Progress 09/01/07 to 08/31/11
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 to 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 were characterized in a previous year. 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. AtFAD/XD and the two individual domains (AtFAD and AtXD) were overexpressed with no HisTag in E. coli; AtFAD/XD and AtFAD formed inlusion bodies and were resolubilized into active proteins. Since the resolubilized recombinant proteins expressed in E. coli had a very low specific activity, we overexpressed AtFAD/XD, AtFAD, and AtXD in the yeast Saccharomyces cerevisiae. The recombinant proteins expressed in yeast are soluble, and have been purified. Their biochemical characterization is in progress. (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 putative FMN hydrolase from pea chloroplasts has been completed. We have accomplished a partial purification of the native protein using hydrophobic interaction (Butyl HP column), hydroxyapatite, gel filtration, and ion exchange (MonoQ column) chromatography. A candidate protein was identified using peptide mass identification. The candidate protein was overexpressed in E. coli as a soluble protein, shown to be a highly active FMN hydrolase, and biochemically characterized. A manuscript describing this work was published in JBC. (3) Investigate the metabolic role of AtFMN/FHy using reverse genetic approaches (overexpression and knock-out). The cDNAs for AtFMN/FHy and its individual domains (AtFMN and AtFHy) were introduced into a Gateway-based plant expression vector, and heterozygous Arabidopsis plants were generated in a previous year. We have screened the antibiotic-resistant plants for the recombinant protein expression using the antibodies obtained from GenScript. We have also selfed the produced heterozygous plants, screened the produced seed to identify homozygous plants, and collected homozygous seed from multiple individual transformants for all three transgenic lines. We are now getting ready to start analyzing the produced transgenics. Selfed T-DNA insertional mutants of AtFMN/FHy produced only heterozygous progeny. Inspection of the siliques revealed that approximately 25% of the seed was aborted, suggesting that AtFMN/FHy is an essential gene. We have rescued the insertional mutants using AtFMN/FHy and AtFMN, showing that the lethal phenotype is due to the missing riboflavin kinase function. 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 AtRibF1, AtRibF2 and AtFAD/XD has been achieved. An FMN hydrolase from chloroplasts has been purified, cloned, and characterized. 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
- 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.
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Progress 09/01/09 to 08/31/10
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 Arabidopsis, and to determine their subcellular localization. Four enzymes are to be investigated: two organellar FAD synthetases (AtRibF1 and AtRibF2), a cytosolic FAD synthetase (AtFAD/XD), and an organellar riboflavin kinase (AtRFK). AtRibF1 and AtRibF2 were characterized in a previous year. 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. AtFAD/XD and the two individual domains (AtFAD and AtXD) were overexpressed with no HisTag in E. coli; AtFAD/XD and AtFAD formed inclusion bodies and were resolubilized into active proteins. Since the resolubilized recombinant proteins expressed in E. coli had a very low specific activity, we overexpressed AtFAD/XD, AtFAD, and AtXD in the yeast Saccharomyces cerevisiae. The recombinant proteins expressed in yeast are soluble, and we are in process of purifying them so that they can be characterized biochemically. (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 putative FMN hydrolase from pea chloroplasts has been completed. We have accomplished a partial purification of the native protein using hydrophobic interaction, hydroxyapatite, gel filtration, and ion exchange chromatography. A candidate protein was identified using nanoLC MS/MS. The candidate protein was overexpressed in E. coli as a soluble protein, and shown to be a highly active FMN hydrolase. A manuscript describing this work is in preparation. (3) Investigate the metabolic role of AtFMN/FHy using reverse genetic approaches. The cDNAs for AtFMN/FHy and its individual domains (AtFMN and AtFHy) were introduced into a Gateway-based plant expression vector, and heterozygous Arabidopsis plants were generated in a previous year. We have screened the antibiotic-resistant plants for the recombinant protein expression using the antibodies obtained from GenScript. We have also selfed the produced heterozygous plants, screened the produced seed to identify homozygous plants, and collected homozygous seed from multiple individual transformants for all three transgenic lines. We are now getting ready to start analyzing the produced transgenics. Selfed T-DNA insertional mutants of AtFMN/FHy produced only heterozygous progeny. Inspection of the siliques revealed that ~25% of the seed was aborted, suggesting that AtFMN/FHy is an essential gene. We have rescued the insertional mutants using AtFMN/FHy and AtFMN, showing that the lethal phenotype is due to the missing riboflavin kinase function, and not due to the missing FMN hydrolase function. Plants rescued with AtFMN will be analyzed to determine the effects of the missing FMN hydrolase function on flavin metabolism. 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
A significant progress towards completing recombinant expression, purification, and biochemical characterization of AtRibF1, AtRibF2 and AtFAD/XD has been achieved. An FMN hydrolase from chloroplasts has been purified, cloned, and characterized. 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
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Progress 09/01/08 to 08/31/09
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 to 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 were characterized in the previous year. 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. We have now overexpressed AtFAD/XD and the two individual domains (AtFAD and AtXD) with no HisTag in E. coli; AtFAD/XD and AtFAD formed inclusion bodies and were resolubilized into active proteins. (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 putative FMN hydrolase from pea chloroplasts has been completed. We have accomplished a partial purification of the native protein using hydrophobic interaction (Butyl HP column), hydroxyapatite, gel filtration, and ion exchange (MonoQ column) chromatography. A candidate protein was identified using peptide mass identification. We are now overexpressing the candidate protein in E. coli to test its catalytic function. (3) Investigate the metabolic role of AtFMN/FHy using reverse genetic approaches (overexpression and knock-out). The cDNAs for AtFMN/FHy and its individual domains (AtFMN and AtFHy) were introduced into a Gateway-based plant expression vector, and heterozygous Arabidopsis plants were generated in the previous year. We have now screened the antibiotic-resistant plants for the recombinant protein expression using the antibodies obtained from GenScript. We have also selfed the produced heterozygous plants, screened the produced seed to identify homozygous plants, and collected homozygous seed from multiple individual transformants for all three transgenic lines. We are now getting ready to start analyzing the produced transgenics. Selfed T-DNA insertional mutants of AtFMN/FHy produced only heterozygous progeny. Inspection of the siliques revealed that ~25% of the seed was aborted, suggesting that AtFMN/FHy is an essential gene. We are now trying to rescue the insertional mutants using AtFMN/FHy and AtFMN. 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 completing recombinant expression, purification, and biochemical characterization of AtRibF1, AtRibF2 and AtFAD/XD has been achieved. Generation of the transgenic plants with increased expression of AtFMN/FHy and its individual domains has been accomplished. Analysis of T-DNA insertion lines AtFMN/FHy 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.
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Progress 09/01/07 to 08/31/08
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 Dr. Francisco J. Sandoval Mr. Joseph Lynch 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.
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