic protein PHYTOALEXIN DEFICIENT4 is required for a slight NHP-mediated increase in NPR1 protein levels and NHP-induced expression of SAR-related genes. Our experiments have unraveled that NHP requires basal SA and components of the SA signaling pathway to induce SAR genes. Still, the mechanism of NHP perception remains enigmatic., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

BackgroundPyridoxine-dependent epilepsy (PDE-ALDH7A1) is an inborn error of lysine catabolism that presents with refractory epilepsy in newborns. Biallelic ALDH7A1 variants lead to deficiency of α-aminoadipic semialdehyde dehydrogenase/antiquitin, resulting in accumulation of piperideine-6-carboxylate (P6C), and secondary deficiency of the important cofactor pyridoxal-5'-phosphate (PLP, active vitamin B6) through its complexation with P6C. Vitamin B6 supplementation resolves epilepsy in patients, but intellectual disability may still develop. Early diagnosis and treatment, preferably based on newborn screening, could optimize long-term clinical outcome. However, no suitable PDE-ALDH7A1 newborn screening biomarkers are currently available.MethodsWe combined the innovative analytical methods untargeted metabolomics and infrared ion spectroscopy to discover and identify biomarkers in plasma that would allow for PDE-ALDH7A1 diagnosis in newborn screening.ResultsWe identified 2S,6S-/2S,6R-oxopropylpiperidine-2-carboxylic acid (2-OPP) as a PDE-ALDH7A1 biomarker, and confirmed 6-oxopiperidine-2-carboxylic acid (6-oxoPIP) as a biomarker. The suitability of 2-OPP as a potential PDE-ALDH7A1 newborn screening biomarker in dried bloodspots was shown. Additionally, we found that 2-OPP accumulates in brain tissue of patients and Aldh7a1-knockout mice, and induced epilepsy-like behavior in a zebrafish model system.ConclusionThis study has opened the way to newborn screening for PDE-ALDH7A1. We speculate that 2-OPP may contribute to ongoing neurotoxicity, also in treated PDE-ALDH7A1 patients. As 2-OPP formation appears to increase upon ketosis, we emphasize the importance of avoiding catabolism in PDE-ALDH7A1 patients.FundingSociety for Inborn Errors of Metabolism for Netherlands and Belgium (ESN), United for Metabolic Diseases (UMD), Stofwisselkracht, Radboud University, Canadian Institutes of Health Research, Dutch Research Council (NWO), and the European Research Council (ERC).

N-hydroxypipecolic acid (NHP) accumulates in the plant foliage in response to a localized microbial attack and induces systemic acquired resistance (SAR) in distant leaf tissue. Previous studies indicated that pathogen inoculation of Arabidopsis (Arabidopsis thaliana) systemically activates SAR-related transcriptional reprogramming and a primed immune status in strict dependence of FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), which mediates the endogenous biosynthesis of NHP. Here, we show that elevations of NHP by exogenous treatment are sufficient to induce a SAR-reminiscent transcriptional response that mobilizes key components of immune surveillance and signal transduction. Exogenous NHP primes Arabidopsis wild-type and NHP-deficient fmo1 plants for a boosted induction of pathogen-triggered defenses, such as the biosynthesis of the stress hormone salicylic acid (SA), accumulation of the phytoalexin camalexin and branched-chain amino acids, as well as expression of defense-related genes. NHP also sensitizes the foliage systemically for enhanced SA-inducible gene expression. NHP-triggered SAR, transcriptional reprogramming, and defense priming are fortified by SA accumulation, and require the function of the transcriptional coregulator NON-EXPRESSOR OF PR GENES1 (NPR1). Our results suggest that NPR1 transduces NHP-activated immune signaling modes with predominantly SA-dependent and minor SA-independent features. They further support the notion that NHP functions as a mobile immune regulator capable of moving independently of active SA signaling between leaves to systemically activate immune responses., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

Exposure of oocytes to specific amino acids during in vitro fertilisation (IVF) improves preimplantation embryo development. Embryos fertilised in medium with proline and its homologue pipecolic acid showed increased blastocyst formation and inner cell mass cell numbers compared to embryos fertilised in medium containing no amino acids, betaine, glycine, or histidine. The beneficial effect of proline was prevented by the addition of excess betaine, glycine, and histidine, indicating competitive inhibition of transport-mediated uptake. Expression of transporters of proline in oocytes was investigated by measuring the rate of uptake of radiolabelled proline in the presence of unlabelled amino acids. Three transporters were identified, one that was sodium-dependent, PROT (SLC6A7), and two others that were sodium-independent, PAT1 (SLC36A1) and PAT2 (SLC36A2). Immunofluorescent staining showed localisation of PROT in intracellular vesicles and limited expression in the plasma membrane, while PAT1 and PAT2 were both expressed in the plasma membrane. Proline and pipecolic acid reduced mitochondrial activity and reactive oxygen species in oocytes, and this may be responsible for their beneficial effect. Overall, our results indicate the importance of inclusion of specific amino acids in IVF medium and that consideration should be given to whether the addition of multiple amino acids prevents the action of beneficial amino acids.

Systemic acquired resistance (SAR) is a mechanism that plants utilize to connect a local pathogen infection to global defense responses. N-hydroxy-pipecolic acid (NHP) and a glycosylated derivative are produced during SAR, yet their individual roles in this process are currently unclear. Here, we report that Arabidopsis thaliana UGT76B1 generated glycosylated NHP (NHP-Glc) in vitro and when transiently expressed alongside Arabidopsis NHP biosynthetic genes in two Solanaceous plants. During infection, Arabidopsis ugt76b1 mutants did not accumulate NHP-Glc and accumulated less glycosylated salicylic acid (SA-Glc) than wild-type plants. The metabolic changes in ugt76b1 plants were accompanied by enhanced defense to the bacterial pathogen Pseudomonas syringae, suggesting that glycosylation of the SAR molecules NHP and salicylic acid by UGT76B1 plays an important role in modulating defense responses. Transient expression of Arabidopsis UGT76B1 with the Arabidopsis NHP biosynthesis genes ALD1 and FMO1 in tomato (Solanum lycopersicum) increased NHP-Glc production and reduced NHP accumulation in local tissue and abolished the systemic resistance seen when expressing NHP-biosynthetic genes alone. These findings reveal that the glycosylation of NHP by UGT76B1 alters defense priming in systemic tissue and provide further evidence for the role of the NHP aglycone as the active metabolite in SAR signaling., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Glucosylation modulates the biological activity of small molecules and frequently leads to their inactivation. The Arabidopsis thaliana glucosyltransferase UGT76B1 is involved in conjugating the stress hormone salicylic acid (SA) as well as isoleucic acid (ILA). Here, we show that UGT76B1 also glucosylates N-hydroxypipecolic acid (NHP), which is synthesized by FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1) and activates systemic acquired resistance (SAR). Upon pathogen attack, Arabidopsis leaves generate two distinct NHP hexose conjugates, NHP-O-β-glucoside and NHP glucose ester, whereupon only NHP-O-β-glucoside formation requires a functional SA pathway. The ugt76b1 mutants specifically fail to generate the NHP-O-β-glucoside, and recombinant UGT76B1 synthesizes NHP-O-β-glucoside in vitro in competition with SA and ILA. The loss of UGT76B1 elevates the endogenous levels of NHP, SA, and ILA and establishes a constitutive SAR-like immune status. Introgression of the fmo1 mutant lacking NHP biosynthesis into the ugt76b1 background abolishes this SAR-like resistance. Moreover, overexpression of UGT76B1 in Arabidopsis shifts the NHP and SA pools toward O-β-glucoside formation and abrogates pathogen-induced SAR. Our results further indicate that NHP-triggered immunity is SA-dependent and relies on UGT76B1 as a common metabolic hub. Thereby, UGT76B1-mediated glucosylation controls the levels of active NHP, SA, and ILA in concert to balance the plant immune status., (© The Author(s) 2021. Published by Oxford University Press on behalf of American Society of Plant Biologists.)

