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For all enzyme reactions, we used room temperature to meet the plants preferred growing temperatures in the original botanical garden. After we finished our measurements, we completed our final analysis using the mean value of the three replicates and calculating the standard deviation. They were then plotted into graphs which showed their development before and after the addition of the enzyme or water (Figures 2-3 as well as 5-6, also see Final Discussion). All of them showed activity. | For all enzyme reactions, we used room temperature to meet the plants preferred growing temperatures in the original botanical garden. After we finished our measurements, we completed our final analysis using the mean value of the three replicates and calculating the standard deviation. They were then plotted into graphs which showed their development before and after the addition of the enzyme or water (Figures 2-3 as well as 5-6, also see Final Discussion). All of them showed activity. | ||
We also did over-night enzyme activity assay. However, while they did not show any significant developments after the first hour, the enzyme reactions seemed to be very fast, mainly within the first minutes. | We also did over-night enzyme activity assay. However, while they did not show any significant developments after the first hour, the enzyme reactions seemed to be very fast, mainly within the first minutes. | ||
− | Further, we wanted to show that the reactions brought out the desired products. For this purpose, we used the HPLC (high performance liquid chromatography) “LaChrom Ultra” from VWR (find more information<a href=" https://at.vwr.com/store/product/10032120/hplc-system-chromaster">here</a>) in combination with the MicroToFQ mass spectrometer from <a href=" https://www.bruker.com/de/products/mass-spectrometry-and-separations/lc-ms/o-tof/microtof-focus-ii/overview.html">Bruker</a>) to determine the structure of the substances. The combination of these allowed us to separate the substances of the reaction mixtures, analyze their molecular weight and compare them with standards. Generally, in an HPLC measurement, substances (or sample mixtures) are pumped through a certain separation column containing a stationary phase that interacts with the analytes. The more interaction, the longer the analyte needs to flow through the complete column. The duration of this flow is measured by a detector so that conclusions about the analytes can be made. | + | Further, we wanted to show that the reactions brought out the desired products. For this purpose, we used the HPLC (high performance liquid chromatography) “LaChrom Ultra” from VWR (find more information<a href="https://at.vwr.com/store/product/10032120/hplc-system-chromaster">here</a>) in combination with the MicroToFQ mass spectrometer from <a href="https://www.bruker.com/de/products/mass-spectrometry-and-separations/lc-ms/o-tof/microtof-focus-ii/overview.html">Bruker</a>) to determine the structure of the substances. The combination of these allowed us to separate the substances of the reaction mixtures, analyze their molecular weight and compare them with standards. Generally, in an HPLC measurement, substances (or sample mixtures) are pumped through a certain separation column containing a stationary phase that interacts with the analytes. The more interaction, the longer the analyte needs to flow through the complete column. The duration of this flow is measured by a detector so that conclusions about the analytes can be made. |
In combination with the MicroTofQ system, a mass spectrometer, not only the duration of flow through can be measured but also the molecular mass of the substances can be estimated. In general, mass spectrometers transfer the analytes into their gas form and ionize them. Afterwards, they are accelerated and transferred to the analysis system that then separates them according to their masses. Combined, these two systems can give valuable statements about the substances included in a reaction mixture. For our purposes, we used parameters for the MicroTofQ like in (Ruwe <i>et al.</i>, 2017) with a measurement in negative mode were the masses would be measured subtracting the mass of an H atom. However, since we wanted to differentiate between different forms of substances with the same mass, we had to try additional measurement methods for the HPLC. Eventually, we used the “Zip-pHILIC” column with a length of 150 mm and a diameter of 2.1 mm from Merck. | In combination with the MicroTofQ system, a mass spectrometer, not only the duration of flow through can be measured but also the molecular mass of the substances can be estimated. In general, mass spectrometers transfer the analytes into their gas form and ionize them. Afterwards, they are accelerated and transferred to the analysis system that then separates them according to their masses. Combined, these two systems can give valuable statements about the substances included in a reaction mixture. For our purposes, we used parameters for the MicroTofQ like in (Ruwe <i>et al.</i>, 2017) with a measurement in negative mode were the masses would be measured subtracting the mass of an H atom. However, since we wanted to differentiate between different forms of substances with the same mass, we had to try additional measurement methods for the HPLC. Eventually, we used the “Zip-pHILIC” column with a length of 150 mm and a diameter of 2.1 mm from Merck. | ||
