The pyrimidine metabolic maps at KEGG were used to trace the pathways of thymidine [5]. The de novo pathway, thymidine kinase converts thymidine to thymidine monophosphate (TMP). In the catabolic direction, TMP is converted to thymidine by the action of 5'-nucleotidase (Fig. 1). Further, conversion of thymidine to thymine by thymidine/pyrimidine phosphorylase. Whereas, nucleoside deoxyribosyltransferase catalyses’ the interconversion of thymidine and thymine.
In the camel thymidine pathway, camels were expected to be devoid of nucleoside deoxyribosyltransferase (Fig. 2). Therefore, there is no interconversion between thymine and thymidine in camel pyrimidine metabolism. Thus, the sole source of thymidine in camels is from TMP by the action of 5'-nucleotidase or potentially by the transport of ready formed thymidine from outside the cells.
In T. evansi, the enzymes 5'-nucleotidase and thymidine/pyrimidine phosphorylase were not confirmed, suggesting interesting differences in thymidine metabolism, compared with camels. TMP cannot be converted to thymidine in T. evansi due to the absence of 5'-nucleotidase. The sole metabolic source of thymidine could be from thymine by the action of nucleoside deoxyribosyltransferase, which is absent in camel (Fig. 3). The list of enzymes of thymidine pathway as well as the predicted content in camel and T. evansi are provided in Tables 1-3.
Camel thymidine/pyrimidine phosphorylase (TP)
Thymidine phosphorylase (EC 2.4.2.4;TP): pyrimidine phosphorylase); thymidine orthophosphate deoxyribosyltransferase; animal growth regulators, blood platelet-derived endothelial cell growth factors; blood platelet-derived endothelial cell growth factor; deoxythymidine phosphorylase; gliostatins; pyrimidine deoxynucleoside phosphorylase; thymidine:phosphate deoxy-D-ribosyltransferase. It is a glycosyltransferase stimulate reversible dephosphorylation of thymidine phosphate supplying thymine and 2-deoxy -D-ribose 1-phosphate. In addition, it catalyzes deoxyribosyltransferase reactions that catalyzed by nucleoside deoxyribosyltransferase in various tissues. The enzyme can also convert thymidine to thymine, which is not used by the trypanosomes [6]. The properties of the phosphorylases differ significantly from prokaryotes to those of the mammalian animals. Giardia lamblia is mainly dependent on salvage synthesis for its pyrimidine requirements. In which, one enzyme of uridine, deoxyuridine and thymidine phosphorylases are responsible for the activities of three enzymes. The enzyme could utilize both uracil and thymine as substrates [7]. Complicated infection with Mycoplasmas reduced the efficiency of anticancer and antiviral nucleoside analogue-based therapies due to the presence of Mycoplasma TP. Impaired activity of TP provoked the elevated uptake and inclusion of deoxyuridine and uracil but thymidine uptake was not affected. Thus, enzymes of Mycoplasma nucleotide synthesis pathway are prospective targets for imminent creation of antibiotics [8]. In Trypanosoma cruzei, a phosphorylase activity was detected but was more specific to uridine phosphorylase without specificity to thymidine or purine phosphorylase [9, 10].
The obtained sequence of camel TP was low quality protein and very short amino acids length compared with human TP. The recorded sequence of camel TP was 202 amino acids, bearing 18.44% similarity rate, compared with 482 amino acids length in human TP (Fig. 4). The same finding applies for the TP sequence in the three camel species, dromedary, Bactrian and ferus camels, in which Camelus ferus showed the shortest length of 162 amino acids (Fig 5). Comparison of TPs from different prokaryotic and eukaryotic sources (Fig. 6) showed a general low similarity rate from 9.5-41.8% among the tested species. Owing to this difference, the camel TPs were forming a monophyletic group that shared common origin with the prokaryotic TPs and not the vertebrate TPs (Fig. 7). The motif search retrieved zero hits by sing the motif finder, pfam and prosite prediction tools, while a phosphorylase domain was predicted by using the NCBI conserved domain search tool.
evansi nucleoside deoxyribosyltransferase (NDRT)
This enzyme was found to be a unique enzyme for T. evansi and not present in camel, suggesting its use as a safe drug target. After searching the gene databases, NDRT could be predicted in some protozoa and bacteria e.g. lactobacillus, Leishmania spp., and Trypanosomes. A previous study investigated the crystal structure of T. brucei NDRT and found that its structure is highly similar to NDRT from Lactobacillus helveticus [11]. In the previous study, several crystal bound compounds were tested against the blood forms of T. brucei and found weak inhibition of parasite growth with IC50 values above 100 µM.
Sequence comparisons between T. evansi, Lactobacillus fecum and Enterococcus fecum NDRTs revealed 17.68-21.21% sequence similarity (Fig. 8). Comparison of T. evansi and T. brucei NDRTs revealed 100% similarity. This may account for sharing the common features in the published structure, function and inhibition.
The models for T. evansi NDRT were built at Swiss-Model server [12]. The model of T. evansi was predicted based on the deposited PDB ID 2a0k, which is the NDRT from T. brucei (Fig. 9). The modeling statistics comprised 99% coverage, 0.6 sequence similarity and 98% sequence identity.