N-hydroxy-pipecolic acid (NHP) activates plant systemic acquired resistance (SAR). Enhanced defense responses are typically accompanied by deficiency in plant development and reproduction. Despite of extensive studies on SAR induction, the effects of NHP metabolism on plant growth remain largely unclear. In this study, we discovered that NHP glycosylation is a critical factor that fine-tunes the tradeoff between SAR defense and plant growth. We demonstrated that a UDP-glycosyltransferase (UGT76B1) forming NHP glycoside (NHPG) controls the NHP to NHPG ratio. Consistently, the ugt76b1 mutant exhibits enhanced SAR response and an inhibitory effect on plant growth, while UGT76B1 overexpression attenuates SAR response, promotes growth, and delays senescence, indicating that NHP levels are dependent on UGT76B1 function in the course of SAR. Furthermore, our results suggested that, upon pathogen attack, UGT76B1-mediated NHP glycosylation forms a "hand brake" on NHP accumulation by attenuating the positive regulation of NHP biosynthetic pathway genes, highlighting the complexity of SAR-associated networks. In addition, we showed that UGT76B1-mediated NHP glycosylation in the local site is important for fine-tuning SAR response. Our results implicate that engineering plant immunity through manipulating the NHP/NHPG ratio is a promising method to balance growth and defense response in crops., (Copyright © 2020 The Author. Published by Elsevier Inc. All rights reserved.)

Pipecolic acid, a non-proteinogenic amino acid, is a metabolite in lysine metabolism and a key chiral precursor in local anesthesia and macrolide antibiotics. To replace the environmentally unfriendly chemical production or preparation procedure of pipecolic acid, many biological synthetic routes have been studied for a long time. Among them, synthesis by lysine cyclodeaminase (LCD), encoded by pipA, has several advantages, including stability of enzyme activity and NAD + self-regeneration. Thus, we selected this enzyme for pipecolic acid biosynthesis in a whole-cell bioconversion. To construct a robust pipecolic acid production system, we investigated important conditions including expression vector, strain, culture conditions, and other reaction parameters. The most important factor was the introduction of multiple pipA genes into the whole-cell system. As a result, we produced 724 mM pipecolic acid (72.4 % conversion), and the productivity was 0.78 g/L/h from 1 M l-lysine after 5 days. This is the highest production reported to date., (Copyright © 2020 Elsevier Inc. All rights reserved.)

BACKGROUND Since venous drainage in acute arterial ischemic stroke has not been thoroughly researched, we evaluate the effect of argatroban, a selective direct thrombin inhibitor, as a therapy to increase the rate of basal vein Rosenthal (BVR) drainage and improve patients' post-stroke outcomes. MATERIAL AND METHODS In this multicenter clinical trial, 60 eligible patients at 4.5 to 48 hours after the stroke onset were recruited. After being randomly allocated into 2 groups, they were treated with standard therapy either alone or with argatroban. RESULTS Compared to the contralateral brain hemisphere, the mean flow velocity (MFV) in BVR drainage was significantly reduced in the stroke-afflicted ipsilateral hemisphere. After treatment with argatroban for 7 days, the MFV from BVR of the ipsilateral hemisphere in the argatroban treated group was significantly increased when compared to the control group. At 90 days after the onset of stroke, the MFV of BVR in the ipsilateral hemisphere was similar in both groups. Compared with controls, the argatroban-treated patients had smaller lesions from baseline to 7 days. Argatroban also improved National Institutes of Health Stroke Scale (NIHSS) scores on day 7 after the onset of stroke. Furthermore, the argatroban group's neurological functions were superior to those of their untreated counterparts after 90 days. No difference was found in the incidence of adverse reactions between the 2 groups. CONCLUSIONS These observations indicate that vein drainage change may contribute to the acute phase of brain edema and the outcomes of ischemic stroke patients.

Pyridoxal phosphate (PLP)-dependent enzymes can catalyze transformations of l-amino acids at α, β, and γ positions. These enzymes are frequently involved in the biosynthesis of nonproteinogenic amino acids as building blocks of natural products and are attractive biocatalysts. Here, we report the discovery of a two-step enzymatic synthesis of (2 S ,6 S )-6-methyl pipecolate 1 , from the biosynthetic pathway of citrinadin. The key enzyme CndF is PLP-dependent and catalyzes the synthesis of ( S )-2-amino-6-oxoheptanoate 3 that is in equilibrium with the cyclic Schiff base. The second enzyme CndE is a stereoselective imine reductase that gives 1 . Biochemical characterization of CndF showed this enzyme performs γ-elimination of O -acetyl-l-homoserine to generate the vinylglycine ketimine, which is subjected to nucleophilic attack by acetoacetate to form the new C γ -C δ bond in 3 and complete the γ-substitution reaction. CndF displays promiscuity toward different β-keto carboxylate and esters. With use of an Aspergillus strain expressing CndF and CndE, feeding various alkyl-β-keto esters led to the biosynthesis of 6-substituted l-pipecolates. The discovery of CndF expands the repertoire of reactions that can be catalyzed by PLP-dependent enzymes.

Plant-parasitic nematodes are devastating pathogens of many important agricultural crops. They have been successful in large part due to their ability to modify host plant metabolomes to their benefit. Both root-knot and cyst nematodes are endoparasites that have co-evolved to modify host plants to create sophisticated feeding cells and suppress plant defenses. In contrast, the ability of migratory ectoparasitic nematodes to modify host plants is unknown. Based on global metabolomic profiling of sting nematodes in African bermudagrass, ectoparasites can modify the global metabolome of host plants. Specifically, sting nematodes suppress amino acids in susceptible cultivars. Upregulation of compounds linked to plant defense have negative impacts on sting nematode population densities. Pipecolic acid, linked to systemic acquired resistance induction, seems to play a large role in protecting tolerant cultivars from sting nematode feeding and could be targeted in breeding programs.

