Reply to: LsBOS utilizes oxalyl-CoA produced by LsAAE3 to synthesize β-ODAP in grass pea.
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SUBMITTER: Edwards A
PROVIDER: S-EPMC11310462 | biostudies-literature | 2024 Aug
REPOSITORIES: biostudies-literature
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Reply to: LsBOS utilizes oxalyl-CoA produced by LsAAE3 to synthesize β-ODAP in grass pea.
Edwards Anne A Jiang Zhouqian Z Nepogodiev Sergey S Rejzek Martin M Martin Cathie C Emmrich Peter M F PMF
Nature communications 20240808 1
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Project description: Not available
| S-EPMC11310188 | biostudies-literature
Project description:Oxalic acid is a small metabolite found in many plants. It serves as protection from herbivores, a chelator of metal ions, a regulator of calcium levels, and additional tasks. However, it is also a strong di-carboxylic acid that can compromise plant viability by reducing cellular pH. Several metabolic pathways have evolved to control oxalate levels in plants by enzymatic degradation. Among them is the pathway that utilizes oxalyl-CoA synthetase (OCS, EC 6.2.1.8) and ATP to convert oxalate to oxalyl-CoA. Oxalyl-CoA can then be degraded to CO2 or utilized as a precursor for the synthesis of other compounds. In grass pea (Lathyrus sativus L.), a grain legume grown in Asia and Africa for human and animal consumption, the neurotoxic compound β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP) is synthesized from oxalyl-CoA and l-α,β-diaminopropionic acid (l-DAPA). Here, we report on the identification and characterization of oxalyl CoA-synthetase from grass pea (LsOCS). The gene encoding LsOCS was amplified from grass pea, and then expressed and purified from E. coli cells as an untagged, monomeric protein of 56 kDa. Its catalytic efficiency with oxalate, K oxalate M = 71.5 ± 13.3 μM, V max = 8.2 ± 0.8 μmole min-1 mg-1, was similar to that of OCS homologs from Arabidopsis thaliana (AtAAE3) and Medicago truncatula (MtAAE3). The enzyme was crystalized in complex with AMP and is the first OCS whose structure was determined in the thioester-forming conformation. Finally, we propose that substituting LsOCS with an oxalate oxidase or decarboxylase may reduce the levels of β-ODAP in grass pea.
| S-EPMC8905533 | biostudies-literature
Project description:Grass pea seeds and seedlings protein extracts were chromatographically fractionated. To identify the β-ODAP synthase enzyme, active fractions, as determined by a colorimetric assay that detects the presence of a derivative of free L-α,β-diaminopropionic acid (L-DAPA), were subjected to tryptic digestion and LC-MS/MS and searched against a database containing translated sequences from a long-read PacBio mRNA sequencing of grass pea seeds and seedlings.
2022-05-19 | PXD030847 | Pride
Project description:Grass pea (Lathyrus sativus L.) is a grain legume commonly grown in Asia and Africa for food and forage. It is a highly nutritious and robust crop, capable of surviving both droughts and floods. However, it produces a neurotoxic compound, β-N-oxalyl-L-α,β-diaminopropionic acid (β-ODAP), which can cause a severe neurological disorder when consumed as a primary diet component. While the catalytic activity associated with β-ODAP formation was demonstrated more than 50 years ago, the enzyme responsible for this activity has not been identified. Here, we report on the identity, activity, 3D structure, and phylogenesis of this enzyme-β-ODAP synthase (BOS). We show that BOS belongs to the benzylalcohol O-acetyltransferase, anthocyanin O-hydroxycinnamoyltransferase, anthranilate N-hydroxycinnamoyl/benzoyltransferase, deacetylvindoline 4-O-acetyltransferase superfamily of acyltransferases and is structurally similar to hydroxycinnamoyl transferase. Using molecular docking, we propose a mechanism for its catalytic activity, and using heterologous expression in tobacco leaves (Nicotiana benthamiana), we demonstrate that expression of BOS in the presence of its substrates is sufficient for β-ODAP production in vivo. The identification of BOS may pave the way toward engineering β-ODAP-free grass pea cultivars, which are safe for human and animal consumption.
