Summary of Study ST000900
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000626. The data can be accessed directly via it's Project DOI: 10.21228/M8RH5S This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST000900 |
| Study Title | Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function |
| Study Summary | NAD(P)H-hydrate epimerase (EC 5.1.99.6) is known to help repair NAD(P)H hydrates (NAD(P)HX), which are damage products existing as R and S epimers. The S epimer is reconverted to NAD(P)H by a dehydratase; the epimerase facilitates epimer interconversion. Epimerase deficiency in humans causes a lethal disorder attributed to NADHX accumulation. However, bioinformatic evidence suggests caution about this attribution by predicting that the epimerase has a second function connected to vitamin B6 (pyridoxal 5'-phosphate and related compounds). Specifically, (i) the epimerase is fused to a B6 salvage enzyme in plants, (ii) epimerase genes cluster on the chromosome with B6-related genes in bacteria, and (iii) epimerase and B6-related genes are coexpressed in yeast and Arabidopsis. The predicted second function was explored in Escherichia coli, whose epimerase and dehydratase are fused and encoded by the yjeF gene. The putative NAD(P)HX epimerase active site has a conserved lysine residue (K192 in E. coli YjeF). Changing this residue to alanine cut in-vitro epimerase activity by ≥95% but did not affect dehydratase activity. Mutant cells carrying the K192A mutation had essentially normal NAD(P)HX levels, showing that the mutation had very little or no effect on NAD(P)HX repair in vivo. However, these cells showed metabolome changes, particularly in amino acids, that exceeded those in cells lacking the entire yjeF gene. The K192A mutant cells also had lower levels of free pyridoxal 5'-phosphate than wild-type cells. These results provide strong circumstantial evidence that the epimerase has a metabolic function beyond NAD(P)HX repair and that this function involves vitamin B6. |
| Institute | University of California, Davis |
| Department | Genome and Biomedical Sciences Facility |
| Laboratory | WCMC Metabolomics Core |
| Last Name | Fiehn |
| First Name | Oliver |
| Address | 1315 Genome and Biomedical Sciences Facility, 451 Health Sciences Drive, Davis, CA 95616 |
| ofiehn@ucdavis.edu | |
| Phone | (530) 754-8258 |
| Submit Date | 2017-11-14 |
| Raw Data File Type(s) | cdf |
| Analysis Type Detail | GC-MS |
| Release Date | 2017-11-20 |
| Release Version | 1 |
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Project:
| Project ID: | PR000626 |
| Project DOI: | doi: 10.21228/M8RH5S |
| Project Title: | Evidence that the metabolite repair enzyme NAD(P)HX epimerase has a moonlighting function |
| Project Summary: | Damaging enzymatic and chemical side-reactions in all organisms convert NADH and NADPH to their hydrates, NADHX and NADPHX, which exist as R and S epimers [1,2]. The hydrates, which inhibit various dehydrogenases [3,4], are reconverted to NAD(P)H by the sequential actions of NADP-(H)X epimerase (EC 5.1.99.6) and NAD(P)HX dehydratase (EC 4.2.1.93) [5-7] (Figure 1A). Both enzymes occur in all domains of life [5]. The dehydratase is specific for the S form of NAD(P)HX [1] and the epimerase facilitates conversion of the R form to the S form used by the dehydratase [5-7]. Over time, if not reconverted to NAD(P)H, the R and S forms of NAD(P)HX give rise spontaneously to cyclic forms of NAD(P)HX, which are not substrates for the epimerase or the dehydratase [1,5] (Figure 1A). Together, the formation and removal of NAD(P)HX constitute an archetypal example of metabolite damage and