Two signal molecules, salicylic acid (SA) and N-hydroxypipecolic acid (NHP), play critical roles in plant immunity. The biosynthetic genes of both compounds are positively regulated by master immune-regulating transcription factors SARD1 and CBP60g. However, the relationship between the SA and NHP pathways is unclear. CALMODULIN-BINDING TRANSCRIPTION FACTOR 1 (CAMTA1), CAMTA2, and CAMTA3 are known redundant negative regulators of plant immunity, but the underlying mechanism also remains largely unknown. In this study, through chromatin immunoprecipitation and electrophoretic mobility shift assays, we uncovered that CBP60g is a direct target of CAMTA3, which also negatively regulates the expression of SARD1, presumably via an indirect effect. The autoimmunity of camta3-1 is suppressed by sard1 cbp60g double mutant as well as ald1 and fmo1, two single mutants defective in NHP biosynthesis. Interestingly, a suppressor screen conducted in the camta1/2/3 triple mutant background yielded various mutants blocking biosynthesis or signaling of either SA or NHP, leading to nearly complete suppression of the extreme autoimmunity of camta1/2/3, suggesting that the SA and NHP pathways can mutually amplify each other. Together, these results reveal that CAMTAs repress the biosynthesis of SA and NHP by modulating the expression of SARD1 and CBP60g, and that the SA and NHP pathways are coordinated to optimize plant immune response., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

The Arabidopsis thaliana Calmodulin-binding Transcription Activator (CAMTA) transcription factors CAMTA1, CAMTA2, and CAMTA3 (CAMTA123) serve as master regulators of salicylic acid (SA)-mediated immunity, repressing the biosynthesis of SA in healthy plants. Here, we show that CAMTA123 also repress the biosynthesis of pipecolic acid (Pip) in healthy plants. Loss of CAMTA123 function resulted in the induction of AGD2-like defense response protein 1 (ALD1), which encodes an enzyme involved in Pip biosynthesis. Induction of ALD1 resulted in the accumulation of high levels of Pip, which brought about increased levels of the SA receptor protein NPR1 without induction of NPR1 expression or requirement for an increase in SA levels. Pip-mediated induction of ALD1 and genes regulating the biosynthesis of SA-CBP60g, SARD1, PAD4, and EDS1-was largely dependent on NPR1. Furthermore, Pip-mediated increase in NPR1 protein levels was associated with priming of Pip and SA biosynthesis genes to induction by low levels of SA. Taken together, our findings expand the role for CAMTA123 in regulating key immunity genes and suggest a working model whereby loss of CAMTA123 repression leads to the induction of plant defense genes and initiation of SAR., (Copyright © 2019 The Author. Published by Elsevier Inc. All rights reserved.)

Systemic acquired resistance (SAR) prepares infected plants for faster and stronger defense activation upon subsequent attacks. SAR requires an information relay from primary infection to distal tissue and the initiation and maintenance of a self-maintaining phytohormone salicylic acid (SA)-defense loop. In spatial and temporal resolution, we show that calcium-dependent protein kinase CPK5 contributes to immunity and SAR. In local basal resistance, CPK5 functions upstream of SA synthesis, perception, and signaling. In systemic tissue, CPK5 signaling leads to accumulation of SAR-inducing metabolite N-hydroxy-L-pipecolic acid (NHP) and SAR marker genes, including Systemic Acquired Resistance Deficient 1 (SARD1) Plants of increased CPK5, but not CPK6, signaling display an 'enhanced SAR' phenotype towards a secondary bacterial infection. In the sard1-1 background, CPK5-mediated basal resistance is still mounted, but NHP concentration is reduced and enhanced SAR is lost. The biochemical analysis estimated CPK5 half maximal kinase activity for calcium, K50 [Ca 2+ ], to be c. 100 nM, close to the cytoplasmic resting level. This low threshold uniquely qualifies CPK5 to decode subtle changes in calcium, a prerequisite to signal relay and onset and maintenance of priming at later time points in distal tissue. Our data explain why CPK5 functions as a hub in basal and systemic plant immunity., (© 2019 The Authors. New Phytologist © 2019 New Phytologist Trust.)

Pipecolic acid (Pip) is an essential component of systemic acquired resistance, priming resistance in Arabidopsis thaliana against (hemi)biotrophic pathogens. Here, we studied the potential role of Pip in bacteria-induced systemic immunity in barley. Exudates of barley leaves infected with the systemic immunity-inducing pathogen Pseudomonas syringae pv. japonica induced immune responses in A. thaliana. The same leaf exudates contained elevated Pip levels compared with those of mock-treated barley leaves. Exogenous application of Pip induced resistance in barley against the hemibiotrophic bacterial pathogen Xanthomonas translucens pv. cerealis . Furthermore, both a systemic immunity-inducing infection and exogenous application of Pip enhanced the resistance of barley against the biotrophic powdery mildew pathogen Blumeria graminis f. sp. hordei . In contrast to a systemic immunity-inducing infection, Pip application did not influence lesion formation by a systemically applied inoculum of the necrotrophic fungus Pyrenophora teres . Nitric oxide (NO) levels in barley leaves increased after Pip application. Furthermore, X. translucens pv. cerealis induced the accumulation of superoxide anion radicals and this response was stronger in Pip-pretreated compared with mock-pretreated plants. Thus, the data suggest that Pip induces barley innate immune responses by triggering NO and priming reactive oxygen species accumulation.

The measurements of lysine metabolites provide valuable information for the rapid diagnosis of pyridoxine-dependent epilepsy (PDE). Here, we aimed to develop a sensitive method to simultaneously quantify multiple lysine metabolites in PDE, including α-aminoadipic semialdehyde (a-AASA), piperideine-6-carboxylate (P6C), pipecolic acid (PA) and α-aminoadipic acid (α-AAA) in plasma, serum, dried blood spots (DBS), urine and dried urine spots (DUS). Fifteen patients with molecularly confirmed PDE were detected using liquid chromatography-mass spectrometry (LC-MS/MS) method. Compared to the control groups, the concentrations of a-AASA, P6C and the sum of a-AASA and P6C (AASA-P6C) in all types of samples from PDE patients were markedly elevated. The PA and a-AAA concentrations ranges overlapped partially between PDE patients and control groups. The concentrations of all the analytes in plasma and serum, as well as in urine and DUS were highly correlated. Our study provided more options for the diverse sample collection in the biochemical tests according to practical requirements. With treatment modality of newly triple therapy investigated, biomarker study might play important role not only on diagnosis but also on treatment monitoring and fine tuning the diet. The persistently elevated analytes with good correlation between plasma and DBS, as well as urine and DUS made neonatal screening using DBS and DUS possible.

