Progress 09/01/08 to 08/31/13
Outputs
Target Audience: Broad scientific community with interest in plant metabolish Changes/Problems:
Nothing Reported
What opportunities for training and professional development has the project provided? Two graduate students and two undergraduate students have been trained. How have the results been disseminated to communities of interest? Two manuscripts have been published. What do you plan to do during the next reporting period to accomplish the goals?
Nothing Reported
Impacts
What was accomplished under these goals?
We have identified the missing pyrimidine reductase on the plant riboflavin biosynthesis pathway, this work has been published. We have also identified two enzymes (At1g79790 and At4g11570) catalyzing dephosphorylation of 5-amino-6-ribitylamino-2,4(1H,3H) pyrimidinedione 5’-phosphate, which complete the riboflavin biosynthesis pathway in plants. We are in the process of generating single and double Arabidopsis knockouts/knockdowns to address the physiological function of these enzymes in plants. The available knockouts for At1g79790 have been screened, and homozygous lines have been identified. These plants have no phenotype when grown under standard greenhouse conditions. Since no knockout lines are available for At4g11570, we are generating knockdowns using the miRNA technology. Wild type Arabidopsis has been transformed to generate single knockdowns, At1g79790 has been transformed to generate the double mutants. Seeds have been collected from the transformed plants, and we are getting ready to start screening the transformed seedlings.
Publications
- Type:
Journal Articles
Status:
Published
Year Published:
2013
Citation:
Hasnain G, Frelin O, Roje S, Ellens KW, Ali K, Guan JC, Garrett TJ, de Cr�cy-Lagard V, Gregory JF 3rd, McCarty DR, Hanson AD. 2013. Identification and characterization of the missing pyrimidine reductase in the plant riboflavin biosynthesis pathway. Plant Physiol. 161:48-56.
- Type:
Journal Articles
Status:
Published
Year Published:
2014
Citation:
Yurgel SN, Rice J, Domreis E, Lynch J, Sa N, Qamar Z, Rajamani S, Gao M, Roje S, Bauer W. 2014. Sinorhizobium meliloti flavin secretion and bacteria-host interaction: Role of the bifunctional RibBA protein. Mol Plant Microbe Interact. Jan 3. [Epub ahead of print]
|
Progress 01/01/12 to 12/31/12
Outputs
OUTPUTS: The objective of this project is to increase our knowledge of the metabolism of flavin nucleotides in plants. Towards that goal, we are investigating enzymes that synthesize and hydrolyze FMN and FAD in the model plant species, Arabidopsis. An enzyme with FAD synthetase activity, AtFAD/XD was cloned, overexpressed in E. coli as AtFAD/XD-HisTag inclusion bodies, and successfully re-solubilized in the last reporting period to yield enzymatically active protein. Because this specific activity was low, and we could not detect enzyme activity that would be associated with the XD domain of unknown function, we pursued protein expression in yeast. Yeast strains overexpressing tagged yeast sequence homologs of AtFAD (ScFAD) and AtXD (Ymr178w) were thus also purchased. ScFAD and Ymr178w expression in these strains was checked using antibodies to the tag. Recombinant ScFAD was purified using Ni-NTA affinity resin; the purified recombinant enzyme was highly active. Subsequently, AtFAD/XD and its individual domains were overexpressed in yeast as soluble proteins, and purified. Ymr178w from yeast was also overexpressed and purified. Biochemical characterization of these enzymes is now in progress. We found that Ymr178w is an active FAD pyrophosphatase that requires potassium ions for activity. Biochemical characterization of this enzyme and determination of the impact of its inactivation on yeast cells have been completed, and a manuscript is in preparation. The FAD synthetase activity of AtFAD/XD has also been characterized. We found that this activity depends on zinc, and does not require the XD domain. The XD domain, which is homologous to Ymr187w, is still under investigation. We have determined that chloroplasts contain three hydrolases capable of catalyzing the missing (in all organisms!) dephosphorylation step on the riboflavin biosynthesis pathway. We recombinantly expressed in E. coli all three enzymes. Scaled-up purification of these enzymes has been completed and the biochemical characterization is in progress. In collaboration with Andrew Hanson, we identified and characterized the missing pyrimidine reductase in the plant riboflavin biosynthesis pathway. PARTICIPANTS: Dr. Renu Rawat, Dr. Francisco J. Sandoval, Mr. Joseph Lynch, Ms. Na Sa TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant progress towards completing recombinant expression, purification, and biochemical characterization of AtFAD/XD has been achieved. Characterization of enzymes catalyzing the missing dephosphorylation step on the riboflavin biosynthesis pathway is in progress. This research is bringing important new insights into how plants synthesize and hydrolyze flavins.