repair [8-10]. The functions of the epimerase and the dehydratase are supported by biochemical evidence from mammals, yeast, Escherichia coli, and plants [5-7], and that of the dehydratase is also supported by genetic and metabolomic evidence from Arabidopsis [6]. The in-vivo function of the NAD(P)HX dehydratase protein seems to match its in-vitro enzymatic activity [6], but the situation for the epimerase protein may be more complex, as several discordant ob3 servations suggest. First, as NAD(P)HX epimers equilibrate spontaneously and quite rapidly (t½ = 40 min) in physiological conditions, it is not entirely clear why epimerase activity is needed [1]. Second, while the epimerase is fused to the dehydratase in most prokaryotes, it is fused in plants to pyridoxine/pyridoxamine phosphate oxidase (PPOX), a salvage enzyme for vitamin B6, i.e. pyridoxal 5′-phosphate (PLP) and related compounds [5-7,11,12]. Since protein fusions imply functional relationshipsbetween the partners [13,14], the PPOX fusion points to a B6 connection, in plants at least. Third, and relatedly, epimerase and dehydratase expression are not correlated in Arabidopsis [7]. Fourth, the mammalian epimerase (NAXE) occurs in extracellular compartments (cerebrospinal fluid, urine, and the plasma of septic patients) [15], which the dehydratase does not – although both enzymes occur in the mitochondria and cytosol [16]. Moreover, NAXE can bind apolipoprotein A-1, a plasma protein that is a major component of the high-density lipoprotein (HDL) complex [15]. This binding is proposed to link NAXE to cholesterol transport, atherosclerosis, and angiogenesis [17,18]. To summarize the above, there are reasons to suspect that NAD(P)HX epimerase is more than just an epimerase, and that it has a moonlighting function [19] related to B6. This suspicion has become medically relevant with the discovery that nonsense or missense mutations in the human epimerasegene NAXE (also called AIBP) lead to a neurodegenerative disease that is lethal in infancy [20, 21]. The etiology of this disease – and potential therapies – have so far been conceived solely in terms of loss of NAD(P)HX epimerase activity [20,21]. While the NAXE protein deficiency and cyclic NADHX accumulation observed in fibroblasts from affected individuals [21] are consistent with lack of epimeraseactivity causing the symptoms, they do not prove that this is the case. Nor is it evident why intracellular buildup of NADHX to the reported level of ~5 pmol/mg protein [21], equivalent to ~1 μM [22], would have such a severe impact in humans given that a similar accumulation in Arabidopsis had no discernable consequences [6] and that 1 μM NADHX corresponds to only ~1% of the NADH pool in typical mammalian cells [23]. These considerations led us to use comparative genomic and genetic approaches to explore the possibility that NAD(P)HX epimerase has a moonlighting function. Our results build a strong circumstantial case that such a function exists, and that it involves B6 metabolism. |
| Institute: | University of Florida |
| Department: | Horticultural Sciences |
| Last Name: | Hanson |
| First Name: | Andrew |
| Address: | Gainesville, FL 32611 |
| Email: | adha@ufl.edu |
| Phone: | 352-273-4856 |
Subject:
| Subject ID: | SU000937 |
| Subject Type: | Cells |
| Subject Species: | Escherichia coli |
| Taxonomy ID: | 562 |
| Species Group: | Microorganism |
Factors:
Subject type: Cells; Subject species: Escherichia coli (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment* |
|---|---|---|
| SA052872 | 170123blvsa14_1 | KA pt mut |
| SA052873 | 170123blvsa06_1 | KA pt mut |