The pepper ( Capsicum annuum ) resistance gene bacterial spot3 ( Bs3 ) is transcriptionally activated by the matching Xanthomonas euvesicatoria transcription-activator-like effector (TALE) AvrBs3. AvrBs3-induced Bs3 expression triggers a rapid and local cell death reaction, the hypersensitive response (HR). Bs3 is most closely related to plant flavin monooxygenases of the YUCCA (YUC) family, which catalyze the final step in auxin biosynthesis. Targeted mutagenesis of predicted NADPH- and FAD-cofactor sites resulted in Bs3 derivatives that no longer trigger HR, thereby suggesting that the enzymatic activity of Bs3 is crucial to Bs3-triggered HR. Domain swap experiments between pepper Bs3 and Arabidopsis ( Arabidopsis thaliana ) YUC8 uncovered functionally exchangeable and functionally distinct regions in both proteins, which is in agreement with a model whereby Bs3 evolved from an ancestral YUC gene. Mass spectrometric measurements revealed that expression of YUC s, but not expression of Bs3 , coincides with an increase in auxin levels, suggesting that Bs3 and YUCs, despite their sequence similarity, catalyze distinct enzymatic reactions. Finally, we found that expression of Bs3 coincides with increased levels of the salicylic acid and pipecolic acid, two compounds that are involved in systemic acquired resistance., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

Allosteric regulators of clinically important enzymes are gaining popularity as alternatives to competitive inhibitors. This is also the case for the proteasome, a major intracellular protease and a target of anti-cancer drugs. All clinically used proteasome inhibitors bind to the active sites in catalytic chamber and display a competitive mechanism. Unfortunately, inevitable resistance associated with this type of inhibition drives the search for non-competitive agents. The multisubunit and multicatalytic "proteolytic machine" such as the proteasome is occasionally found to be affected by agents with other primary targets. For example the immunosuppressive agent rapamycin has been shown to allosterically inhibit the proteasome albeit at levels far higher than its mTOR related efficacy. As part of an ongoing program to search for novel proteasome-targeting pharmacophores, we identified the binding domain of rapamycin as required for proteasome inhibition even without the macrocyclic context of the parent compound. By subsequent structure-activity relationship studies, we generated a pipecolic ester derivative compound 3 representing a new class of proteasome inhibitors. Compound 3 affects the core proteasome activities and proliferation of cancer cells with low micromolar/high nanomolar efficacy. Molecular modeling, atomic force microscopy imaging and biochemical data suggest that compound 3 binds into one of intersubunit pockets in the proteasomal α ring and destabilizes the α face and the gate. The α face is used as a docking area for proteasome-regulating protein modules and the gate is critical for controlling access to the catalytic chamber. Thus, the pipecolic ester template elicits a new and attractive mechanism for proteasome inhibition distinct from classical competitive drugs.

l-lysine catabolic routes in plants include the saccharopine pathway to α-aminoadipate and decarboxylation of lysine to cadaverine. The current review will cover a third l-lysine metabolic pathway having a major role in plant systemic acquired resistance (SAR) to pathogen infection that was recently discovered in Arabidopsis thaliana. In this pathway, the aminotransferase AGD2-like defense response protein (ALD1) α-transaminates l-lysine and generates cyclic dehydropipecolic (DP) intermediates that are subsequently reduced to pipecolic acid (Pip) by the reductase SAR-deficient 4 (SARD4). l-pipecolic acid, which occurs ubiquitously in the plant kingdom, is further N-hydroxylated to the systemic acquired resistance (SAR)-activating metabolite N-hydroxypipecolic acid (NHP) by flavin-dependent monooxygenase1 (FMO1). N-hydroxypipecolic acid induces the expression of a set of major plant immune genes to enhance defense readiness, amplifies resistance responses, acts synergistically with the defense hormone salicylic acid, promotes the hypersensitive cell death response and primes plants for effective immune mobilization in cases of future pathogen challenge. This pathogen-inducible NHP biosynthetic pathway is activated at the transcriptional level and involves feedback amplification. Apart from FMO1, some cytochrome P450 monooxygenases involved in secondary metabolism catalyze N-hydroxylation reactions in plants. In specific taxa, pipecolic acid might also serve as a precursor in the biosynthesis of specialized natural products, leading to C-hydroxylated and otherwise modified piperidine derivatives, including indolizidine alkaloids. Finally, we show that NHP is glycosylated in Arabidopsis to form a hexose-conjugate, and then discuss open questions in Pip/NHP-related research., (© 2018 The Authors The Plant Journal © 2018 John Wiley & Sons Ltd.)

Pipecolic acid (Pip), a non-proteinaceous product of lysine catabolism, is an important regulator of immunity in plants and humans alike. In plants, Pip accumulates upon pathogen infection and has been associated with systemic acquired resistance (SAR). However, the molecular mechanisms underlying Pip-mediated signaling and its relationship to other known SAR inducers remain unknown. We show that in plants, Pip confers SAR by increasing levels of the free radicals, nitric oxide (NO), and reactive oxygen species (ROS), which act upstream of glycerol-3-phosphate (G3P). Plants defective in NO, ROS, G3P, or salicylic acid (SA) biosynthesis accumulate reduced Pip in their distal uninfected tissues although they contain wild-type-like levels of Pip in their infected leaves. These data indicate that de novo synthesis of Pip in distal tissues is dependent on both SA and G3P and that distal levels of SA and G3P play an important role in SAR. These results also suggest a unique scenario whereby metabolites in a signaling cascade can stimulate each other's biosynthesis depending on their relative levels and their site of action.

Systemic acquired resistance (SAR) is a global response in plants induced at the site of infection that leads to long-lasting and broad-spectrum disease resistance at distal, uninfected tissues. Despite the importance of this priming mechanism, the identity and complexity of defense signals that are required to initiate SAR signaling is not well understood. In this paper, we describe a metabolite, N -hydroxy-pipecolic acid ( N -OH-Pip) and provide evidence that this mobile molecule plays a role in initiating SAR signal transduction in Arabidopsis thaliana We demonstrate that FLAVIN-DEPENDENT MONOOXYGENASE 1 (FMO1), a key regulator of SAR-associated defense priming, can synthesize N -OH-Pip from pipecolic acid in planta , and exogenously applied N -OH-Pip moves systemically in Arabidopsis and can rescue the SAR-deficiency of fmo1 mutants. We also demonstrate that N -OH-Pip treatment causes systemic changes in the expression of pathogenesis-related genes and metabolic pathways throughout the plant and enhances resistance to a bacterial pathogen. This work provides insight into the chemical nature of a signal for SAR and also suggests that the N -OH-Pip pathway is a promising target for metabolic engineering to enhance disease resistance., Competing Interests: The authors declare no conflict of interest.

