Open Access Thesis
Date of Award
Steven M. Firestine
De novo purine biosynthesis is divergent depending upon the organism. In bacteria, yeasts and plants, 5-aminoimidazole ribonucleotide (AIR) is converted to 4-carboxyaminoimidazole ribonucleotide (CAIR) by N5-carboxyaminoimidazole ribonucleotide (N5-CAIR) synthetase (PurK) and N5-CAIR mutase (Class I PurE). In animals, AIR is converted into CAIR by AIR carboxylase (Class II PurE). The distinction makes PurK and Class I PurE good targets in design of antimicrobial drugs. N5-CAIR is chemically unstable with t1/2 of about 0.9 minutes at pH 7.8 and 37 °C. If the production of N5-CAIR is not regulated relative to its conversion to CAIR, ATP utilization can rapidly become non-stoichiometric. One mechanism to study this problem is to investigate how the problem is dealt with under conditions in which N5-CAIR is even more unstable, such as at higher temperature. Thermotoga maritima, originally isolated from geothermal heated marine sediment, is a thermophilic bacterium whose optimal growth temperature is 80°C. The TM0446 and TM0447 genes of Thermotoga maritima encode a phosphoribosylaminoimidazole mutase (PurE) and synthetase (PurK) individually. We cloned genes and expressed both proteins. Enzyme kinetic analyses for each enzyme indicate that these enzymes follow the same pathway as those found in E. coli. Studies of the stoichiometry of the reaction reveal that at elevated temperature, the reactions are stoichiometric as long as the ratio of PurE and PurK is 1:1. When this ratio is lower, ATP consumption becomes non-stoichiometric. We have also examined whether there is a complex formed between these enzymes. Our data indicate that each enzyme is capable of regulating the activity of the other. This suggests that a complex is formed by these two enzymes.
Zhao, Mingyang, "Functional Studies Of 5-Carboxyaminoimidazole Ribonucleotide Synthetase And Mutase From Thermotoga Maritima" (2014). Wayne State University Theses. 361.