After we knew that all proteins had been extracted properly, our next step was to test their functionality. Therefore, we used the plate-reader “Tecan infinite® 200” and the program “Tecan i-control, 18.104.22.168”. For all enzyme reactions, we used room temperature to meet the physiological conditions of these plant enzymes. Values were plotted to show the absorption of the main substrate before and after the addition of the enzyme or water, respectively (Figures 2-3 as well as 5-6, also see Final Discussion). Also all of them showed only strong activity within the first hour, we performed over-night enzyme activity assays to reach the final end point. To verify the reaction product, we used the HPLC (high performance liquid chromatography) ”LaChrom Ultra”
in combination with the MicroToFQ mass spectrometer
. The combination of these separation systems allowed us to separate the substances of the reaction mixtures, analyze their molecular weight and compare them with standards. For our purposes, we used parameters for the MicroTofQ like in (Ruwe et al., 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 acetonitril 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. Since our main goal was to produce iso-GMP or iso-Guanosine using the purified enzymes of Croton tiglium
, we focused on the main promising candidate enzymes: both iso-forms of GMPS ( BBa_K220160
) and CDA( Part BBa_K220162
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 used six replicates with 196 µL of the mixture and measured it for about 20 min (measurement all 30 sec) with the Tecan infinite® 200. 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.
As it can be seen in Figure 2, the absorption of cytidine at 282 nm began to continuously decrease after the addition of the cytidine deaminase, whereas the absorption remained more or less constant when only water was added. With these results, the activity of our extracted cytidine deaminase could be proven.
After confirming general activity of CDA, 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:
Afterwards we set up new activity assays, using 196 µL of the reaction mixture in six of the well plate’s holes. After measuring the absorbance at 282 nm, we added 4 µL of either water or the enzyme (6 µg) to three biological replicates each, continuing the (previous) measurements for about an hour.
The reaction of the cytidine deaminase with xanthosine showed diverse results (Figure 3). Here, also a slight decrease of the xanthosine concentration could be seen, which, however, was not significant.
Figure (3): Enzyme activity assay for the reaction of the cytidine deaminase with xanthosine as a substrate.The reaction was set up at room temperature, 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 difference was observed.
The HPLC-MicroTofQ Measurements could only make up the xanthosine and various other substances. However, there were no significant masses and peaks for guanosine or iso-guanosine. (Figure 4)
Figure (4): HPLC-MicroTofQ measurement for the products of the reaction of CDA with xanthosine. Measurement at 40 °C. Even if many different masses could be detected, none of these could be matched to guanosine or iso-guanosine. For these, a peak should be at about 282 g/mol.
So, with only a slight decrease of the absorbance and no detectable products in the HPLC, it seems reliable that there is only a very small amount of xanthosine converted to isoguanosine, since the reaction is not specific to the CDA and thus rare. However, supplementary tests and experiments with different reaction mixtures would be needed to further analyze it.
We set up the reaction mixture of the two isoforms of the GMPS following a protocol for the enzyme activity assay by Abbott, J., Newell, J., Lightcap, C. et al.(2006). 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
Due to their instability, XMP and ATP were always added freshly. After the samples were set up, we measured them with the Tecan infinite® 200 reader for about 20 minutes at an absorbance of 290 nm. Afterwards, 4 µL of either water or 4 µL (6 µg) of the isoforms of the GMPS (isoform1: BBa_K220160
and isoform 2: BBa_K220161
) were each added to three samples. The measurement was continued for approximately an hour. The activity assays of isoforms 1 and 2 both proved that the GMPS enzymes are working correctly, reducing the amount of XMP in the reaction mixture significantly. Therefore, the absorption at 290 nm decreased a lot after adding the enzyme to the solution of isoform 1 of GMPS, whereas the initial decrease was weaker for the codon-optimized isoform 2. However, both decreased the amount of XMP about the same within the hour in which their reaction was measured. Thus, it can be said that both, isoform 1 and isoform 2 are working as expected (See Figure 5 and Figure 6 for comparison)
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.
As described earlier, it took some time to figure out the right requirements for the HPLC-MicroTofQ measurements, since iso-GMP and GMP have the exact same mass and are thus only separable by their structure. However, with the method chosen in the end, it was possible to identify analytes that seem to represent iso-GMP. Therefore, at first, the general substances within the reaction mix had to be figured out to ensure that only those representing GMP/iso-GMP will be included in the analyses. The general analysis of all substances included showed significant values for all the interesting substrates and products that should be within the reaction mix, including AMP, ADP and ATP, some remaining traces of XMP and of course GMP/iso-GMP (Figures 7 and 8).
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. Measurement at 40 °C. Next to the substrates, ATP and XMP, also resulting substances like AMP and GMP can be found.
We then compared the resulting form of GMP with a GMP-standard (10^-5 diluted solution) and the exact measurements of the HPLC. For both, isoform 2 and isoform 1 of GMPS the peaks of the substance’s flow-through found at the molecular mass of GMP and iso-GMP (approximately 363.22 g/mol, in the graph at approximately 362 g/mol because of the missing H due to the measurement method) were significantly shifted to the right compared to the standard. Thus, the form of GMP that is created with the enzyme reactions of the two isoforms of GMPS and the gene synthesis has to be another form of GMP, most likely iso-GMP. (Figure 9)
In conclusion, we did not only figure out the synthesis pathways in Croton tiglium but could even recreate a part of it, showing that the enzymes expressed in Croton tiglium are more likely to generate a different form of GMP (presumably iso-GMP).
Figure (9): HPLC-MicroTofQ measurement comparing the GMP standard and the reaction products’ flow-through. In red the product of isoform 2 of GMPS. In blue, the one found for isoform 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.