Publications
- Qiu Y., J.Xi, L.Du, S.Roje, B.W.Poovaiah 2012. A dual regulatory role of Arabidopsis calreticulin 2 in plant innate immunity. Plant Journal. 69(3):489-500.
- Hasnain G., O.Frelin, S.Roje, K.W.Ellens, K.Ali, J.C.Guan, T.J.Garett, V.de Crecy-Lagard, J.F.Gregory, D.R.McCarty, A.D.Hanson 2012. Identification and characterization of the missing pyrimidine reductase in the plant riboflavin biosynthesis pathway. Plant Physiology. 161(1):48-56.
- Hung C.Y., L.Fan, F.Kittur, K.Sun, J.Qiu, S.Tang, B.M.Hilliday, B.Xiao, K.O.Burkey, L.P.Bush, M.Conckling, S.Roje, J.Xie 2012. Alteration of the alkaloid profile in genetically modified tobacco reveals a role of methylenetetrahydrofolate reductase in nicotine N-demethylation. Plant Physiology. Epub ahead of print.
- Fernie A.R., H.Bauwe, M.Eisenhut, A.Florian, D.T.Hanson, M.Hagemann, O.Keech, M.Mielewczik, Z.Nikoloski, C.Peterhansel, S.Roje, R.Sage, S.Timm, S.Von Cammerer, A.P.Weber, P.Westhoff 2012. Perspectives on plant photorespiratory metabolism. Plant Biology. Epub ahead of print.
|
Progress 01/01/11 to 12/31/11
Outputs
OUTPUTS: The objective of this project is to increase the knowledge of the metabolism of flavin nucleotides in plants. Towards that goal, we are investigating enzymes that synthesize and hydrolyze FMN and FAD in the model species, Arabidopsis. An enzyme with FAD synthetase activity, AtFAD/XD was cloned, overexpressed in E. coli as AtFAD/XD-HisTag inclusion bodies, and successfully re-solubilized to yield enzymatically active protein in a previous year. Because the specific activity of the resolubilized enzyme was low, and we could not detect enzyme activity that would be associated with the XD domain of unknown function, we pursued protein expression in yeast. In a previous year, yeast strains overexpressing tagged yeast sequence homologs of AtFAD (ScFAD) and AtXD (Ymr178w) were purchased from a stock center. ScFAD and Ymr178w expression in these strains was checked using antibodies to the tag. The recombinant ScFAD was purified using Ni-NTA affinity resin. The purified recombinant enzyme was highly active. Subsequently, AtFAD/XD and its individual domains were overexpressed in yeast as soluble proteins, and purified. Biochemical characterization of these enzymes is now in progress. We have determined that chloroplasts contain an FMN hydrolase belonging to the haloacid dehalogenase family of enzymes. We recombinantly expressed in E. coli all haloacid dehalogenases containing putative organellar transit peptides we could identify in the Arabidopsis genome, and assayed them for FMN hydrolase activity. We found that the enzyme encoded by the gene, At1g79790, is a highly active and very specific FMN hydrolase. This enzyme was biochemically characterized, and a manuscript describing this work has been published in JBC. We also recombinantly expressed the haloacid dehalogenases putatively localized in the cytosol. Scaled-up purification of these enzymes for biochemical characterization is in progress. PARTICIPANTS: Dr. Renu Rawat, Dr. Francisco J. Sandoval, Mr. Joseph Lynch TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
A significant progress towards completing recombinant expression, purification, and biochemical characterization of AtFAD/XD has been achieved. A novel FMN hydrolase from chloroplasts has been identified and characterized. This research is bringing important new insights into how plants synthesize and hydrolyze FMN and FAD.
