Methane is a greenhouse gas and a major contributor to climate change. Methanogenic Archaea produce more than 1 billion tons of this gas each year through methanogenesis, the anaerobic reduction of CO₂ to methane. Coenzyme B (CoB) is one of eight coenzymes required for methanogenesis and it is unique to methanogens. Therefore, this coenzyme is a potential target for inhibiting methanogenesis. To further elucidate the CoB biosynthetic pathway, genes from Methanocaldococcus jannaschii were cloned and expressed in an effort to identify the CoB homoaconitase. From this study, the MJ0499-MJ1277 pair of proteins was identified as the methanogen isopropylmalate isomerase involved in leucine and isoleucine biosynthesis. The MJ1003-MJ1271 pair of proteins was characterized as the homoaconitase required for CoB biosynthesis. This enzyme exhibited broad substrate specificity, catalyzing the isomerization of cis-unsaturated tri-carboxylates with [gamma]-chains of 1-5 methylenes in length. Previously characterized homoaconitases only catalyzed half of the predicted reactions in the isomerization of homocitrate. The MJ1003-MJ1271 proteins function as the first homoaconitase described to catalyze the full isomerization of homocitrate to homoisocitrate. Also, the CoB homoaconitase was identified as specific for (R)-homocitrate and cis-unsaturated intermediates, contrary to a previous study that suggested the substrate specificity of this enzyme included (S)-homocitrate and trans-homoaconitate. The M. jannaschii isopropylmalate isomerase and homoaconitase share more than 50% sequence identity and catalyze analogous reactions. Site directed mutagenesis of the MJ1271 protein was used to identify residues involved in substrate specificity. Arg26 of MJ1271 was critical for the specificity of the CoB homoaconitase. Mutation of this residue to the analogous residue in the M. jannaschii isopropylmalate isomerase, Val28, altered the substrate specificity of the homoaconitase to include the substrates of isopropylmalate isomerase. These homologs of aconitase require a [4Fe-4S] cluster for coordinating their respective substrates at the enzyme active site. However, methanogens lack most of the proteins required for iron-sulfur cluster assembly. Therefore, genes homologous to the Salmonella enterica ApbC iron-sulfur scaffold protein were characterized from methanogens. The MMP0704, MJ0283, and SSO0460 proteins from Methanococcus maripaludis, M. jannaschii, and Solfolobus solfataricus, respectively, were identified as scaffold proteins involved in methanogen iron-sulfur cluster biosynthesis. / text
| Identifer | oai:union.ndltd.org:UTEXAS/oai:repositories.lib.utexas.edu:2152/23478 |
| Date | 11 March 2014 |
| Creators | Drevland, Randy Michael |
| Source Sets | University of Texas |
| Detected Language | English |
| Type | Thesis |
| Format | application/pdf |
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