Part:BBa_K2201062

CDA

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. The cytidine deaminase (CDA) seemed to be of immense potential. The CDA, which belongs to the class of hydrolases acting on carbon-nitrogen bonds different from peptide bonds <a href=" http://www.genome.jp/dbget-bin/www_bget?ec:3.5.4.5">(see KEGG)</a> is usually applied to deaminate cytidine to uridine. However, there is also the possibility of the reverse reaction catalyzed by CDA. A reaction from xanthosine to iso-GMP might be possible. The best matching sequence for CDA in the transcriptome assembly encodes 535 amino acids . The putative gene product has a molecular mass of 33.95 kDa.

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 600
Illegal BglII site found at 695
23
COMPATIBLE WITH RFC[23]
25
COMPATIBLE WITH RFC[25]
1000
COMPATIBLE WITH RFC[1000]

Enzyme activity assays

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.

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.

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:

without xanthosine, without CDA
with xanthosine, without CDA
without xanthosine , with CDA
with xanthosine, with CDA

These mixtures were then compared to estimate the absorption rate for xanthosine.

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

We hereby could figure out the absorption rate at which xanthosine can be measured (B) as well as ensure that the peak was independent from the CDA (A). Further on, we could identify the absorbance of CDA at about 254-260 nm (A and C). (Figure 1) )

Figure (1): Results of the analysis of the absorbance of xanthosine at different nanometers.
AAll measurements made with the Tecan infinite® 200 at room temperature. The difference between a mixture with and without xanthosine (red) can clearly be made up at about 282 nm.

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.

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.

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.