| S-EPMC9061259 | biostudies-literature
Project description:Grass pea (Lathyrus sativus L.), a protein-rich pulse crop, is often overlooked due to its association with neurolathyrism and its neurotoxin, β-ODAP. The study aims to compare the β-ODAP content, chemical, and functional properties of four BARI varieties and two local varieties of grass pea seed flour. The findings presented that the β-ODAP content of BARI varieties grass pea flour was significantly (p < 0.05) lower than local varieties, and the least amount of β-ODAP was found in BARI-3 varieties (0.086 %), which is below the safe level (0.15 %) for consumption. The safe level of neurotoxin was also found in the BARI-1 variety (0.13 %), but local varieties grass pea flour of Pabna and Tangail showed a significantly higher (p < 0.05) value of 0.39 and 0.49 % β-ODAP content, respectively. There were no significant differences in protein content among BARI and local varieties, with the highest value of 26.58 % protein content found in the BARI-2 variety. In terms of functional properties, the BARI-5 variety had the highest water absorption capacity (2.92 ml/g) and oil absorption capacity (1.48 ml/g). The grass pea BARI variety, with its high oil absorption capacity and low β-ODAP content, can be utilized in food formulations for bakery products, sausages, and functional ingredients.
| S-EPMC11417204 | biostudies-literature
Project description:Grass pea (Lathyrus sativus L.) is an important legume crop grown mainly in South Asia and Sub-Saharan Africa. This underutilized legume can withstand harsh environmental conditions including drought and flooding. During drought-induced famines, this protein-rich legume serves as a food source for poor farmers when other crops fail under harsh environmental conditions; however, its use is limited because of the presence of an endogenous neurotoxic nonprotein amino acid β-N-oxalyl-l-α,β-diaminopropionic acid (β-ODAP). Long-term consumption of Lathyrus and β-ODAP is linked to lathyrism, which is a degenerative motor neuron syndrome. Pharmacological studies indicate that nutritional deficiencies in methionine and cysteine may aggravate the neurotoxicity of β-ODAP. The biosynthetic pathway leading to the production of β-ODAP is poorly understood, but is linked to sulfur metabolism. To date, only a limited number of studies have been conducted in grass pea on the sulfur assimilatory enzymes and how these enzymes regulate the biosynthesis of β-ODAP. Here, we review the current knowledge on the role of sulfur metabolism in grass pea and its contribution to β-ODAP biosynthesis. Unraveling the fundamental steps and regulation of β-ODAP biosynthesis in grass pea will be vital for the development of improved varieties of this underutilized legume.
| S-EPMC5372542 | biostudies-literature
Project description:BackgroundGrass pea (Lathyrus sativus) is an underutilised crop with high tolerance to drought and flooding stress and potential for maintaining food and nutritional security in the face of climate change. The presence of the neurotoxin β-L-oxalyl-2,3-diaminopropionic acid (β-L-ODAP) in tissues of the plant has limited its adoption as a staple crop. To assist in the detection of material with very low neurotoxin toxin levels, we have developed two novel methods to assay ODAP. The first, a version of a widely used spectrophotometric assay, modified for increased throughput, permits rapid screening of large populations of germplasm for low toxin lines and the second is a novel, mass spectrometric procedure to detect very small quantities of ODAP for research purposes and characterisation of new varieties.ResultsA plate assay, based on an established spectrophotometric method enabling high-throughput ODAP measurements, is described. In addition, we describe a novel liquid chromatography mass spectrometry (LCMS)-based method for β-L-ODAP-quantification. This method utilises an internal standard (di-13C-labelled β-L-ODAP) allowing accurate quantification of β-L-ODAP in grass pea tissue samples. The synthesis of this standard is also described. The two methods are compared; the spectrophotometric assay lacked sensitivity and detected ODAP-like absorbance in chickpea and pea whereas the LCMS method did not detect any β-L-ODAP in these species. The LCMS method was also used to quantify β-L-ODAP accurately in different tissues of grass pea.ConclusionsThe plate-based spectrophotometric assay allows quantification of total ODAP in large numbers of samples, but its low sensitivity and inability to differentiate α- and β-L-ODAP limit its usefulness for accurate quantification in low-ODAP samples. Coupled to the use of a stable isotope internal standard with LCMS that allows accurate quantification of β-L-ODAP in grass pea samples with high sensitivity, these methods permit the identification and characterisation of grass pea lines with a very low ODAP content. The LCMS method is offered as a new 'gold standard' for β-L-ODAP quantification, especially for the validation of existing and novel low- and/or zero-β-L-ODAP genotypes.