Following a previous microbial inoculation, plants can induce broad-spectrum immunity to pathogen infection, a phenomenon known as systemic acquired resistance (SAR). SAR establishment in Arabidopsis thaliana is regulated by the Lys catabolite pipecolic acid (Pip) and flavin-dependent-monooxygenase1 (FMO1). Here, we show that elevated Pip is sufficient to induce an FMO1-dependent transcriptional reprogramming of leaves that is reminiscent of SAR. In planta and in vitro analyses demonstrate that FMO1 functions as a pipecolate N-hydroxylase, catalyzing the biochemical conversion of Pip to N-hydroxypipecolic acid (NHP). NHP systemically accumulates in plants after microbial attack. When exogenously applied, it overrides the defect of NHP-deficient fmo1 in acquired resistance and acts as a potent inducer of plant immunity to bacterial and oomycete infection. Our work has identified a pathogen-inducible L-Lys catabolic pathway in plants that generates the N-hydroxylated amino acid NHP as a critical regulator of systemic acquired resistance to pathogen infection., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Salicylic acid (SA) and pipecolic acid (Pip) play important roles in plant immunity. Here we analyzed the roles of transcription factors TGACG-BINDING FACTOR 1 (TGA1) and TGA4 in regulating SA and Pip biosynthesis in Arabidopsis thaliana. We quantified the expression levels of SYSTEMIC ACQUIRED RESISTANCE DEFICIENT 1 (SARD1) and CALMODULIN-BINDING PROTEIN 60g (CBP60g), which encode two master transcription factors of plant immunity, and the accumulation of SA and Pip in tga1-1 tga4-1 mutant plants. We tested whether SARD1 and CBP60g are direct targets of TGA1 by chromatin immunoprecipitation-polymerase chain reaction (ChIP-PCR). In addition to promoting pathogen-induced SA biosynthesis, we found that SARD1 and CBP60g also positively regulated Pip biosynthesis by targeting genes encoding key biosynthesis enzymes of Pip. TGA1/TGA4 were required for full induction of SARD1 and CBP60g in plant defense. ChIP-PCR analysis showed that SARD1 was a direct target of TGA1. In tga1-1 tga4-1 mutant plants, the expression levels of SARD1 and CBP60g along with SA and Pip accumulation following pathogen infection were dramatically reduced compared with those in wild-type plants. Consistent with reduced expression of SARD1 and CBP60g, pathogen-associated molecular pattern (PAMP)-induced pathogen resistance and systemic acquired resistance were compromised in tga1-1 tga4-1. Our study showed that TGA1 and TGA4 regulate Pip and SA biosynthesis by modulating the expression of SARD1 and CBP60g., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Corynebacterium glutamicum is used for the million-ton scale production of amino acids and has recently been engineered for production of the cyclic non-proteinogenic amino acid L-pipecolic acid (L-PA). In this synthetic pathway L-lysine was converted to L-PA by oxidative deamination, dehydration and reduction by L-lysine 6-dehydrogenase (deaminating) from Silicibacter pomeroyi and pyrroline 5-carboxylate reductase from C. glutamicum. However, production of L-PA occurred as by-product of L-lysine production only. Here, the author show that abolishing L-lysine export by the respective gene deletion resulted in production of L-PA as major product without concomitant lysine production while the specific growth rate was reduced due to accumulation of high intracellular lysine concentrations. Increasing expression of the genes encoding L-lysine 6-dehydrogenase and pyrroline 5-carboxylate reductase in C. glutamicum strain PIPE4 increased the L-PA titer to 3.9 g L -1 , and allowed faster growth and, thus, a higher volumetric productivity of 0.08 ± 0.00 g L -1 h -1 respectively. Secondly, expression of heterologous genes for utilization of glycerol, xylose, glucosamine, and starch in strain PIPE4 enabled L-PA production from these alternative carbon sources. Third, in a glucose/sucrose-based fed-batch fermentation with C. glutamicum PIPE4 L-PA was produced to a titer of 14.4 g L -1 with a volumetric productivity of 0.21 g L -1 h -1 and an overall yield of 0.20 g g -1 ., (Copyright © 2017 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The nonprotein amino acid pipecolic acid (Pip) regulates plant systemic acquired resistance and basal immunity to bacterial pathogen infection. In Arabidopsis ( Arabidopsis thaliana ), the lysine (Lys) aminotransferase AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) mediates the pathogen-induced accumulation of Pip in inoculated and distal leaf tissue. Here, we show that ALD1 transfers the α-amino group of l-Lys to acceptor oxoacids. Combined mass spectrometric and infrared spectroscopic analyses of in vitro assays and plant extracts indicate that the final product of the ALD1-catalyzed reaction is enaminic 2,3-dehydropipecolic acid (DP), whose formation involves consecutive transamination, cyclization, and isomerization steps. Besides l-Lys, recombinant ALD1 transaminates l-methionine, l-leucine, diaminopimelate, and several other amino acids to generate oxoacids or derived products in vitro. However, detailed in planta analyses suggest that the biosynthesis of 2,3-DP from l-Lys is the major in vivo function of ALD1. Since ald1 mutant plants are able to convert exogenous 2,3-DP into Pip, their Pip deficiency relies on the inability to form the 2,3-DP intermediate. The Arabidopsis reductase ornithine cyclodeaminase/μ-crystallin, alias SYSTEMIC ACQUIRED RESISTANCE-DEFICIENT4 (SARD4), converts ALD1-generated 2,3-DP into Pip in vitro. SARD4 significantly contributes to the production of Pip in pathogen-inoculated leaves but is not the exclusive reducing enzyme involved in Pip biosynthesis. Functional SARD4 is required for proper basal immunity to the bacterial pathogen Pseudomonas syringae Although SARD4 knockout plants show greatly reduced accumulation of Pip in leaves distal to P. syringae inoculation, they display a considerable systemic acquired resistance response. This suggests a triggering function of locally accumulating Pip for systemic resistance induction., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Background: The six-carbon circular non-proteinogenic compound L-pipecolic acid is an important chiral drug intermediate with many applications in the pharmaceutical industry. In the present study, we developed a metabolically engineered strain of Escherichia coli for the overproduction of L-pipecolic acid from glucose., Results: The metabolic pathway from L-lysine to L-pipecolic acid was constructed initially by introducing lysine cyclodeaminase (LCD). Next, L-lysine metabolic flux from glucose was amplified by the plasmid-based overexpression of dapA, lysC, and lysA under the control of the strong trc promoter to increase the biosynthetic pool of the precursor L-lysine. Additionally, since the catalytic efficiency of the key enzyme LCD is limited by the cofactor NAD + , the intracellular pyridine nucleotide concentration was rebalanced by expressing the pntAB gene encoding the transhydrogenase, which elevated the proportion of LCD with bound NAD + and enhanced L-pipecolic acid production significantly. Further, optimization of Fe 2+ and surfactant in the fermentation process resulted in 5.33 g/L L-pipecolic acid, with a yield of 0.13 g/g of glucose via fed-batch cultivation., Conclusions: We expanded the metabolic pathway for the synthesis of the chiral pharmaceutical intermediate L-pipecolic acid in E. coli. Using the engineered E. coli, a fast and efficient fermentative production of L-pipecolic acid was achieved. This strategy could be applied to the biosynthesis of other commercially and industrially important chiral compounds containing piperidine rings.

