Diversidade e evolução do nono e décimo passos da via biossintética de purinas em procariotos

Abstract

The purine biosynthetic pathway (PBP) starts from phosphoribosyl pyrophosphate (PRPP) which is converted to Inosine monophosphate (IMP) in 10 enzymatic steps, in 4 of these steps the enzymes involved may vary according to the taxonomic group. In Bacteria and eukaryotes the ninth and tenth steps are performed by the PurH enzyme and in the archaeas by the PurP and PurO respectively. PurH has two domains, the AICARFT that performs the ninth step and the IMPCH that performs the tenth step, these domains have recently been found in species of the Archaea Domain as independent unfused genes. The PurP and PurO are analogous to the PurH domains and until then considered signatures of the Archaea Domain. In the first chapter of this work a study of the distribution of the genes related to the last steps of the PBP (purH, purP, purO and the genes coding for AICARFT and IMPCH named purV and purJ, respectively) was carried out on 1405 completely sequenced genomes of the Domains Archaea and Bacteria. Analyzes of comparative genomics indicated that PurH was the evolutionary solution of the Bacteria Domain to catalyze the enzymatic reactions of the last stages of PBP. Homologues of the purO, purV and purJ genes were found in bacterial genomes indicating that these genes are not exclusive to the Archaea Domain. It was also observed that there is a taxonomic pattern for the occurrence of these genes and that in some species they are in the same genomic context as other genes of the PBP. In spite of presenting the same conservation pattern of the primary structure, the PurO homologs of the Domains Archaea and Bacteria are not related and formed distinct groups in the phylogenetic tree, due to this we can infer that the PurO already existed in the common ancestor of all living beings. On the other hand the PurP seems to have arisen after the divergence of archaeas, and its isoforms were originated from events of gene duplication. The objective of the work presented in the second chapter was to understand the evolutionary history of the PurVs and PurJs and their evolutionary relations with the AICARFT and IMPCH domains of PurHs. The comparative enomics were performed with 2735 genomes, although the number of analyzed genomes was almost double, the results observed for the distribution of purH, purV, purJ, purP and purO in the prokaryotic lines were similar to those presented in the first chapter. The amino acid analysis of the active site showed that despite some variations, there are conserved amino acids between AICARFTs/PurVs and IMPCHs/PurJs, including amino acids which have been described as essential for AICARFT activity. According to the literature most of these variations are physically-chemically possible, in addition, they are equivalent between AICARFTs/PurVs and IMPCHs/PurJs. The topology of the phylogenetic tree of the PurHs shows a clear separation between Gram positive and Gram negative suggesting that the current PurHs originated from a single event of gene fusion. Phylogenetic analyzes indicated that archaeal PurHs are phylogenetically related to Gram Negative PurHs, indicating that they were acquired by horizontal transfer. This result is consistent with the hypothesis that the event of gene fusion that originated the PurH occurred in the Bacteria Domain. From the phylogeny we can infer that the PurV probably originated from the ancestral domain that originated PurH and PurJ from breaks of the PurH gene along the evolution and diversification of prokaryotic lines.

CAPÍTULO 1 = The last two steps of the purine biosynthetic pathway may be catalysed by different enzymes in prokaryotes. The genes that encode these enzymes include homologs of purH, purP, purO and those encoding the AICARFT and IMPCH domains of PurH, here named purV and purJ, respectively. In Bacteria these reactions are mainly catalysed by the domains AICARFT and IMPCH of PurH. In Archaea these reactions may be carried out by PurH and also by PurP and PurO, both considered signatures of this domain and analogous to the AICARFT and IMPCH domains of PurH, respectively. These genes were searched for in 1,405 completely sequenced prokaryotic genomes publicly available. Our analyses revealed taxonomic patterns for the distribution of these genes and anticorrelations in their occurrence. The analyses of bacterial genomes revealed the existence of genes coding for PurV, PurJ and PurO, which may no longer be considered signatures of the domain Archaea. Although genetically unrelated, the PurOs of Archaea and Bacteria show a high level of conservation in the amino acids of the active sites of the protein, allowing us to conclude that these enzymes are analogs. The gene purO was present in the common ancestor of all living beings, whereas the gene encoding PurP emerged after the divergence of Archaea and Bacteria and their isoforms originated in duplication events in the common ancestor of phyla Crenarchaeota and Euryarchaeota. The results reported here expand our understanding of the diversity and evolution of the last two steps of the purine biosynthetic pathway in prokaryotes.

CAPÍTULO 2 = The study of the purine biosynthetic pathway (PBP) has contributed significantly to the development of anticancer and , antimicrobial drugs, providing the production of compounds of commercial value from microorganisms, and also in the agricultural area, since their enzymes are directly related to factors such as growth and virulence of phytopathogens. Phosphoribosyl-pyrophosphate (PRPP) is the precursor of the purine pathway, it is converted to inosine monophosphate (IMP) through 10 enzymatic steps. The ninth and tenth steps of PBP are originally performed by PurH in Bacteria and by PurP and PurO in Archaea, respectively. In Chapter 1 we report the existence of PurO, PurV and PurJ in the Bacterial Domain, new enzymes of the PBP that are probably also related to the ninth and tenth passage of the pathway. As discussed earlier, PurV and PurJ are homologous to the AICARFT and IMPCH domains of PurH, however, it is not yet known what their origin would have been if they originated from the ancestral domains that gave rise to PurH or from the PurH gene, due to this the objective of this work was first to better understand the diversity of the genes involved with the ninth and tenth steps of the PBP in the prokaryotic genomes, to find evidence that allow us to infer that PurV and PurJ are active in the pathway and to understand the evolutionary relation between they are the PurH. A comparative genomic was performed with 2735 completely sequenced prokaryotic genomes, searching for the purH, purV, purJ, purP and purO genes. Although the number of genomes is almost double that of chapter 1 the results of comparative genomics were basically the same. An analysis of the amino acids of the active site of the AICARFTs and IMPCHs of all the recovered PurHs and PurVs and PurJs recovered was performed, with it being possible to observe that despite some variations there is conservation of amino acids from the PurV and PurJ active site including amino acids that were described as essential for the activity of the enzyme. The conservation pattern of the primary structure of AICARFTs/PurVs and IMPCH/PurJ is similar, they present basically the same conserved regions. In the PurH tree it was possible to observe a clear division between Gram positive and Gram negative. Of the 32 phyla sampled only 8 were monophyletic for PurH, while monophyly was also observed at lower rates such as family, order or class. The Archaea PurHs were dispersed and probably would have been acquired from lateral gene transfer. The topology that the PurJs tree with the PurHs IMPCH domains took, reflects that the purJs did not have a single origin and probably arose from purH breaks, since some of them clustered with the IMPCHs of the same edge. The PurV on the other hand formed distinct groups in the tree and are not related to the PurHs AICARFTs, indicating that they probably originated from the ancestral domain.