Publications
- Rawat, R., F.J.Sandoval, Z.Wei, R.Winkler, and S.Roje. 2011. An FMN Hydrolase of the Haloacid Dehalogenase Superfamily Is Active in Plant Chloroplasts.. The Journal of Biological Chemistry. 286(49):42091-8.
|
Progress 01/01/10 to 12/31/10
Outputs
OUTPUTS: The objective of this project is to increase the knowledge of the metabolism of flavin nucleotides in plants. Towards that goal, we are investigating enzymes that synthesize and hydrolyze FMN and FAD in the model species, Arabidopsis. An enzyme with FAD synthetase activity, AtFAD/XD, was cloned, overexpressed in E. coli as AtFAD/XD-HisTag inclusion bodies, and successfully re-solubilized to yield enzymatically active protein in the previous year. Because the specific activity of the resolubilized enzyme was low, and we could not detect enzyme activity that would be associated with the XD domain of unknown function, we are now pursuing protein expression in yeast. In the previous year, yeast strains overexpressing tagged yeast sequence homologs of AtFAD (ScFAD) and AtXD (Ymr178w) were purchased from a stock center. ScFAD and Ymr178w expression in these strains was checked using antibodies to the tag. The recombinant ScFAD was purified using Ni-NTA affinity resin. The purified recombinant enzyme was highly active. In this year, AtFAD/XD and its individual domains were overexpressed in yeast as soluble proteins, and we are now in the process of optimizing purification of those proteins. We are also purifying Ymr178w and trying to establish its biochemical function because we think that this would help us determine the function of AtXD. Yeast cells overexpressing Ymr178w have a perturbed ratio of individual flavin species (riboflavin, FMN FAD), suggesting a role of this protein in flavin metabolism. Purification of a riboflavin kinase from Arabidopsis that is immunologicaly distinct from the previously characterized AtFMN/FHy is in progress. We have determined that chloroplasts contain an FMN hydrolase belonging to the haloacid dehalogenase family of enzymes. We recombinantly expressed all haloacid dehalogenases containing putative organellar transit peptides we could identify in the Arabidopsis genome, and assayed them for FMN hydrolase activity. We found that the enzyme encoded by the gene At1g79790 is a highly active and very specific FMN hydrolase. A manuscript is in preparation. PARTICIPANTS: Dr. Renu Rawat, Dr. Francisco J. Sandoval, Mr. Joseph Lynch. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.
Impacts
Significant progress towards completing recombinant expression, purification, and biochemical characterization of AtRibF1, AtRibF2 and AtFAD/XD has been achieved. A novel FMN hydrolase has been identified and characterized. Purification of a novel riboflavin kinase is in progress. Generation of the transgenic plants with increased expression of AtFMN/FHy and its individual domains has been accomplished. Analysis of T-DNA insertion lines supports AtFMN/FHy being an essential gene. This research is bringing important new insights into how plants synthesize and hydrolyze FMN and FAD.
