The use of a plant enzyme for rapid and sensitive analysis of naturally-occurring folates in food by liquid chromatography-tandem mass spectrometry.

A rapid, sensitive and reproducible method for analysis of naturally-occurring folates and folic acid in food has been developed and validated. A single-enzyme extraction step, in which a pure recombinant enzyme of plant origin (Arabidopsis thaliana) was used, enabled fast and reproducible deglutamylation during folate extraction within the incubation time of 1 h. Six commonly occurring folate forms (tetrahydrofolate, 5,10-methenyltetrahydrofolate, 10-formylfolic acid, 5-formyltetrahydrofolate, folic acid and 5-methyltetrahydrofolate) were detected and quantified in 9 min using liquid chromatography-tandem mass spectrometry (LC-MS/MS). 13C5-labeled 5-formyltetrahydrofolate, 13C5-labeled folic acid and 13C5-labeled 5-methyltetrahydrofolate were used as internal standards for the quantification. The method is described by a calibration curve (R2>0.99 and trueness 85-115%), a limit of quantification at 0.1 µg/100 g, trueness at 80-120% in spiked samples and certified reference materials, and a precision <10%. However, the precision in quantification of tetrahydrofolate was not within the acceptable limits due to the lack of use of the corresponding internal standard. An interconversion study of unstable formyl forms was performed which showed that 50% of 5,10-methenyltetrahydrofolate is converted to 5-formyltetrahydrofolate during the analysis. The developed LC-MS/MS method is a candidate for a future standard method for folate analysis in food.

High hydrostatic pressure treatments trigger de novo carotenoid biosynthesis in papaya fruit (Carica papaya cv. Maradol).

High hydrostatic pressure (HHP) processing is a non-thermal technology reported to increase desirable metabolites in plant foods. This work evaluated changes in carotenoid accumulation in fresh-cut papaya fruit as affected by HHP treatment (50-400 MPa for 3-60 min) and during subsequent storage at 4 °C; simultaneously, transcriptional activities of carotenoid biosynthetic genes and oxidative stress markers were evaluated. LC-MS analyses revealed that HHP treatment increased carotenoid precursors and carotenes contents following processing and storage: lycopene levels increased up to 11-fold compared to the non-treated samples, and H2O2 and lipid peroxidation were concomitantly increased. qRT-PCR of intact RNA showed that the amount of phytoene desaturase transcripts increased after HHP treatment, and that they were correlated with carotene accumulation. This is the first study to show that HHP treatment triggers de novo carotenoid biosynthesis, which is regulated at the transcriptional level, possibly by inducing oxidative stress signaling in fruit tissue.

MTHFD1 controls DNA methylation in Arabidopsis.

DNA methylation is an epigenetic mechanism that has important functions in transcriptional silencing and is associated with repressive histone methylation (H3K9me). To further investigate silencing mechanisms, we screened a mutagenized Arabidopsis thaliana population for expression of SDCpro-GFP, redundantly controlled by DNA methyltransferases DRM2 and CMT3. Here, we identify the hypomorphic mutant mthfd1-1, carrying a mutation (R175Q) in the cytoplasmic bifunctional methylenetetrahydrofolate dehydrogenase/methenyltetrahydrofolate cyclohydrolase (MTHFD1). Decreased levels of oxidized tetrahydrofolates in mthfd1-1 and lethality of loss-of-function demonstrate the essential enzymatic role of MTHFD1 in Arabidopsis. Accumulation of homocysteine and S-adenosylhomocysteine, genome-wide DNA hypomethylation, loss of H3K9me and transposon derepression indicate that S-adenosylmethionine-dependent transmethylation is inhibited in mthfd1-1. Comparative analysis of DNA methylation revealed that the CMT3 and CMT2 pathways involving positive feedback with H3K9me are mostly affected. Our work highlights the sensitivity of epigenetic networks to one-carbon metabolism due to their common S-adenosylmethionine-dependent transmethylation and has implications for human MTHFD1-associated diseases.

Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB).

Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molecules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-products. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK) in many sequenced bacterial genomes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that overexpression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxymethylpterin and other pterins in cells and medium, and-unexpectedly-to a 46% increase in total folate content. In silico modeling of the folate biosynthesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynthesis by folates or their damage products.

Metabolic engineering of folate and its precursors in Mexican common bean (Phaseolus vulgaris L.).

Folate (vitamin B9) deficiency causes several health problems globally. However, folate biofortification of major staple crops is one alternative that can be used to improve vitamin intakes in populations at risk. We increased the folate levels in common bean by engineering the pteridine branch required for their biosynthesis. GTP cyclohydrolase I from Arabidopsis (AtGchI) was stably introduced into three common bean Pinto cultivars by particle bombardment. Seed-specific overexpression of AtGCHI caused significant increases of up to 150-fold in biosynthetic pteridines in the transformed lines. The pteridine boost enhanced folate levels in raw desiccated seeds by up to threefold (325 µg in a 100 g portion), which would represent 81% of the adult recommended daily allowance. Unexpectedly, the engineering also triggered a general increase in PABA levels, the other folate precursor. This was not observed in previous engineering studies and was probably caused by a feedforward mechanism that remains to be elucidated. Results from this work also show that common bean grains accumulate considerable amounts of oxidized pteridines that might represent products of folate degradation in desiccating seeds. Our study uncovers a probable different regulation of folate homoeostasis in these legume grains than that observed in other engineering works. Legumes are good sources of folates, and this work shows that they can be engineered to accumulate even greater amounts of folate that, when consumed, can improve folate status. Biofortification of common bean with folates and other micronutrients represents a promising strategy to improve the nutritional status of populations around the world.

Folate levels and polyglutamylation profiles of papaya (Carica papaya cv. Maradol) during fruit development and ripening.

Folates are essential micronutrients for humans, and their deficiency causes several detrimental effects on human health. Papaya fruit is an important natural source of some micronutrients. This paper presents a first complete characterization of folate derivatives accumulated in cv. Maradol papaya during fruit development and ripening processes. During postharvest ripening, the fruit accumulated up to 24.5% of the daily folate recommended dietary allowance (RDA) for an adult in a 1 cup (145 g) portion. Tetrahydrofolate (THF) and 5-methyl-THF were the predominant folate classes observed. Surprisingly, an unusually long polyglutamylation profile of tentatively up to 17 glutamates linked to 5-methyl-THF was detected; to the authors' knowledge, this very long polyglutamyl tail has not been reported for any organism, and it is probably characteristic of this plant species. This polyglutamylation degree changed throughout fruit development and ripening, showing the largest differences at the onset of ripening. This work raises questions about the functional role of folate derivatives in fruit development.

Kinetic characterization of a glycoside hydrolase family 44 xyloglucanase/endoglucanase from Ruminococcus flavefaciens FD-1.

Two forms of Ruminococcus flavefaciens FD-1 endoglucanase B, a member of glycoside hydrolase family 44, one with only a catalytic domain and the other with a catalytic domain and a carbohydrate binding domain (CBM), were produced. Both forms hydrolyzed cellotetraose, cellopentaose, cellohexaose, carboxymethylcellulose (CMC), birchwood and larchwood xylan, xyloglucan, lichenan, and Avicel but not cellobiose, cellotriose, mannan, or pullulan. Addition of the CBM increased catalytic efficiencies on both CMC and birchwood xylan but not on xyloglucan, and it decreased rates of cellopentaose and cellohexaose hydrolysis. Catalytic efficiencies were much higher on xyloglucan than on other polysaccharides. Hydrolysis rates increased with increasing cellooligosaccharide chain length. Cellotetraose hydrolysis yielded only cellotriose and glucose. Hydrolysis of cellopentaose gave large amounts of cellotetraose and glucose, somewhat more of the former than of the latter, and much smaller amounts of cellobiose and cellotriose. Cellohexaose hydrolysis yielded much more cellotetraose than cellobiose and small amounts of glucose and cellotriose, along with a low and transient amount of cellopentaose.