Known sequences for specific enzymes from related species were subjected to a
uniprot, we used
BLAST search against the transcriptome assembly. We identified several putative pathways for the isoG biosynthesis based on existing databases like
uniprot, we used
KEGG.
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’. 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.
Another interesting enzyme from the purine metabolism is the Inosine monophosphate-dehydrogenase (IMPDH) that matched three sequences in the transcriptome assembly. IMPDH is an enzyme from the 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 encoded by sequences in the transcriptome assembly have a molecular mass of 53-58 kDa and amino acid lengths between 500 to 550. In the purine metabolism, IMPDH is the catalyst of the synthesis of XMP out of inosine monophosphate (IMP). Therefore, it could enable the biosynthesis of an isoform of XMP that might then even be a substrate for the production of iso-GMP.
Furthermore, 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
(see KEGG) 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.
Aside from these enzymes, the adenylosuccinate synthetase (ADSS) could be an interesting candidate. The ADSS belongs to the class of ligases, which are forming carbon-nitrogen bonds
(see KEGG). Only one matching sequence was identified in the transcriptome assembly. The encoded gene product has a molecular weight of 53.32 kDa and a size of 489 amino acids. In
C. tiglium, it is expected to catalyze the reaction of IMP to adenylosuccinate that will then be further processed into AMP.
Finally, we identified the enzyme xanthine dehydrogenase(XDH) as promising candidate. The XDH converts 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. The encoded gene products are expected to have a molecular mass of 64.12 kDa and a size of 587 amino acids.