For the mobile phase, we used Ammoniumbicarbonat (pH 9.3) and Actonitril in a ratio of 27 % to 73 %. This was used in isocratic mode with a flow-through of 0.2ml/min. The injection volume was set to 2 µL of the reaction mixture from the corresponding enzyme assay. The separations took place at 40 °C. | For the mobile phase, we used Ammoniumbicarbonat (pH 9.3) and Actonitril in a ratio of 27 % to 73 %. This was used in isocratic mode with a flow-through of 0.2ml/min. The injection volume was set to 2 µL of the reaction mixture from the corresponding enzyme assay. The separations took place at 40 °C. |
Revision as of 21:13, 1 November 2017
Short Summary
RNA extraction
Library Preparation
Sequencing
Table 1: Sequenced tissue-specfic libraries
Tissue | Number of sequenced fragments | |
---|---|---|
Young leaves | 24,704,122 | |
Stem | 20,032,1422 | |
Inflorescence | 22,804,752 | |
Seed | 43,447,889 | |
Root | 28,007,089 |
Data Processing
Transcriptome assembly
Expression quantification
Results
Usage of the Data Generated by the Trinity Assembly
Identification of Candidate Genes
Firstly, there is the guanosine monophosphate synthase (GMPS), an enzyme from the class of ligases that form carbon-nitrogen-bonds with glutamine as an amido-N-donor acceptors (see KEGG for more information). It is also known as Guanosine monophosphate synthetase and is abbreviated with GuaA. GMPS is important for us since it is needed for the amination of XMP (xanthosine monophosphate) to create GMP and possibly iso-GMP in the case of Croton tiglium. Besides, GMPS can be found in many organisms apart from Croton tiglium, including Homo sapiens and E.coli. Comparing the found sequences of the GMPS with the trinity assembly allowed us to figure out two slightly different sequences for it. These sequences have the size of 314 amino acids and molecular mass of 59.46 kDa. For our project, the GMPS is of special interest as it may be able to not only catalyze the reaction of XMP to GMP but also to iso-GMP.
Another interesting enzyme from the purine metabolism is the Inosine monophosphate-dehydrogenase (IMPDH) that matched with three trinity sequences. IMPDH is an enzyme fromthe class of the Oxydoreductases, which are acting on CH-OH groups of donors with NAD+ or NADP+ as acceptors (see KEGG). The different forms of IMPDH found within the trinity assembly have a molecular mass of 53-58 kDa depending on the exact sequence and a length of approximately 500 to 550 amino acids. In the purine metabolism, IMPDH is the catalysator of the synthesis of XMP out of inosine monophosphate (IMP). For C. tiglium it means that it could possibly enable the biosynthesis of an isoform of XMP that might then even be a substrate for the production of iso-GMP.
Further on, the cytidine deaminase seemed to be of immense potential. The CDA, which belongs to the class of hydrolases acting on carbon-nitrogen bonds different from peptide bonds(see KEGG)., is usually used to deaminate cytidine to uridine. However, there is also the possibility of the reverse reaction catalyzed by CDA. For our purposes, we thought of a reaction from xanthosine to iso-GMP. The found amino acid sequence for CDA from trinity assembly is 535 amino acids long and has a molecular mass of 33.95 kDa.
Aside from these enzymes, we thought of the adenylosuccinate synthetase as an interesting aspect of the purine pathway. The ADSS belonging to the class of ligases, which are forming carbon-nitrogen bonds (see KEGG), was recreated from the trinity assembly with one sequence only. This has a molecular weight of 53.32 kDa and a size of 489 amino acids. In Croton tiglium, it is supposed to catalyze the reaction of IMP to adenylosuccinate that will then be further processed into AMP.
Finally, we focused on the enzyme xanthine dehydrogenase(XDH). The XDH will usually convert xanthine into urate that will be further processed afterwards. XDH is an enzyme from the class of oxidoreductases that is acting on CH or CH2 groups with NAD+ or NADH+ as an acceptor (see KEGG), and could even be matched with six sequences of the trinity assembly. It has a molecular mass of 64.12 kDa and a size of 587 amino acids.
Extraction of Enzyme DNA out of the cDNA Library
Protein purification
Estimation of the Protein Concentration
Table (2): Concentrations of the proteins, estimated with Roti® Nanoquant. Multiple values are the result of multiple protein extractions of those proteins due to their importance.
Protein name | Concentration in mg/mL | |
---|---|---|
GMPS iso-form 1 | 4.1775 2.1507 1.4610 |
|
GMPS iso-form 2 | 1.3497 | |
IMPDH form 1 | 4.4007 | |
IMPDH form 2 | 4.1763 | |
ADSS | 4.2616 | |
XDH | 4.1302 1.9349 |
|
CDA | 5,3092 1,8054 1,3881 |
Investigation of enzyme activity
CDA
First, we set up an enzyme activity assay for CDA with cytidine to ensure its activity following the protocol by Robert M. Cohen and Richard Wolfenden from 1971 that stated that the disappearance of cytidine can be measured in relation to the decrease of absorption at 282 nm. Therefore, we set up the following reaction mixture containing 50 mM TRIS-HCl buffer (pH 7.5) and 0.167 mM cytidine as a substrate.We created six identical measurement samples with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan. We then paused the measurement program to add 4 µL (6 µg) of the previously extracted CDA or 4 µL of water to three samples each. Then, we immediately continued the measurement for about an hour.
