The hyperthermophilic archaeon, Pyrococcus furiosus depends on the element tungsten for growth since tungsten-containing enzymes such as aldehyde ferredoxin oxidoreductase (AOR) are key to its metabolism. Crystal structure analysis of the tungsten cofactor in AOR indicated that the tungsten cofactor exists as part of a tricyclic pterin moiety analogous to molybdopterin cofactor present in molybdoenzymes such as nitrate reductase. Molybdopterin cofactor synthesis has been well characterized in Escherichia coli with the identification of at least 14 genes that participate in this process. Analysis of the P. furiosus genome revealed that it has homologs to all cofactor synthesis genes except for modE, a transcriptional regulator of molybdopterin cofactor synthesis, and mogA, a putative molybdo-chelator. Two of the molybdenum cofactor biosynthesis genes, moeA and moeB, involved in activation of molybdenum and in donation of sulfur to the pterin ring structure, respectively, each have two homologs in P. furiosus (MoeA, 45%, MoeA2, 44%, MoeB, 50%, and MoeB2, 47% similar to E. coli MoeA and MoeB respectively). The MoeA and MoeB homologs were targeted for initial functional activity studies to determine if they participate in cofactor formation. The activity studies entailed complementing E. coli strains mutant in moeA or moeB with recombinant P. furiosus homologs in an in vitro system and assaying for restoration of the molydoenzyme nitrate reductase (NR) activity. Partial complementation of defects in E. coli moeA and moeB were observed for assays including P. furiosus MoeA2 and MoeB2 which supported 13.1 nmole NO2-?min-1?mg-1 (10µg MoeA2) and 19.6 nmole NO2-?min-1?mg-1 (100µg MoeB2) activity respectively. These specific activities represent 10.1% and 15.1% of wild type E.coli nitrate reductase activity (130 nmole NO2-?min-1?mg-1) Only negligible restoration of nitrate reductase activity was observed when P. furiosus MoeA or MoeB was included in the assay, with specific activities 0.16 nmole NO2-?min-1?mg-1 (1µg MoeA) and 0.74 NO2-?min-1?mg-1 (50µg MoeB). Partial complementation of the E. coli moeA mutant was also observed for in vitro trimethyl amine oxide (TMAO) reductase assays where 10 g MoeA2 supported a specific activity of 40.97 nmole TMAO reduced?min-1?mg-1 and 10 g MoeB2 supported a specific activity of 6.3 nmole TMAO reduced?min-1?mg-1 compared to 2,374 nmole TMAO reduced?min-1?mg-1 for the wild type E. coli. When TMAO assays were conducted in the presence of tungsten rather than molybdenum, the wild type E. coli had a specific activity of 1,297 nmole TMAO reduced-?min-1?mg-1. E. coli moeA mutant had an activity of 2.07 nmole TMAO reduced -?min-1?mg-1 when supplemented with 10 g MoeA2 and the E. coli moeB mutant supported 6.01 nmole TMAO reduced-?min-1?mg-1 when supplemented with 100 g MoeB2. The partial nature of the complementation seen in these studies is likely due in part to the use of sub optimal assay temperatures (37C), as required for E. coli NR, well below the optimum temperature of 95C seen for most P. furiosus enzymes. Nevertheless, these complementation assays demonstrate that P. furiosus MoeA2, and MoeB2 homologs likely function as MoeA and MoeB in tungsten cofactor synthesis in P. furiosus.
| Identifer | oai:union.ndltd.org:NCSU/oai:NCSU:etd-11202002-162819 |
| Date | 20 November 2002 |
| Creators | Malotky, Erica Louise |
| Contributors | James Brown, Stephen J. Libby, Amy Grunden |
| Publisher | NCSU |
| Source Sets | North Carolina State University |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://www.lib.ncsu.edu/theses/available/etd-11202002-162819/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to NC State University or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
Page generated in 0.0018 seconds