Publications
- No publications reported this period
|
Progress 01/01/09 to 12/31/09
Outputs
OUTPUTS: The objective of this project is to increase the knowledge of the metabolism of flavin nucleotides in plants. Towards that goal, we are investigating enzymes that synthesize and hydrolyze FMN and FAD in the model species Arabidopsis. An enzyme with FAD synthetase activity, AtFAD/XD was cloned, overexpressed in E. coli as AtFAD/XD-HisTag inclusion bodies, and successfully re-solubilized to yield enzymatically active protein in the previous year. Because the specific activity of the resolubilized enzyme was low, and we could not detect enzyme activity that would be associated with the XD domain of unknown function, we are now pursuing protein expression in yeast. Yeast strains overexpressing tagged yeast sequence homologs of AtFAD (ScFAD) and AtXD (Ymr178w) were purchased from a stock center. ScFAD and Ymr178w expression in these strains was checked using antibodies to the tag. The recombinant ScFAD was purified using Ni-NTA affinity resin. The purified recombinant enzyme was highly active. We are now planning to overexpress AtFAD/XD and its individual domains in yeast, and compare the activity levels of the enzymes produced in yeast vs. those produced in E. coli. We are also planning to try establishing the biochemical function of Ymr178w to help determine the function of AtXD. Yeast cells overexpressing Ymr178w have perturbed ratios of individual flavin species (riboflavin, FMN FAD), suggesting a role of this protein in flavin metabolism. Purification of a riboflavin kinase from Arabidopsis that is immunologicaly distinct from the previously characterized AtFMN/FHy is in progress. We have determined that chloroplasts contain an FMN hydrolase belonging to the haloacid dehalogenase family of enzymes. We are planning to recombinantly express all haloacid dehalogenases containing putative organellar transit peptides we could identify in the Arabidopsis genome (17 enzymes). We have obtained the cDNAs for these enzymes from ABRC, and are in the process of inserting them into an expression vector. PARTICIPANTS: Dr. Renu Rawat, Dr. Francisco J. Sandoval, Mr. Joseph Lynch TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Significant progress toward increasing the overall understanding of enzymes that synthesize and hydrolyze FMN and FAD in plants has been achieved. Purification of a novel riboflavin kinase is in progress. This research is bringing important new insights into how plants synthesize and hydrolyze FMN and FAD.
Publications
- Rhee, H.W., S.J.Choi, S.H.Yoo, Y.O.Jang, H.H.Park, R.M.Pinto, J.C.Cameselle, F.J.Sandoval, S.Roje, K.Han, D.S.Chung, J.Suh, and J.I.Hong. 2009. A bifunctional molecule as an artificial flavin mononucleotide cyclase and a chemosensor for selective fluorescent detection of flavins. Journal of the American Chemical Society. 131(29):10107-10112.
|
Progress 01/01/08 to 12/31/08
Outputs
OUTPUTS: The progress accomplished is described under each Specific Aim: (1) Determine how plants synthesize FMN and FAD. The objective is to clone, functionally overexpress, and characterize Rf kinases and FAD synthetases from the model plant Arabidopsis; and determine their subcellular localization and pattern of mRNA expression. Four enzymes are to be investigated under this specific aim. Those enzymes are: two organellar FAD synthetases (AtRibF1 and AtRibF2), a cytosolic FAD synthetase (AtFAD/XD), and an organellar riboflavin kinase (AtRFK). AtRibF1 and AtRibF2 cDNAs were cloned using a PCR-based approach. The recombinant AtRibF1 and AtRibF2 proteins were overexpressed in E. coli, purified, and biochemically characterized. Fluorescence microscopy of GFP-tagged proteins localized AtRibF1 and AtRibF2 in plastids. A low-level expression throughout plant body was shown using digital Northern blot analysis (Genevestigator). A manuscript describing these results was published in JBC. AtFAD/XD was cloned using a PCR-based approach. The His-Tagged protein overexpressed in E. coli formed inclusion bodies. The AtFAD-HisTag inclusion bodies were successfully re-solubilized to yield enzymatically active protein using the iFOLD Protein Refolding System 1 (Novagen). We are now overexpressing AtFAD with no HisTag for the biochemical characterization. (2) Determine how plastids hydrolyze FMN and FAD. Our working hypothesis is that plastids contain a hydrolase specific for FMN and a pyrophosphatase specific for FAD. The objective is to purify, clone, and biochemically characterize these two enzymes. Purification of the FMN hydrolase from pea chloroplasts is in progress. We have accomplished a partial purification of the native protein using hydrophobic interaction (Butyl HP column), chromatofocusing (MonoP column), and ion exchange (MonoQ column) chromatography. We are in the process of setting up a purification scheme that would yield sufficient protein for the N-terminal sequencing. (3) Investigate the metabolic role of AtFMN/FHy using reverse genetic approaches. The objectives are to overexpress this enzyme and its individual domains in the cytosol in Arabidopsis, and to generate Arabidopsis plants with null or reduced activity of this enzyme using the existing T-DNA insertional mutants or RNAi. We will then analyze the generated transgenic plants for changes in the relevant metabolites and mRNA transcripts, and in growth. The cDNAs for AtFMN/FHy and its individual domains (AtFMN and AtFHy) were introduced into a Gateway-based plant expression vector. Arabidopsis plants were transformed using the floral dip method. The transgenic seed has been recovered from the transformed plants at an expected rate (~1% of the seed is antibiotic-resistant) for all three constructs. Antibodies for AtFMN and AtFHy have been received from GenScript, and we are getting ready to screen the antibiotic-resistant plants for the recombinant protein expression using these antibodies. PARTICIPANTS: Dr. Renu Rawat (Postdoctoral Research Associate) Dr. Francisco J. Sandoval (Associate) Mr. Joseph Lynch (Molecular Plant Sciences Graduate Student) TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
Significant progress towards completing recombinant expression, purification, and biochemical characterization of AtRibF1, AtRibF2 and AtFAD/XD has been achieved. This research is bringing important new insights into how plants synthesize and hydrolyze FMN and FAD. Generation of the transgenic plants with increased or reduced expression of AtFMN/FHy and its individual domains will enable determination of the metabolic role of this enzyme.
