Browsing by Subject "Methanogens"
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Item Advances in applications of modern biotechnology methods in methanogens(2014-08) Williams, Bianca Aleceya; Contreras, Lydia M.Methanogens are autotrophic Archaea that produce methane as a product of their anaerobic metabolism. They are the largest producers of global methane, contributing over 60% of the total methane budget each year. Methane is an extremely potent greenhouse gas, with emissions providing the second-largest contribution to historical global temperature increases after carbon dioxide. Methanogens have become extremely important industrially as because they are used in the production of biofuels, as well as in treating industrial waste for industrial processes. This report will focus on those successful genetic methods and modifications that have been developed for methanogens and how they have started to contribute to understanding methanogen biochemistry.Item Biosynthesis of coenzyme M and the catabolism of halogenated aromatic compounds(2012-12) Taylor, Stephanie Michelle 1985-; Whitman, Christian P.Methanogens, members of the domain Archaea, are unique in their ability to reduce carbon substrates to methane. Coenzyme M (CoM) is required in all methanogenic pathways. The biosynthesis of this coenzyme has been well studied in Class I Methanogens, but in Class II Methanogens, such as Methanosarcina acetivorans, little is known. The first step in the biosynthetic pathway might be catalyzed by cysteate synthase (CS), which converts phosphoserine to cysteate by the addition of sulfite. The 46 kDa enzyme was successfully purified from inclusion bodies and characterized. The identity of the product was confirmed by liquid chromatography-mass spectrometry (LC-MS) results as well as by derivatization of the reaction product coupled with high pressure liquid chromatography (HPLC) analysis. Kinetic analysis showed that the enzyme has a K [subscript m] of 0.43 mM for its substrate, phosphoserine, and a K [subscript m] of 0.05 mM for its required nucleophile, sulfite. Four compounds were found to be inhibitors and IC₅₀ values were determined. The results show that CS carries out a new reaction and narrows the gap in our knowledge of Class II Methanogen CoM biosynthesis. In the second part of this dissertation, five enzymes in a newly discovered but poorly characterized pathway for the degradation of halogenated aromatic compounds in Leptothrix cholodnii SP-6 were examined. The pathway reportedly culminates in the production of 2-chloroacetaldehyde, a well-known alkylating agent. In order to determine if 2-chloroacetaldehyde is produced and how the organism survives in its presence, the pathway intermediates are being identified. To this end, 4-oxalocrotonate tautomerase (4-OT), 4-oxalocrotonate decarboxylase (4-OD), vinylpyruvate hydratase (VPH), pyruvate aldolase (PA) and acetaldehyde dehydrogenase (AAD) were cloned, expressed and characterized. 4-OT was found to process the 5-(chloro)-2-hydroxymuconate, but only when the equilibrium was shifted by the addition of 4-OD and VPH. Steady state kinetic analysis showed that while there is a slight decrease in K [subscript m] for the halogenated substrate when compared to the non-halogenated substrate, indicating a difference in binding. There is also a 30-fold decrease in the turnover number, indicating a preference for the non-halogenated substrate. The identity of the product, 5-(chloro)-2-oxo-4-hydroxypentanoate, was verified by ¹H NMR spectroscopy. A stereochemical analysis was also carried out.Item Coenzyme B, amino acid, and iron-sulfur cluster biosynthesis in methanogenic archaea(2009-08) Drevland, Randy Michael; Graham, David E.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.