Connecting proline metabolism and signaling pathways in plant senescence

The amino acid proline has a unique biological role in stress adaptation. Proline metabolism is manipulated under stress by multiple and complex regulatory pathways and can profoundly influence cell death and survival in microorganisms, plants, and animals. Though the effects of proline are mediated by diverse signaling pathways, a common theme appears to be the generation of reactive oxygen species (ROS) due to proline oxidation being coupled to the respiratory electron transport chain. Considerable research has been devoted to understand how plants exploit proline metabolism in response to abiotic and biotic stress. Here, we review potential mechanisms by which proline metabolism influences plant senescence, namely in the petal and leaf. Recent studies of petal senescence suggest proline content is manipulated to meet energy demands of senescing cells. In the flower and leaf, proline metabolism may influence ROS signaling pathways that delay senescence progression. Future studies focusing on the mechanisms by which proline metabolic shifts occur during senescence may lead to novel methods to rescue crops under stress and to preserve post-harvest agricultural products

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in \u3ci\u3eEscherichia coli\u3c/i\u3e

The oxidation of L-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and _1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the _katG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity

Proline Biosynthesis Is Required for Endoplasmic Reticulum Stress Tolerance in \u3ci\u3eSaccharomyces cerevisiae\u3c/i\u3e

Background: Proline is an important amino acid for stress resistance in different organisms. Results: Depletion of proline biosynthesis disrupts redox homeostasis and increases sensitivity to endoplasmic reticulum (ER) stress in yeast. Conclusion: Proline biosynthesis is critical for maintaining the intracellular redox environment and the UPR during ER stress. Significance: Proline metabolism is shown to have an important role in ER stress tolerance that was previously unknown

Identification and Characterization of Oxalate Oxidoreductase, a Novel Thiamine Pyrophosphate-dependent 2-Oxoacid Oxidoreductase That Enables Anaerobic Growth on Oxalate

Moorella thermoacetica is an anaerobic acetogen, a class of bacteria that is found in the soil, the animal gastrointestinal tract, and the rumen. This organism engages the Wood-Ljungdahl pathway of anaerobic CO2 fixation for heterotrophic or autotrophic growth. This paper describes a novel enzyme, oxalate oxidoreductase (OOR), that enables M. thermoacetica to grow on oxalate, which is produced in soil and is a common component of kidney stones. Exposure to oxalate leads to the induction of three proteins that are subunits of OOR, which oxidizes oxalate coupled to the production of two electrons and CO2 or bicarbonate. Like other members of the 2-oxoacid: ferredoxin oxidoreductase family, OOR contains thiamine pyrophosphate and three [Fe4S4] clusters. However, unlike previously characterized members of this family, OOR does not use coenzyme A as a substrate. Oxalate is oxidized with a kcat of 0.09 s-1and a Km of 58 µM at pH 8. OOR also oxidizes a few other 2-oxoacids (which do not induce OOR) also without any requirement for CoA. The enzyme transfers its reducing equivalents to a broad range of electron acceptors, including ferredoxin and the nickel-dependent carbon monoxide dehydrogenase. In conjunction with the well characterized Wood- Ljungdahl pathway, OOR should be sufficient for oxalate metabolism by M. thermoacetica, and it constitutes a novel pathway for oxalate metabolism

Mutant Study of Sinorhizobium meliloti Proline Utilization A (PutA)

The purpose of this project is to purify and characterize the reaction kinetics of mutant versions the enzyme Proline Utilization A (PutA) in Sinorhizobium meliloti. The enzyme catalyzes the first step in proline metabolism. It has two active sites. The first is proline dehydrogenase (PRODH) which converts proline to pyrroline-5-carboxylate (P5C). The second is P5C dehydrogenase (P5CDH) which converts P5C to glutamate. Although many bacterial organisms have PutA, there are still significant interspecies variations, resulting in an entire family of PutA enzymes. The main difference is the length of the amino acid sequence. This affects the protein’s structure or its shape, and the protein’s kinetics or how it behaves in reactions. In order to have a complete understanding of proline metabolism, all the variations of PutA must be characterized both structurally and kinetically. The version of PutA found in S. meliloti (SmPutA) has been categorized structurally but not kinetically. This project aims to fill this gap in our knowledge of proline metabolism and PutA from S. meliloti

First Evidence for Substrate Channeling between Proline Catabolic Enzymes \u3ci\u3eA VALIDATION OF DOMAIN FUSION ANALYSIS FOR PREDICTING PROTEIN-PROTEIN INTERACTIONS\u3c/i\u3e

