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44382-G4
Thiamine-Dependent Decarboxylation Reactions in Methanogenic Coenzyme M Biosynthesis
Specialized anaerobic microorganisms reduce single-carbon compounds to produce all the biologically generated methane on Earth. These methanogens use coenzyme M (CoM; 2-mercaptoethanesulfonate) as the terminal methyl carrier. Although methanogens grow on different one-carbon and acetate substrates, all rely on CoM for methane production. Thus 2-bromoethanesulfonate, a CoM analog, has been the most effective inhibitor of methanogenesis. To identify more enzyme targets for inhibiting methanogenesis (and therefore reducing methane production) we are studying the pathway for CoM biosynthesis in the marine methanogens Methanocaldococcus jannaschii and Methanococcus maripaludis, as well as the freshwater methanogen Methanosarcina acetivorans.
The fourth step in CoM biosynthesis in M. jannaschii and M. maripaludis is catalyzed by sulfopyruvate decarboxylase, a two-subunit, thiamine-dependent enzyme encoded by the comD and comE genes. This protein was previously expressed in Escherichia coli, purified, and shown to be poorly soluble and sensitive to oxygen. It required refolding and dithionite treatment for activity. We have developed a reliable expression system for producing a soluble form of the M. jannaschii ComDE protein. The affinity-purified protein efficiently catalyzes sulfopyruvate decarboxylation in the absence of dithionite, although it requires unusually high levels of thiamine pyrophosphate and anoxic conditions for maximal activity. To identify the cause of this oxygen sensitivity we are using electrospray mass spectrometry to identify side-products or protein modifications that could affect activity.
Testing the function of the sulfolactate decarboxylase in vivo requires a mutant strain that lacks the native comDE genes. To disrupt the comD gene in M. maripaludis, we constructed an integration vector that will produce an in-frame deletion in that organism. DNA flanking the upstream and downstream regions of comD was amplified by PCR from genomic DNA. The fragments were digested at an XbaI site introduced during PCR, and then ligated together and cloned in integration vector pCRPrtNeo to produce vector pSN07. A similar procedure cloned flanking regions of the comA gene into pCRPrtNeo producing vector pDG274. (The comA gene encodes phosphosulfolactate synthase, the first gene in CoM biosynthesis). Both vectors were transformed into M. maripaludis str. 900, and we obtained neomycin-resistant recombinant merodiploids. These cells contain both the mutant and wild-type copies of comD or comA. We are currently selecting for double recombinants that have lost the native genes and become resistant to the purine analog 8-azahypoxanthine. Screening these resistant strains by PCR will differentiate between strains that have reverted to a wild-type genotype, and those that have lost comD and comA genes. These mutants should be CoM auxotrophs, verifying the genes' functions in vivo, and establishing platforms for future complementation experiments by sulfopyruvate decarboxylase variants.
Genome sequences of the Methanosarcinales are missing the first three genes used by the Methanococcales in CoM biosynthesis. We have begun culturing Methanosarcina acetivorans str. C2A cells for experiments to identify the CoM biosynthetic pathway in that organism. Labeled precursor molecules will be added to cells and cell-free extracts to determine incorporation into CoM. We have also identified genes from this organism that could form an alternative pathway to produce sulfopyruvate. These proteins will be heterologously expressed in E. coli and assayed for the predicted activity in vitro.
