Biochemical and molecular characterization of tetrahydromethanopterin-dependent enzymes in methanogenesis

The biosynthetic pathway of methanogenesis proceeds via several bound intermediates. One of the coenzymes involved in this conversion is tetrahydromethanopterin (H$\sb4$MPT), a structural and biochemical analog of tetrahydrofolate (THF). Enzymes mediating these reactions in Methanobacterium thermoautotrophicum were subjected to biochemical and molecular analysis to determine their relatedness to the analogous THF-dependent reactions in non-methanogens.Purification of the 5,10-methenyl-H4MPT cyclohydrolase (CYC) revealed that this enzyme was distinct and separable from the enzymes catalyzing the previous formyl group transfer reaction and subsequent reduction reaction. This is an important evolutionary consideration with respect to the THF-utilizing enzymes. Extensive biochemical analysis of the cyclohydrolase was performed to characterize the enzyme's analogy to its THF-utilizing counterpart. This work suggested a functional conservation within the pterin binding sites of different pterin enzymes. To investigate this further, the gene encoding the formyl-MFR:H$\sb4$MPT formyltransferase (FTR) was cloned and sequenced. No similarity with other pterin enzymes was found; FTR apparently possesses a novel pterin binding site.FTR was expressed as a catalytically functional enzyme in E. coli. As the first such success with a methane biosynthetic enzyme, this is an encouraging invitation to further manipulations. Surprisingly, ftr is transcribed as a monocistronic message; promoter and terminator sequences were identified.Biochemical analysis of methyltransfer reactions in this organism, compounded with additional observations relating to other enzymes involved in methanogenesis, led to the proposition of a methanogenic metabolon, the methanogenosome, where methanogenic reactions are physically integrated, but separated from enzyme reactions not involved in methanogenesis.U of I OnlyETDs are only available to UIUC Users without author permissio