Bioinformatics Analysis of the De Novo Purine Biosynthesis Pathway in Ammonifex degensii

1. Bioinformatics Analysis of the De Novo Purine Biosynthesis Pathway in Ammonifex degensii Megan Samuel, Rebecca Roggelin, Whitney Dropsey • The pathway for De Novo Purine Biosynthesis involves 15 steps in many organisms, although only 13 steps are essential (Figure 1). Either PurN or Pur T are necessary, but not both. PurK is not essential for function of A. degensii. In our particular pathway PurN, phosphoribosylglycinamide formyltransferase, was present. PurN was chosen because when observing the pathway for closely related organisms, they all used PurN. Figure 1: This image shows the complete pathway for De Novo Purine Biosynthesis. The red circles show the missing pathways and the red lines illustrate which are not necessary. • For 9 of the genes involved in this pathway, the Critica, Glimmer, and RAST gene calls agreed on the start codon position and these were confirmed by Blastp hits to homologs of close relatives. For PurF(917), Critica confirmed that the start codon was correct while Glimmer and RAST said it was 5-13 amino acids off. The remaining genes had possible wrong start codons (Table 1). Table 1. Possible new start codons Gene #Original Start Codon Possible New Start Codon 1819 293043 293038 917 121930 (RAST/Glimmer) 121891 (critica) 289 11306 10718 920 124537 124436 • The 13 genes of interest are found in 3 apparent operons (Table2 , Table 3, Figure 2). The only operon that makes regulatory sense at the moment is the one that includes PurF, PurD, PurM, PurE, PurC, PurB and PurH. Figure 2. This image shows the largest operon in our pathway containing PurF, PurD, PurM, PurE, PurC, PurB, and PurH. • Table 2. Potential Operon containing PurL, PurQ, PurS genes • Gene#Gene Name • PurS: Phosphoribosylformylglycinamidine synthase, PurS subunit • PurL: Phosphoribosylformylglycinamidine synthase, synthetase subunit • Phosphoribosylformylglycinamidine synthase, glutamine a,odptramsferase subunit • After using the BLAST program to compare the sequences of Ammonifex with its closely related organisms, we found poor E Values. For Moorella, the E Value was 1e -08. Because of this reading, we decided to BLAST it against all organisms, giving us a good match with the Dehalococcoides family. It is possible that the purN gene for Ammonifex came in from the outside and is not the same purN as its closely related organisms. One explanation for why Ammonifex and Dehalococcoides may have the same purN pathway is because they live in the same type of thermophilic environment. Figure 3. Phylogenetic tree (cladogram) of PurN protein sequences from Ammonifex, its closest relatives, and other organisms that share the same PurN protein. A multiple sequence alignment was carried out using the program DIALIGN and the cladogram was constructed using the UPGMA distance method. The closely related organism are underlined in red and the organisms with the same PurN pathway are underlined in green. A project of the funded by the U.S. Dept. of Energy Joint Genome Institute and the