Publications
- Sandoval, F.J., Y.Zhang, and S.Roje. 2008. Flavin nucleotide metabolism in plants: monofunctional enzymes synthesize FAD in plastids. The Journal of Biological Chemistry. 283:30890--30900.
|
Progress 01/01/07 to 12/31/07
Outputs
1. Cloning and characterization of the putative serine hydroxymethyl transferase from Arabidopsis. AtSHMT1, AtSHMT2, and AtSHMT3 were expressed in E. coli and purified. The expression problems with AtSHMT2 were solved by replacing the first 330 nucleotides of the AtSHMT2 cDNA with a synthetic sequence optimized for expression in E. coli. Biochemical characterization of AtSHMT1, AtSHMT2, and AtSHMT3 is in progress. AtSHMT4 was purified to electrophoretic homogeneity, and its biochemical characterization will start in 2008. 2. Subcellular localization of putative organellar SHMTs in Arabidopsis. Confocal microscopy of Arabidopsis protoplasts transformed with the AtSHMT3-GFP fusion confirmed that AtSHMT3 is localized in plastids. The AtSHMT1-GFP and AtSHMT2-GFP fusion constructs for determining subcellular targeting have been prepared. Confocal microscopy to determine subcellular localization of these proteins will be carried out in 2008. 3. Testing the hypothesis that
plastids engineered to overexpress MS and/or MTHFR will accumulate folate and/or soluble Met. Tissue-culture grown primary transformants overexpressing the plastidial methionine synthase (MS) were screened, but no increase in MS activity compared to the wild type plants was detected. We estimated that, as plastidial MS activity is probably <5% of the total in the wild type plants, most of the MS activity being cytosolic, the transgenics have less than 20-fold increase in the MS activity in plastids. Thus, it was necessary to assay MS activity in isolated chloroplasts from wild type and the transgenics. We couldn't prepare good chloroplasts from the tissue-culture grown plants. Thus, we back-crossed the primary transformants with the wild type, and collected seed from the cross. We are now germinating the seed on MS medium containing kanamycin to select for seedlings containing the transgene. Kanamycin-resistant seedlings are transferred to soil, and grown for use in analyses. We are
producing good quality protoplasts from the soil-grown plants, and preparing subcellular fractions from these protoplasts. Chloroplasts from transgenic plants have up to four times more methionine synthase activity than those from the wild-type plants. Soluble amino acids were determined in leaves of the wild type tobacco and seven nuclear MS transformants. The MS transgenics contain up to two-fold more Met, and up to five-fold less Thr than the wild type plants. This is unusual because a parallel increase in Met and Thr was observed in all previous genetic manipulations of Met biosynthesis. We hypothesize that overexpression of MS in plastids reduces availability of homocysteine for Met and S-adenosylmethionine (SAM) synthesis in the cytosol. As SAM is an activator of threonine synthase, plants producing less SAM are also expected to produce less Thr. To test this hypothesis, we will measure SAM in the transgenic plants and the wild-type. Nuclear and plastidial transformants
overexpressing chimeric methylenetetrahydrofolate reductase (MTHFR) were generated, but no increase in NADPH-dependent MTHFR activity was detected in these plants. Dr. Maliga's lab is now transforming tobacco with E. coli MTHFR.