| S-EPMC6849181 | biostudies-literature
Project description:Acyl activating enzyme 3 (AAE3) encodes oxalyl-CoA synthetase involved in oxalate degradation. In this study, we investigated the role of AAE3 (SlAAE3) in the fruit quality of tomato (Solanum lycopersicum). The purified recombinant SlAAE3 protein from Escherichia coli exhibited a high activity toward oxalate, with a K m of 223.8 ± 20.03 μm and V max of 7.908 ± 0.606 μmol mg-1 protein min-1. Transient expression of SlAAE3-green fluorescent protein (GFP) fusion proteins suggests that SlAAE3 is a soluble protein without specific subcellular localization. The expression of SlAAE3 is both tissue- and development-dependent, and increased during fruit ripping. The Slaae3 knockout mutants had improved fruit quality as evidenced by the increased sugar-acid ratio and mineral nutrient content. To find the mechanism by which SlAAE3 affects fruit quality, transcriptome, and metabolome were employed on SlAAE3 over-expressed line and wide type fruits. The transcriptomic and metabolic profiles indicated that SlAAE3 in fruits mainly functions at 20 days post-anthesis (20 DPA) and mature green (MG) stages, resulting in up-regulation of amino acid derivatives, nucleotides, and derivatives, but down-regulation of lipid compounds. However, differentially expressed genes (DEGs) were mainly enriched at redox pathways. Taken together, both in vivo and in vitro results suggest that SlAAE3-encoded protein acts as an oxalyl-CoA synthetase, which also participates in redox metabolism. These data provide a further understanding of the mechanism by which SlAAE3 participates in tomato fruit quality.
| S-EPMC9301600 | biostudies-literature
Project description: Not available
| S-EPMC8427657 | biostudies-literature
Project description:BackgroundOne of the most common diseases of the urinary tract is stones of this system, including kidney stones. About 70%-80% of kidney stones are calcium oxalate. Oxalyl-CoA decarboxylase is a single polypeptide included of 568 amino acids which play a key role in oxalate degradation.Materials and methodsThe aim of current study is high-level expression of oxalyl-CoA decarboxylase in Escherichia coli BL21 (DE3). To achieve this aim, oxalyl-CoA decarboxylase gene was cloned upon pET-30a (+) with T7 promoter. The vector containing the oxalyl-CoA decarboxylase gene was transformed into E. coli and the expression of the gene was examined on a laboratory scale and fermentor. Atfirst, the effect of temperature, culture medium, and induction time on oxalyl-CoA decarboxylase expression at three levels was examined.ResultsThe obtained data showed that the highest expression was related to the terrific broth culture medium and temperature of 32°C with an inducer concentration of 1 mM. Under this situation the ultimate cells dry weight and the final oxalyl-CoA decarboxylase expression were 2.46 g/l and 36% of total protein, respectively. Then induction time was optimized in a bench bioreactor and productivity of oxalyl-CoA decarboxylase was calculated. Under optimized condition the cell density, biomass productivity and oxalyl-CoA decarboxylase concentration reached 4.02 g/l, 0.22 g/l/h, and 0.7 g/l which are one of the highest reported rates.ConclusionThis study demonstrated that high levels of oxalyl-CoA decarboxylase can be achieved by optimizing the expression conditions.
| S-EPMC9926027 | biostudies-literature