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44382-G4
Thiamine-Dependent Decarboxylation Reactions in Methanogenic Coenzyme M Biosynthesis

David E. Graham, University of Texas (Austin)

Methanogens produce more than 400 Tg of methane each year, corresponding to 80% of global methane production from all sources. This methane is a valuable energy source, as well as a greenhouse gas. To develop new ways of controlling methanogenesis, we are elucidating the biosynthetic pathways for two coenzymes that these archaea require to produce methane. Coenzyme M (CoM; 2-mercaptoethanesulfonate) is the terminal methyl carrier. Coenzyme B (CoB; containing 7-mercaptoheptanoyl-threonine phosphate) reduces the methyl-CoM thioether bond in a reaction catalyzed by the methyl-CoM reductase enzyme. Treatments that block the biosynthesis of CoM or CoB could be selective inhibitors of methanogenesis.

Figure 1. Biosynthesis of CoM in the Methanococcales (A) and the Methanosarcinales (B).

We previously identified the pathway that Methanocaldococcus jannaschii uses to make CoM (Fig. 1A). The third enzyme in this pathway uses a thiamine pyrophosphate (TPP) coenzyme to catalyze the decarboxylation of sulfopyruvate, producing sulfoacetaldehyde. We have developed a system to express and purify soluble sulfopyruvate decarboxylase enzyme (ComDE) from the methanogen Methanosarcina acetivorans. We have shown that high TPP levels are required for ComDE activity in the absence of dithionite. Ongoing studies will determine whether TPP acts as a cofactor or substrate in these reactions, and why this enzyme is inactivated by oxygen.

The genome sequence of Methanosarcina acetivorans has no homologs of the comA, comB or comC genes that we identified in M. jannaschii. In this project, we have recently identified a new pathway for CoM biosynthesis in this microorganism that uses phosphoserine to make cysteate, which can be transaminated to produce sulfopyruvate (Fig. 1B). From this methanogen, we have cloned and expressed a novel enzyme, cysteate synthase, that evolved from the threonine synthase enzyme. This enzyme catalyzes the elimination of phosphate from phosphoserine and the nucleophilic addition of sulfite to a dehydroalanine intermediate, forming L-cysteate. Mass spectral data and phosphate analyses confirm this activity. We previously described the methanogen aspartate aminotransferase that efficiently produces sulfopyruvate from cysteate.

These experiments demonstrated that sulfopyruvate biosynthesis evolved convergently in the Methanococcales and in the Methanosarcinales lineages. Consequently, the sulfopyruvate decarboxylase protein is the best target for designing a broad-spectrum inhibitor of methanogen CoM biosynthesis.

Figure 2. Proposed pathway for the biosynthesis of HS-HTP and an extended form of CoB.

In contrast to CoM, CoB has only been found in methanogens. The structure of CoB was originally reported to be 7-mercaptoheptanoylthreonine phosphate (HS-HTP) (Fig. 2A). A subsequent report identified a UDP-disaccharide headgroup bound to HS-HTP through a labile phosphoanhydride bond (Fig. 2B). In this project, we identified and characterized all of the enzymes from Methanococcus maripaludis that are required to make the nucleotide-sugar precursors for this headgroup: UDP-N-acetylglucosamine (GlcNAc) and UDP-N-acetylmannosaminuronate (ManNAcA). These enzymes will be valuable tools to make substrates for future studies of CoB biosynthesis. Other enzymes identified in this project have already proven useful for the analytical determination of UDP-N-acetylglucosamine concentrations in cells.

The heptanoate moiety of CoB is produced by a series of 2-oxoacid elongation reactions, which extend 2-oxoglutarate to 2-oxosuberate (Fig. 2A). The three enzymes that catalyze these reactions evolved from the isopropylmalate pathway that most organisms use for leucine biosynthesis. We have identified and characterized the two hydro-lyase enzymes from M. jannaschii that have isopropylmalate isomerase (IPMI) and homoaconitase (HACN) activities. Through this project, we identified the first homoaconitase enzyme that catalyzes both half-reactions –completely converting (R)-homocitrate to (2R,3S)-homoisocitrate. This iron-sulfur protein efficiently binds all of the different chain length analogs that are intermediates in the three cycles of 2-oxoacid elongation shown in Fig. 2A.

PRF funding for this project has benefitted a number of graduate and undergraduate students, as well as one ACS Project SEED high school student. By providing supplies and stipends this grant has helped these students pursue independent research in microbial biochemistry and develop skills in problem solving and experimental design. This grant has provided valuable startup funds to the PI, who has garnered competitive grants from NIH and NSF based on preliminary data from this work.

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