| Abstract [eng] |
Natural modified nucleotides are present in various kinds of nucleic acids and are most diverse in tRNA. Functions of the nucleotide modifications span from providing structural stability to implications in the regulatory pathways of the cell. The vast variety of these modifications makes it difficult to elucidate the functions and biosynthesis of every single one of them. Nonetheless, the biosynthetic pathways are now rather well understood, albeit not all of them are deciphered completely. On the other hand, the studies of modified nucleic acid degradation are limited: the biodegradation of modified nucleotides is understood mainly to the point of formation of nucleosides, even their subsequent conversion into heterocyclic bases is seldom described. How exactly these modified heterocyclic bases and carbohydrates that result from cleavage of nucleosides are returned into the metabolism is largely unknown. The studies of nucleotide modifications are very versatile. Therefore, this thesis will have a glimpse at the elaborate biosynthesis of archaeal wyosine derivatives, as well as focus on searching for novel enzymatic activities that could elucidate the biodegradation of modified uridines. The aims of this study were: 1. To demonstrate the methyltransferase activities of aTrm5a proteins from Pyrococcus abyssi and Nanoarchaeum equitans. 2. To create a system for identification of novel enzymes, involved in conversion of modified uracil/uridine derivatives into uracil. 3. To identify enzymes involved in the conversion of 2-thiouracil into uracil. 4. To identify enzymes involved in the conversion of 2'-O-methyluridine into uracil. 5. To identify enzymes capable of deamination of isocytosine into uracil. It was demonstrated that aTrm5a proteins from P. abyssi (PAB2272) and N. equitans (NEQ228) display a dual tRNAPhe:m1G/imG2 methyltransferase activity. While the ability of PAB2272 to catalyse the methylation of guanine yielding 1-methylguanosine (m1G) has been demonstrated previously, the conversion of 4-demethylwyosine (imG-14) into isowyosine (imG2), catalysed by the same enzyme, has been proposed, but not previously demonstrated. The intricate biosynthetic pathways of wyosine derivatives in archaea are further clarified with these results. These findings also illustrate that two joint methylation reactions might become independent during the course of evolution which in turn might lead to the development of sequential metabolic pathways. Novel enzymatic activities were found using a screening strategy that employs E. coli uracil auxotroph and the metagenomic libraries. Nine proposed enzymes were identified. Three novel DUF523s have been assigned a function of desulfuration of 2-thiouracil in vivo for the first time. It is also evident that these proteins exhibit properties associated with Fe-S clusters. A 2'-O-methyluridine hydrolase (RK9NH) has been identified together with an aldolase (RK9DPA) – probably forming a part of an operon that is involved in the degradation of 2'-O-methylated nucleosides. The RK9NH is functional in vivo and in vitro. The RK9NH nucleoside hydrolase could be engineered to enzymatically produce 2'-O-methylated nucleosides that are of great demand as raw materials for production of nucleic acid-based drugs. Moreover, RK9NH nucleoside hydrolase converts fluorinated uridine derivatives into 5-fluorouracil, a well-known cancer therapy drug. Three novel isocytosine deaminases have been identified in vivo. Two of these deaminases (Vcz and URA3) were purified and are functional in vitro: not only isocytosine, but also 8-oxoguanine is a substrate of these enzymes. Moreover, these deaminases act upon 5-fluoroisocytosine, producing 5-fluorouracil. Therefore, RK9NH, Vcz and URA3 could possibly be employed in cancer therapy. |