After the general activity of the CDA was tested, we set up a possible reaction with xanthosine instead of cytidine, all other components being the same. However, since there was no real literature on this reaction, we first had to figure out the absorption rate at which xanthosine can be measured. This was done using a general spectrum analysis of different mixtures, three samples each:
- without xanthosine, without cda
- with xanthosine, without cda
- without xanthosine , with cda
- with xanthosine, with cda
- 1+3: difference between a reaction mixture with and without cda
- 1+2: difference between a reaction mixture with and without xanthosine
- 2+4: difference between no reaction and a possible reaction
Figure (1): Results of the analysis of the absorbance of xanthosine at different nanometers.
All measurements made with the Tecan at room tempreature. The difference between a mixture with and without xanthosine(red) can clearly be made up at about 282 nnm.
Figure (2): Enzyme activity assay for the reaction of the cytidine deaminase with cytidine.The reaction took place at room temperatue. Three biological replicates were used each. After the addition of water, the absorbance at 282 nm stayed the same whereas it decreased after the addition of the CDA.
Figure (3): Enzyme activity assay for the reaction of the cytidine deaminase with xanthosine as a substrate.The reaction was set up at room temperatue, using three biological replicates each. After adding CDA to the reaction mixture, a slight decrease in the absorbance at 282 nm was visible. However, as there is also a very small decrease for the addition of water, no significant statement can be made.
Figure (4): HPLC-MicroTofQ measurement for the products of the reaction of CDA with xanthosine. Reaction conditions as described earlier. Even if many different masses could be found, none of these could be matched to guanosine or iso-guanosine. For these, a peak should be at about 282 g/mol..
GMPS
We set up the reaction mixture of the two iso- forms of the GMPS following a protocol for the enzyme activity assay by Abbott, J., Newell, J., Lightcap, C. et a.l. We also regarded the original paper from 1985 that stated the absorbance at 290 nm for the given amount of XMP within the mixture. For that, we set up the following reaction mixture:- 60 mM HEPES
- 5mM ATP
- 0.2mM XMP
- 20mM MgCL2
- 200mM NH4CL
- 0.1mM DTT
- 0.8mM EDTA
- Filled up with ddH2O
Figure (5): Enzyme activity assay of iso-form 2 of the guanosine monophosphate synthetases.The reaction was set up at room temperature using three biological replicates. A significant decrease in the absorption at 290 nm can be made up after the addition of the synthetized GMPS whereas the negative control with water stays at the same absorption.
Figure (6):Enzyme activity assay of iso-form1 of the guanosine monophosphate synthetases. . Three biological replicates were used. The reaction was set up at room temperature. A significant decrease in the absorption at 290 nm can be made up after the addition of the GMPS whereas the negative control with water stays at the same absorption.
Figure (7): HPLC-MicroTofQ measurement for the substances within the reaction mixture of the fully extracted GMPS. Reaction conditions as described earlier. Next to the substrates, ATP and XMP, also resulting substances like AMP and GMP can be found.
Figure (8): HPLC-MicroTofQ measurement for the substances within the reaction mxture of the GMPS with the synthetized sequence. Reaction conditions as described earlier. Next to the substrates, ATP and XMP, also resulting substances like AMP and GMP can be found.
Figure (9): HPLC-MicroTofQ measurement comparing the GMP standard and the reaction products’ flow-through. In red the product of the gene synthesis. In blue, the one found for iso-form 1 of GMPS, in green the standard. Even though the standard as well as the mixtures contained compounds that have the same molecular mass, they show different behaviors on the HPLC. The ordinary GMP was significantly faster than the one generated in the enzyme reactions. Thus, the form of GMP that results from the reactions is likely to be iso-GMP.
References
- “Cytidine Deaminase from E.coli – Purificarion Properties and Inhibiton by the potential transition state analog 3,4,5,6-tetrahydrouridine” by Robert M. Cohen and Richard Wolfenden, published in “The Journal of Biological Chemistry” on December 25, 1971
- “The Effects of Removing the GAT Domain from E.coli GMP Synthetase“ (Abbott, J., Newell, J., Lightcap, C. et al. Protein J (2006) 25: 483. https://doi.org/10.1007/s10930-006-9032-5)
- “GMP Synthetase " by Howard Zalkin, https://doi.org/10.1016/S0076-6879(85)13037-5)