Impacts
Significant progress towards completing recombinant expression, purification, and biochemical characterization of SHMTs has been achieved. This research will clarify the role of polyglutamylation in regulation of SHMT activity in plants. Analyses of the transgenic plants over-expressing methionine synthase in plastids are beginning to yield new insights into the role of these organelles in regulation of amino acid biosynthesis in plants.
Publications
- Roje, S. 2007. Vitamin B biosynthesis in plants. Phytochemistry. 68(14):1904-21.
- Roje, S. 2007. The Chlamydomonas genome reveals the evolution of key animal and plant functions. Merchant SS, Prochnik SE, Vallon O, Harris EH, Karpowicz SJ, et al.. Science. 318(5848):245-50.
- Cross, J., F.J.Sandoval, and S.Roje. 2007. An HPLC-based fluorometric assay for cobalamin-independent methionine synthase. Analytical Biochemistry. 360(14):157-159.
|
Progress 01/01/06 to 12/31/06
Outputs
Cloning and characterization of the putative SHMTs from Arabidopsis. AtSHMT1 fused to a HisTag has been over-expressed in E. coli. Purification is in progress. AtSHMT2 fused to a HisTag has been over-expressed in E. coli. This construct contains a synthetic sequence encoding the first 330 nucleotides of the AtSHMT2 cDNA because the native sequence was poorly expressed in E. coli. Purification is in progress. AtSHMT3 and AtSHMT4 have been purified to electrophoretic homogeneity. Their biochemical characterization is in progress. AtSHMT5 has been over-expressed in E. coli. Despite the high expression level, no SHMT activity was detected with the monoglutamylated tetrahydrofolate (THF). We will assay this enzyme using the polyglutamylated THF substrates as we synthesize them. We will also try re-activating the enzyme in the presence of plant protein extracts, in case that a factor missing from E. coli is required for its activity. Subcellular localization of putative
organellar SHMTs in Arabidopsis. C-terminal GFP fusions of AtSHMT1 (putatively mitochondrial protein) and AtSHMT3 (putatively plastidial protein) have been introduced into a Gateway vector for transient expression in protoplasts. C-terminal GFP fusion of AtSHMT3 has also been introduced into a binary Gateway vector for stable transformation. Testing the hypothesis that plastids engineered to overexpress MS and/or MTHFR will accumulate folate and/or soluble Met. Nuclear transformants over-expressing the plastidial methionine synthase (MS) have been generated by Dr. Pal Maliga and shipped to the PI's lab for analysis. No plastidial transformants were recovered, suggesting that a high-level expression of plastidial MS is toxic to plants. The nuclear transformants over-expressing the plastidial MS were screened using an HPLC assay we developed. No statistically significant changes in MS activity compared to the wild type plants were detected. As plastidial MS activity is only a minor
fraction (probably <5%) of the total in the wild type plants, most of the MS activity being cytosolic, we estimate that the transgenics have less than a 20-fold increase in the MS activity in plastids. We will isolate chloroplasts from the transgenic plants and the wild-type, and assay MS activity in chloroplasts. Soluble amino acids were measured in leaves of the wild type tobacco and fifteen nuclear MS transformants. Most MS transgenics appear to contain less methionine, threonine, isoleucine, and S-methylmethionine than the wild type plants. Based on the amino acid profile of the transgenics, we hypothesize that MS over-expression in plastids reduces availability of homocysteine for methionine and S-adenosylmethionine synthesis in the cytosol. To test this hypothesis, we will isolate protoplasts from selected transgenic lines and the wild type, fractionate the protoplasts into the organellar and cytosolic fractions, and measure the amino acids in the two fractions. Nuclear and
plastidial transformants over-expressing the chimeric methylenetetrahydrofolate reductase have been generated by Dr. Pal Maliga and shipped to the PI's lab for analysis.
Impacts
A significant progress towards completing recombinant expression, purification, and biochemical characterization of SHMTs has been achieved. This research will clarify the role of polyglutamylation in regulation of SHMT activity in plants. Analyses of the transgenic plants over-expressing methionine synthase in plastids are beginning to yield new insights into the role of these organelles in regulation of amino acid biosynthesis in plants.