Thyroid hormones have long been known to play an essential role in brain growth and development, with cytoplasmic thyroid hormone binding proteins (THBPs) playing a critical role in thyroid hormone bioavailability. A major mammalian THBP is μ-crystallin (CRYM), which was originally characterized by its ability to strongly bind thyroid hormones in an NADPH-dependent fashion. However, in 2011 it was discovered that CRYM is also an enzyme, namely ketimine reductase (KR), which catalyzes the NAD(P)H-dependent reduction of -C=N- (imine) double bonds of a number of cyclic ketimine substrates including sulfur-containing cyclic ketimines. The enzyme activity was also shown to be potently inhibited by thyroid hormones, thus suggesting a novel reciprocal relationship between enzyme catalysis and thyroid hormone bioavailability. KR is involved in a number of amino acid metabolic pathways. However, the best documented biological function of KR is its role as a ∆ 1 -piperideine-2-carboxylate (P2C) reductase in the pipecolate pathway of lysine metabolism. The pipecolate pathway is the main L-lysine degradation pathway in the adult brain, whereas the saccharopine pathway predominates in extracerebral tissues and in infant brain, suggesting that KR has evolved to perform specific and important roles in neural development and function. The potent regulation of KR activity by thyroid hormones adds further weight to this suggestion. KR is also involved in L-ornithine/L-glutamate/L-proline metabolism as well as sulfur-containing amino acid metabolism. This review describes the pipecolate pathway and recent discoveries related to mammalian KR function, which have important implications in normal and pathological brain functions.

Systemic acquired resistance (SAR) is an immune response induced in the distal parts of plants following defense activation in local tissue. Pipecolic acid (Pip) accumulation orchestrates SAR and local resistance responses. Here, we report the identification and characterization of SAR-DEFICIENT4 (SARD4), which encodes a critical enzyme for Pip biosynthesis in Arabidopsis thaliana Loss of function of SARD4 leads to reduced Pip levels and accumulation of a Pip precursor, Δ 1 -piperideine-2-carboxylic acid (P2C). In Escherichia coli, expression of the aminotransferase ALD1 leads to production of P2C and addition of SARD4 results in Pip production, suggesting that a Pip biosynthesis pathway can be reconstituted in bacteria by coexpression of ALD1 and SARD4. In vitro experiments showed that ALD1 can use l-lysine as a substrate to produce P2C and P2C is converted to Pip by SARD4. Analysis of sard4 mutant plants showed that SARD4 is required for SAR as well as enhanced pathogen resistance conditioned by overexpression of the SAR regulator FLAVIN-DEPENDENT MONOOXYGENASE1. Compared with the wild type, pathogen-induced Pip accumulation is only modestly reduced in the local tissue of sard4 mutant plants, but it is below detection in distal leaves, suggesting that Pip is synthesized in systemic tissue by SARD4-mediated reduction of P2C and biosynthesis of Pip in systemic tissue contributes to SAR establishment., (© 2016 American Society of Plant Biologists. All rights reserved.)

The Gram-positive Corynebacterium glutamicum is widely used for fermentative production of amino acids. The world production of L-lysine has surpassed 2 million tons per year. Glucose uptake and phosphorylation by C. glutamicum mainly occur by the phosphotransferase system (PTS) and to lesser extent by inositol permeases and glucokinases. Heterologous expression of the genes for the high-affinity glucose permease from Streptomyces coelicolor and Bacillus subtilis glucokinase fully compensated for the absence of the PTS in Δhpr strains. Growth of PTS-positive strains with glucose was accelerated when the endogenous inositol permease IolT2 and glucokinase from B. subtilis were overproduced with balanced translation initiation rates using plasmid pEKEx3-IolTBest. When the genome-reduced C. glutamicum strain GRLys1 carrying additional in-frame deletions of sugR and ldhA to derepress glycolytic and PTS genes and to circumvent formation of L-lactate as by-product was transformed with this plasmid or with pVWEx1-IolTBest, 18 to 20 % higher volumetric productivities and 70 to 72 % higher specific productivities as compared to the parental strain resulted. The non-proteinogenic amino acid L-pipecolic acid (L-PA), a precursor of immunosuppressants, peptide antibiotics, or piperidine alkaloids, can be derived from L-lysine. To enable production of L-PA by the constructed L-lysine-producing strain, the L-lysine 6-dehydrogenase gene lysDH from Silicibacter pomeroyi and the endogenous pyrroline 5-carboxylate reductase gene proC were overexpressed as synthetic operon. This enabled C. glutamicum to produce L-PA with a yield of 0.09 ± 0.01 g g(-1) and a volumetric productivity of 0.04 ± 0.01 g L(-1) h(-1).To the best of our knowledge, this is the first fermentative process for the production of L-PA from glucose.

The causal agent of the soybean sudden death syndrome (SDS), Fusarium virguliforme, remains in infected roots and secretes toxins to cause foliar SDS. In this study we investigated the xylem sap, roots, and leaves of F. virguliforme-infected and -uninfected soybean seedlings for any changes in a set of over 3,000 metabolites following pathogen infection by conducting GC/MS and LC/MS/MS, and detected 273 biochemicals. Levels of many intermediates of the TCA cycle were reduced suggesting suppression of this metabolic pathway by the pathogen. There was an increased accumulation of peroxidated lipids in leaves of F. virguliforme-infected plants suggesting possible involvement of free radicals and lipoxygenases in foliar SDS development. Levels of both isoflavone conjugates and isoflavonoid phytoalexins were decreased in infected roots suggesting degradation of these metabolites by the pathogen to promote root necrosis. The levels of the plant immunity inducer pipecolic acid (Pip) and the plant hormone salicylic acid (SA) were significantly increased in xylem sap (in case of Pip) and leaves (in case of both Pip and SA) of F. virguliforme-infected soybean plants compared to the control plants. This suggests a major signaling role of Pip in inducing host defense responses in above ground parts of the F. virguliforme-infected soybean. Increased accumulation of pipecolic acid in foliar tissues was associated with the induction of GmALD1, the soybean homolog of Arabidopsis ALD1. This metabolomics study generated several novel hypotheses for studying the mechanisms of SDS development in soybean., (Copyright © 2015 Elsevier Ireland Ltd. All rights reserved.)

