Part:BBa_K2201060

GMPS iso1

Usage and Biology

Croton tiglium (L.), also commonly known as the „Croton oil plant“ is a plant of the family of Euphorbiaceae that was first described in 1753 by Carl Linnaeus. Its origin lays in the south-eastern asian countries such as India or Thailand, where it can grow up to 7m tall. In nature, it occurs in many habitats ranging from 300-1500m height as well as from shrublands to forests. The plant we used was kindly provided by the botanic garden of the Philipps University in Marburg. Seeds of this plant were originally given to the botanical garden Marburg in 1986. They were then provided by the botanical garden Giessen. Besides the fact that all components of croton tiglium are poisonous, it is widely used as an herb of the traditional chinese medicine since 2000 years. There, it found its usage as an oil to treat skin diseases and intoxications and as a laxative. Further, the plant was used against cancerous diseases due to its anti-tumoric effects that were proven in 1994 (Kim et al).

In difference to animals, plants usually have a second metabolism that does not interact with any chemicals needed for primary functions like growth or development. However, the seondary metabolism is still connected to the primary metabolism as it uses some of the substances produces within. The products that emerge from the second metabolism offer a wide range of possible usages for humanity due to their great diversity. However, the pants mostly use them as protection against herbivores and pathogens. Some metabolites are toxic whereas others do attract parasites and predators as a protection against the herbivores. Besides from many other secondary metabolites, croton tiglium produces iso-guanosine as well as iso-guanosine monophosphatase (GMP). In 1932, iso-guanosine was first isolated from croton tiglum (Cherbuliez et al 1932). Today, it also commercially available as crotonoside. Iso-guanosine was object of many reaearches and is found to have a lot of effects onto biological processes including antitumorous effects (Kim et al 1942) For us, one of the main aspects that is interesting on iso-guanosine is its use as an unnatural base within organisms.

As guanosine as well as GMP are generated within the purine metabolism, we identified several possible putative pathways for the isoG biosynthesis. So, 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’. GMPS is needed for the amination of XMP (xanthosine monophosphate) to create GMP and possibly iso-GMP in the case of C. tiglium. Besides, GMPS can be found in many organisms apart from Croton tiglium, including Homo sapiens and E. coli. The transcriptome assembly contained two sequences that displayed a strong similarity to known GPMS encoding sequences. These sequences encode peptides of 314 amino acids and molecular mass of approximately 59.46 kDa. GMPS is a promising candidate, since it may not only be able to catalyze the reaction of XMP to GMP but also to iso-GMP. In nature, there are two isoforms of the GMPS. Within this part, the first isoform can be found.

Sequence and Features

Assembly Compatibility:

10
COMPATIBLE WITH RFC[10]
12
COMPATIBLE WITH RFC[12]
21
INCOMPATIBLE WITH RFC[21]
Illegal BglII site found at 1190
Illegal BglII site found at 1313
Illegal BglII site found at 1523
Illegal BamHI site found at 9
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
INCOMPATIBLE WITH RFC[1000]
Illegal BsaI.rc site found at 755
Illegal BsaI.rc site found at 823
Illegal SapI site found at 1438

Enzyme Activity Assays

To test the functionality of the enzyme, we used the plate-reader “Tecan infinite® 200” and the program “Tecan i-control, 1.10.4.0”. 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.

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: <a href=" https://parts.igem.org/Part:BBa_K2201060"> BBa_K220160</a> and isoform 2: <a href=" https://parts.igem.org/Part:BBa_K2201061">BBa_K220161</a>) 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. Thus, it can be said that isoform 1 is working as expected (See Figure 1 for comparison) https://static.igem.org/mediawiki/2017/7/7f/T--Bielefeld-CeBiTec--GMPSSelf.svg

Figure (1):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.

Product Estimation

To verify the reaction product, we used the HPLC (high performance liquid chromatography) La Chrom Ultra (https://at.vwr.com/store/product/10032120/hplc-system-chromaster) in combination with the MicroToFQ mass spectrometer (https://www.bruker.com/de/products/mass-spectrometry-and-separations/lc-ms/o-tof/microtof-focus-ii/overview.html). 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 including this one.

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 (Figure 2).

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.

We then compared the resulting form of GMP with a GMP-standard (10^-5 diluted solution) and the exact measurements of the HPLC. For 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) was significantly shifted to the right compared to the standard. Thus, the form of GMP that is created with the enzyme reaction of this isoform of GMPS has to be another form of GMP, most likely iso-GMP. (Figure 9)

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 (this), 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.

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).