Publications
- Roje, S. 2006. S-adenosyl-L-methionine: beyond the universal methyl group donor.. Phytochemistry. 67(15):1686-1698.
|
Progress 01/01/05 to 12/31/05
Outputs
Full-length cDNAs for two cytosolic (AtSHMT4 and -5) and two mitochondrial (AtSHMT1 and -2) SHMTs were obtained from the Arabidopsis Biological Resource Center (ABRC), verified by sequencing, and subcloned into expression vectors. Presence of the soluble recombinant proteins was confirmed by SDS-PAGE for all four enzymes. AtSHMT4 was purified to electrophoretic homogeneity by ion exchange and size exclusion chromatography. Determination of the biochemical properties of the recombinant enzyme is in progress. The Km value of AtSHMT4 for serine in the presence of monoglutamylated tetrahydrofolate was determined using the standard radio assay for SHMT. This assay measures generation of 14C-methylene-tetrahydrofolate from 14C-serine. We determined that the standard radio assay for SHMT is not sufficiently sensitive to determine the Km values of SHMTs for the polyglutamylated folate substrates; as a result, we developed a novel HPLC-based SHMT assay. This new assay measures
formation of glycine from serine. The substrate and product are derivatized into fluorescent adducts using 4-fluoro-7-nitro-2,1,3-benzoxadiazole (NBD-F). The fluorescent amino acid derivatives are then separated by HPLC, and quantified by fluorescence detection. We estimate that this method is at least 100 times more sensitive than the standard radio assay. Pentaglutamylated tetrahydrofolate was synthesized from pteroylpentaglutamic acid, and was purified by ion exchange chromatography. We determined that the synthesized substrate is stable for at least 30 days when stored in a buffer containing 200 mM 2-mercaptoethanol at -80 degC. Full-length cDNAs for plastidial methionine synthase and yeast-plant hybrid MTHFR were sent to Dr. Pal Maliga. Dr. Maligas laboratory made the methionine synthase constructs for plastidial and nuclear transformation. Construction of the vectors for hybrid MTHFR overexpression is in progress. Transgenic calli overexpressing the plastidial methionine
synthase were generated by direct transformation of plastids. Regeneration of the homogeneously transplastomic lines is in progress. Generation of the nuclear transformants overexpressing methionine synthase is also in progress. Because MTHFR requires FAD, we are also investigating biosynthesis of this cofactor in plants. We published a manuscript (Sandoval and Roje 2005) describing cloning and biochemical characterization of riboflavin kinase/FMN hydrolase, a bifunctional enzyme unique to plants. This enzyme catalyzes biosynthesis and hydrolysis of FMN, the direct precursor of FAD.
Impacts
This research will contribute to the body of knowledge required to engineer folate-enriched plants.
Publications
- Sandoval, FJ and Roje, S (2005). An FMN hydrolase is fused to a riboflavin kinase homolog in plants. Journal of Biological Chemistry 280, 38337-38345.
|
Progress 01/01/04 to 12/31/04
Outputs
Full-length cDNAs for the plastidial and nuclear isozymes of serine hydroxymethyltransferase (SHMT) have been cloned by RT-PCR. The full-length cDNAs for two mitochondrial and two cytosolic SHMTs have been ordered from ABRC. Plastidial SHMT has been functionally expressed in E. coli. Biochemical characterization of the recombinant enzyme is in progress. The full-length cDNA for the plastidial isozyme of methionine synthase (MS) has been cloned by RT-PCR. This cDNA, and the cDNA for the hybrid methylenetetrahydrofolate reductase (MTHFR), have been mailed to Dr. Pal Maliga (Rutgers). Dr. Maliga will over-express the two enzymes in plants, and send the transgenic plants to my laboratory for the biochemical analyses to determine whether the transgenic plants are enriched in folates and/or methionine.
Impacts
This project will provide a better understanding of the one-carbon metabolism in plants, and will ultimately lead to the generation of plants enriched in folates and methionine.
Publications
- No publications reported this period
|
|