Hydroxypipecolic acids are bioactive compounds widely distributed in nature and are valuable building blocks for the organic synthesis of pharmaceuticals. We have found a novel hydroxylating enzyme with activity toward L-pipecolic acid (L-Pip) in a filamentous fungus, Fusarium oxysporum c8D. The enzyme L-Pip trans-4-hydroxylase (Pip4H) of F. oxysporum (FoPip4H) belongs to the Fe(II)/α-ketoglutarate-dependent dioxygenase superfamily, catalyzes the regio- and stereoselective hydroxylation of L-Pip, and produces optically pure trans-4-hydroxy-L-pipecolic acid (trans-4-L-HyPip). Amino acid sequence analysis revealed several fungal enzymes homologous with FoPip4H, and five of these also had L-Pip trans-4-hydroxylation activity. In particular, the homologous Pip4H enzyme derived from Aspergillus nidulans FGSC A4 (AnPip4H) had a broader substrate specificity spectrum than other homologues and reacted with the L and D forms of various cyclic and aliphatic amino acids. Using FoPip4H as a biocatalyst, a system for the preparative-scale production of chiral trans-4-L-HyPip was successfully developed. Thus, we report a fungal family of L-Pip hydroxylases and the enzymatic preparation of trans-4-L-HyPip, a bioactive compound and a constituent of secondary metabolites with useful physiological activities., (Copyright © 2016, American Society for Microbiology. All Rights Reserved.)

We investigated the relationships of the two immune-regulatory plant metabolites, salicylic acid (SA) and pipecolic acid (Pip), in the establishment of plant systemic acquired resistance (SAR), SAR-associated defense priming, and basal immunity. Using SA-deficient sid2, Pip-deficient ald1, and sid2 ald1 plants deficient in both SA and Pip, we show that SA and Pip act both independently from each other and synergistically in Arabidopsis thaliana basal immunity to Pseudomonas syringae. Transcriptome analyses reveal that SAR establishment in Arabidopsis is characterized by a strong transcriptional response systemically induced in the foliage that prepares plants for future pathogen attack by preactivating multiple stages of defense signaling and that SA accumulation upon SAR activation leads to the downregulation of photosynthesis and attenuated jasmonate responses systemically within the plant. Whereas systemic Pip elevations are indispensable for SAR and necessary for virtually the whole transcriptional SAR response, a moderate but significant SA-independent component of SAR activation and SAR gene expression is revealed. During SAR, Pip orchestrates SA-dependent and SA-independent priming of pathogen responses in a FLAVIN-DEPENDENT-MONOOXYGENASE1 (FMO1)-dependent manner. We conclude that a Pip/FMO1 signaling module acts as an indispensable switch for the activation of SAR and associated defense priming events and that SA amplifies Pip-triggered responses to different degrees in the distal tissue of SAR-activated plants., (© 2016 American Society of Plant Biologists. All rights reserved.)

L-Pipecolic acid is a key component of biologically active molecules and a pharmaceutically important chiral building block. It can be stereoselectively produced from L-lysine by a two-step bioconversion involving L-lysine α-oxidase and ∆(1)-piperideine-2-carboxylae (Pip2C) reductase. In this study, we focused on an L-lysine α-oxidase from Scomber japonicus that was originally identified as an apoptosis-inducing protein (AIP) and applied the enzyme to one-pot fermentation of L-pipecolic acid in Escherichia coli. A synthetic gene coding for an AIP was expressed in E. coli, and the recombinant enzyme was purified and characterized. The purified enzyme was determined to be a homodimer with a molecular mass of 133.9 kDa. The enzyme essentially exhibited the same substrate specificity as the native enzyme. Optimal temperature and pH for the enzymatic reaction were 70 °C and 7.4, respectively. The enzyme was stable below 60 °C and at a pH range of 5.5-7.5 but was markedly inhibited by Co(2+). To establish a one-pot fermentation system for the synthesis of optically pure L-pipecolic acid from DL-lysine, an E. coli strain carrying a plasmid encoding AIP, Pip2C reductase from Pseudomonas putida, lysine racemase from P. putida, and glucose dehydrogenase from Bacillus subtilis was constructed. The one-pot process produced 45.1 g/L of L-pipecolic acid (87.4 % yield from DL-lysine) after a 46-h reaction with high optical purity (>99.9 % enantiomeric excess).

Enzymatic regio- and stereoselective hydroxylation are valuable for the production of hydroxylated chiral ingredients. Proline hydroxylases are representative members of the nonheme Fe(2+)/α-ketoglutarate-dependent dioxygenase family. These enzymes catalyze the conversion of L-proline into hydroxy-L-prolines (Hyps). L-Proline cis-4-hydroxylases (cis-P4Hs) from Sinorhizobium meliloti and Mesorhizobium loti catalyze the hydroxylation of L-proline, generating cis-4-hydroxy-L-proline, as well as the hydroxylation of L-pipecolic acid (L-Pip), generating two regioisomers, cis-5-Hypip and cis-3-Hypip. To selectively produce cis-5-Hypip without simultaneous production of two isomers, protein engineering of cis-P4Hs is required. We therefore carried out protein engineering of cis-P4H to facilitate the conversion of the majority of L-Pip into the cis-5-Hypip isomer. We first solved the X-ray crystal structure of cis-P4H in complex with each of L-Pro and L-Pip. Then, we conducted three rounds of directed evolution and successfully created a cis-P4H triple mutant, V97F/V95W/E114G, demonstrating the desired regioselectivity toward cis-5-Hypip.

Inherited deficiencies of the L-lysine catabolic pathway cause glutaric aciduria type I and pyridoxine-dependent epilepsy. Dietary modulation of cerebral L-lysine metabolism is thought to be an important therapeutic intervention for these diseases. To better understand cerebral L-lysine degradation, we studied in mice the two known catabolic routes -- pipecolate and saccharopine pathways -- using labeled stable L-lysine and brain peroxisomes purified according to a newly established protocol. Experiments with labeled stable L-lysine show that cerebral L-pipecolate is generated along two pathways: i) a minor proportion retrograde after ε-deamination of L-lysine along the saccharopine pathway, and ii) a major proportion anterograde after α-deamination of L-lysine along the pipecolate pathway. In line with these findings, we observed only little production of saccharopine in the murine brain. L-pipecolate oxidation was only detectable in brain peroxisomes, but L-pipecolate oxidase activity was low (7 ± 2μU/mg protein). In conclusion, L-pipecolate is a major degradation product from L-lysine in murine brain generated by α-deamination of this amino acid.

Adverse side effects of drugs are often caused by the interaction of drug molecules to targets other than the intended ones. In this study, we investigated the off-target interactions of some commercially available drugs with human α-thrombin. The drugs used in the study were selected from Super Drug Database based on the structural similarity to a known thrombin inhibitor argatroban. Interactions of these drugs with thrombin were initially checked by in silico docking studies and then confirmed by thrombin inhibition assay using a fluorescence microplate-based method. Results show that the three commonly used drugs piperacillin (anti-bacterial), azlocillin (anti-bacterial), and metolazone (anti-hypertensive and diuretic) have thrombin inhibitory activity almost similar to that of argatroban. The Ki values of piperacillin, azlocillin, and metolazone with thrombin are .55, .95, and .62 nM, respectively. The IC50 values of piperacillin, azlocillin, and metolazone with thrombin are 1.7, 2.9, and 1.92 nM, respectively. This thrombin inhibitory activity might be a reason for the observed side effects of these drugs related to blood coagulation and other thrombin activities. Furthermore, these compounds (drugs) may be used as anti-coagulants as such or with structural modifications.