| SA052874 | 170123blvsa09_1 | KA pt mut |
| SA052875 | 170123blvsa12_1 | KA pt mut |
| SA052876 | 170123blvsa02_1 | KA pt mut |
| SA052877 | 170123blvsa15_1 | KA pt mut |
| SA052878 | 170123blvsa03_1 | wild type |
| SA052879 | 170123blvsa11_1 | wild type |
| SA052880 | 170123blvsa10_1 | wild type |
| SA052881 | 170123blvsa05_1 | wild type |
| SA052882 | 170123blvsa16_1 | wild type |
| SA052883 | 170123blvsa01_1 | wild type |
| SA052884 | 170123blvsa17_1 | yjeF KO |
| SA052885 | 170123blvsa04_1 | yjeF KO |
| SA052886 | 170123blvsa08_1 | yjeF KO |
| SA052887 | 170123blvsa07_1 | yjeF KO |
| SA052888 | 170123blvsa13_1 | yjeF KO |
| SA052889 | 170123blvsa18_1 | yjeF KO |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO000931 |
| Collection Summary: | Bacterial cultures were grown overnight in M9 minimal medium plus 0.2% glucose and used to inoculate 2 mL of fresh medium to an optical density of 0.05 at 600 nm. Cultures were grown at 37°C with shaking for 5-6 h until optical density reached 1.7 ± 0.1 at 600 nm, then an equivalent of 1 mL culture at an optical density of 2.0 was collected in 1.5-mL Eppendorf tubes, centrifuged at 16,000 × g for 15 s; the pellet was frozen in liquid nitrogen. The harvesting procedure was completed in <30 s. Samples were stored at -80°C. |
| Sample Type: | Cell |
Treatment:
| Treatment ID: | TR000951 |
| Treatment Summary: | A= E. coli 12MG1655 (WT) B = E. coli yjeF gene knock-out C = E. coli point mutant K192A |
Sample Preparation:
| Sampleprep ID: | SP000944 |
| Sampleprep Summary: | 1. Add 0.5mL of extraction solvent to tube, gently pipet to remove all cells, transfer cells to 2mL eppendorf tube. Repeat for a total of 1mL extraction solvent + cells in 2mL eppendorf tube. 2. Add 2 small stainless steel grinding beads to eppendorf tube 3. Use the GenoGrinder to grind for 30 seconds at 1,250 rpm. 4. Centrifuge at 14,000xg for 5 minutes. 5. Transfer supernatant to a fresh 2mL eppendorf tube. 6. Add 1mL of extraction solvent to tube containing cell pellet + beads, and repeat steps 3 and 4. 7. Collect supernatant, and combine with supernatant collected in step 5. Total volume of extracted sample will be approximately 2mL. 8. Dry down 50uL of extracted sample in 1.5mL eppendorf tube for GC-TOF analysis. 9. Store backups in -20 or -80C. |
Combined analysis:
| Analysis ID | AN001466 |
|---|---|
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Agilent |
| Column | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
| MS Type | EI |
| MS instrument type | GC Ion Trap |
| MS instrument name | Varian 210-MS GC Ion Trap |
| Ion Mode | POSITIVE |
| Units | Counts |
Chromatography:
| Chromatography ID: | CH001030 |
| Instrument Name: | Agilent |
| Column Name: | Restek Rtx-5Sil (30m x 0.25mm,0.25um) |
| Column Pressure: | 7.7 PSI (initial condition) |
| Column Temperature: | 50 - 330°C |
| Flow Rate: | 1 ml/min |
| Injection Temperature: | 50°C ramped to 250°C by 12°C/s |
| Sample Injection: | 0.5 uL |
| Oven Temperature: | 50°C for 1 min, then ramped at 20°C/min to 330°C, held constant for 5 min |
| Transferline Temperature: | 230°C |
| Washing Buffer: | Ethyl Acetate |
| Sample Loop Size: | 30 m length x 0.25 mm internal diameter |
| Randomization Order: | Excel generated |
| Chromatography Type: | GC |
MS:
| MS ID: | MS001354 |
| Analysis ID: | AN001466 |
| Instrument Name: | Varian 210-MS GC Ion Trap |
| Instrument Type: | GC Ion Trap |
| MS Type: | EI |
| Ion Mode: | POSITIVE |
| Ion Source Temperature: | 250°C |
| Ionization Energy: | 70eV |
| Mass Accuracy: | Nominal |
| Scan Range Moverz: | 85-500 |
| Scanning Cycle: | 17 Hz |
| Scanning Range: | 80-500 Da |
| Skimmer Voltage: | 1850 |