Background: PRODH and P5CDH from Thermus thermophilus are monofunctional enzymes in proline catabolism. Results: Steady-state kinetics and intermediate trapping data show the PRODH and P5CDH reactions are coupled by a channeling step. Conclusion: Substrate channeling in monofunctional enzymes is achieved via weak interactions. Significance: Evidence for substrate channeling between monofunctional proline catabolic enzymes is shown and confirms the Rosetta Stone hypothesis

Evidence for Hysteretic Substrate Channeling in the Proline Dehydrogenaseand ∆\u3csup\u3e1\u3c/sup\u3e-Pyrroline-5-carboxylate Dehydrogenase Coupled Reaction of Proline UtilizationA(PutA)

Background: PutA from Escherichia coli is a bifunctional enzyme and transcriptional repressor in proline catabolism. Results: Steady-state and transient kinetic data revealed a mechanism in which the two enzymatic reactions are coupled by an activation step. Conclusion: Substrate channeling in PutA exhibits hysteretic behavior. Significance: This is the first kinetic model of bi-enzyme activity in PutA and reveals a novel mechanism of channeling activation

Evidence for Pipecolate Oxidase in Mediating Protection Against Hydrogen Peroxide Stress

Pipecolate, an intermediate of the lysine catabolic pathway, is oxidized to Δ1-piperideine-6-carboxylate (P6C) by the flavoenzyme lpipecolate oxidase (PIPOX). P6C spontaneously hydrolyzes to generate α-aminoadipate semialdehyde, which is then converted into α-aminoadipate acid by α-aminoadipatesemialdehyde dehydrogenase. l-pipecolate was previously reported to protect mammalian cells against oxidative stress. Here, we examined whether PIPOX is involved in the mechanism of pipecolate stress protection. Knockdown of PIPOX by small interference RNA abolished pipecolate protection against hydrogen peroxide-induced cell death in HEK293 cells suggesting a critical role for PIPOX. Subcellular fractionation analysis showed that PIPOX is localized in the mitochondria of HEK293 cells consistent with its role in lysine catabolism. Signaling pathways potentially involved in pipecolate protection were explored by treating cells with small molecule inhibitors. Inhibition of both mTORC1 and mTORC2 kinase complexes or inhibition of Akt kinase alone blocked pipecolate protection suggesting the involvement of these signaling pathways. Phosphorylation of the Akt downstream target, forkhead transcription factor O3 (FoxO3), was also significantly increased in cells treated with pipecolate, further implicating Akt in the protective mechanism and revealing FoxO3 inhibition as a potentially key step. The results presented here demonstrate that pipecolate metabolism can influence cell signaling during oxidative stress to promote cell survival and suggest that the mechanism of pipecolate protection parallels that of proline, which is also metabolized in the mitochondria

Proline Metabolism Increases katG Expression and Oxidative Stress Resistance in \u3ci\u3eEscherichia coli\u3c/i\u3e

The oxidation of L-proline to glutamate in Gram-negative bacteria is catalyzed by the proline utilization A (PutA) flavoenzyme, which contains proline dehydrogenase (PRODH) and _1-pyrroline-5-carboxylate (P5C) dehydrogenase domains in a single polypeptide. Previous studies have suggested that aside from providing energy, proline metabolism influences oxidative stress resistance in different organisms. To explore this potential role and the mechanism, we characterized the oxidative stress resistance of wild-type and putA mutant strains of Escherichia coli. Initial stress assays revealed that the putA mutant strain was significantly more sensitive to oxidative stress than the parental wild-type strain. Expression of PutA in the putA mutant strain restored oxidative stress resistance, confirming that depletion of PutA was responsible for the oxidative stress phenotype. Treatment of wild-type cells with proline significantly increased hydroperoxidase I (encoded by katG) expression and activity. Furthermore, the _katG strain failed to respond to proline, indicating a critical role for hydroperoxidase I in the mechanism of proline protection. The global regulator OxyR activates the expression of katG along with several other genes involved in oxidative stress defense. In addition to katG, proline increased the expression of grxA (glutaredoxin 1) and trxC (thioredoxin 2) of the OxyR regulon, implicating OxyR in proline protection. Proline oxidative metabolism was shown to generate hydrogen peroxide, indicating that proline increases oxidative stress tolerance in E. coli via a preadaptive effect involving endogenous hydrogen peroxide production and enhanced catalase-peroxidase activity