We have conducted biochemical studies with commercial available pyrroline-5-carboxylate (P5C) reductase (PYCR1) to investigate whether this enzyme plays a role in L-lysine degradation. Our recent studies with antiquitin/ALDH7A1 deficient fibroblasts revealed an alternative genesis of L-pipecolic acid, and we then hypothesized that PYCR1 was responsible for the conversion of Δ(1)-piperideine-6-carboxylate (P6C) into pipecolic acid. We here present evidence that PYCR1 is indeed able to produce L-pipecolic acid from P6C preparations, and the observed K m for this conversion is of the same magnitude as the K m described for the conversion of P5C to L-proline by PYCR1. Urine samples from antiquitin deficient individuals, who accumulate P6C, were also incubated with PYCR1 which resulted in a marked decrease of P6C and a huge increase of L-pipecolic acid as measured by LC-MS/MS, confirming that indeed PYCR1 generates L-pipecolic acid from P6C.

Lysine is an essential amino acid for human nutrition, which is generally low in cereal diets. Its biosynthesis via the aspartate-pathway and catabolism is controlled by complex feedback mechanisms. Recently, aspartate-derived amino acids were found to be elevated during pathogen infection in Arabidopsis and a lysine catabolite, pipecolic acid, was identified as critical regulator of systemic acquired resistance. Pipecolic acid is mobile in plants, functions as an intensifier of defense responses and mediates systemic acquired resistance establishment via signal amplification. The altered pathogen defense in several mutants with altered homeostasis of aspartate-derived amino acids, such as lysine, had already provided a genetic link with amino acid homeostasis. Furthermore, the modification of amino acid transport and distribution within tissues not only affected the plant growth performance, but also the plant-pathogen interaction. The ectopic overexpression of a gene encoding a high affinity importer with preference to basic amino acids, such as lysine, cationic amino acid transporter1 (CAT1), improved the disease resistance to a hemibiotrophic bacterial pathogen in Arabidopsis via a constitutively activated salicylic acid pathway. The importance of Asp-derived amino acid homeostasis for plant systemic acquired resistance and on overall plant growth performance may be relevant to resistance and nutritional quality breeding. Whether nitrogen fertilization has an impact on crop pest control management via amino acid homeostasis is briefly discussed.

The trisubstituted enolate- and C-C bond-forming capacities of engineered carboxymethylproline synthases CMPSs are coupled with the malonyl-CoA synthetase MatB to enable stereoselective preparation of 5- and 6-membered N-heterocycles functionalised with alkyl-substituted carboxymethyl side chains, starting from achiral alkyl-substituted malonic acids and L-amino acid semialdehydes. The results illustrate the biocatalytic utility of crotonases in tandem enzyme-catalysed reactions for stereoselective synthesis.

A linear supramolecular ternary polymer was fabricated by iteratively threading cyclodextrin with suberyl dicholine and end-capping with bis-calixarenes, showing desired cholinesterase response.

Metabolic signals orchestrate plant defenses against microbial pathogen invasion. Here, we report the identification of the non-protein amino acid pipecolic acid (Pip), a common Lys catabolite in plants and animals, as a critical regulator of inducible plant immunity. Following pathogen recognition, Pip accumulates in inoculated Arabidopsis thaliana leaves, in leaves distal from the site of inoculation, and, most specifically, in petiole exudates from inoculated leaves. Defects of mutants in AGD2-LIKE DEFENSE RESPONSE PROTEIN1 (ALD1) in systemic acquired resistance (SAR) and in basal, specific, and β-aminobutyric acid-induced resistance to bacterial infection are associated with a lack of Pip production. Exogenous Pip complements these resistance defects and increases pathogen resistance of wild-type plants. We conclude that Pip accumulation is critical for SAR and local resistance to bacterial pathogens. Our data indicate that biologically induced SAR conditions plants to more effectively synthesize the phytoalexin camalexin, Pip, and salicylic acid and primes plants for early defense gene expression. Biological priming is absent in the pipecolate-deficient ald1 mutants. Exogenous pipecolate induces SAR-related defense priming and partly restores priming responses in ald1. We conclude that Pip orchestrates defense amplification, positive regulation of salicylic acid biosynthesis, and priming to guarantee effective local resistance induction and the establishment of SAR.

The mammalian degradation of lysine is believed to proceed via two distinct routes, the saccharopine and the pipecolic acid routes, that ultimately converge at the level of alpha-aminoadipic semialdehyde (alpha-AASA). alpha-AASA dehydrogenase-deficient fibroblasts were grown in cell culture medium supplemented with either L-[alpha-(15)N]lysine or L-[epsilon-(15)N]lysine to explore the exact route of lysine degradation. L-[alpha-(15)N]lysine was catabolised into [(15)N]saccharopine, [(15)N]alpha-AASA, [(15)N]Delta(1)-piperideine-6-carboxylate, and surprisingly in [(15)N]pipecolic acid, whereas L-[epsilon-(15)N]lysine resulted only in the formation of [(15)N]saccharopine. These results imply that lysine is exclusively degraded in fibroblasts via the saccharopine branch, and pipecolic acid originates from an alternative precursor. We hypothesize that pipecolic acid derives from Delta(1)-piperideine-6-carboxylate by the action of Delta(1)-pyrroline-5-carboxylic acid reductase, an enzyme involved in proline metabolism.

Human butyrylcholinesterase hydrolyzes long chain dicholine esters more rapidly than short chain dicholine esters. The active site of butyrylcholinesterase is deeply buried within the enzyme molecule and there is limited space for binding of large compounds. Our goal was to understand how butyrylcholinesterase accommodates long chain dicholine esters to make them better substrates than short chain dicholine esters. For this purpose we studied the rate of hydrolysis of adipyldicholine (n=4) and sebacyldicholine (n=8) with mass spectrometry, a method that allowed monitoring the dicholine substrates, the monocholine intermediates, the dicarboxylic acid and choline products. It was shown that hydrolysis of adipyldicholine involves two consecutive steps, dicholine ester hydrolysis followed by relatively slow monocholine ester hydrolysis. However, sebacyldicholine was hydrolyzed at both choline ester sites, though hydrolysis of dicholine was faster than hydrolysis of monocholine. Sebacyldicholine was completely converted to sebacic acid and choline within 90 min, whereas only 15% of the adipyldicholine was converted to adipic acid in this time. Molecular modeling indicated that these dicholine esters can bind to butyrylcholinesterase in two energetically equivalent alternative conformations that may theoretically lead to hydrolysis. The long chain dicholine ester makes closer contact than the short chain ester between one of its carbonyl carbons and the catalytic Ser198, thus explaining why long-chain dicholine esters are hydrolyzed more rapidly by butyrylcholinesterase.