Coenzyme M is an essential coenzyme for methanogenesis. The proposed biosynthetic pathway consists of five steps, of which the fourth step is catalyzed by sulfopyruvate decarboxylase (ComDE). Disruption of the gene comE by transposon mutagenesis resulted in a partial coenzyme M auxotroph, which grew poorly in the absence of coenzyme M and retained less than 3% of the wild type level of coenzyme M biosynthesis. Upon coenzyme M addition, normal growth of the mutant was restored. Moreover, complementation of the mutation with the wild type comE gene in trans restored full growth in the absence of coenzyme M. These results confirm that ComE plays an important role in coenzyme M biosynthesis. The inability to yield a complete CoM auxotroph suggests that either the transposon insertion failed to completely inactivate the gene or M. maripaludis possesses a promiscuous activity that partially complemented the mutation. 1. Introduction Hydrogenotrophic methanogens, such as Methanococcus maripaludis, possess a specialized metabolism. In a process known as methanogenesis, they reduce CO2 to CH4 using H2 or formate as the electron donor [1]. While other methanoarchaea can use acetate, methylamine, and other methyl-group-containing compounds [1], coenzyme M (CoM), the smallest known organic cofactor, plays a key role as the last methyl carrier in all methanogens [2]. Thus, methane is formed upon the reduction of methyl-CoM with coenzyme B (CoB) as an electron donor by the methyl-CoM reductase. The oxidation of CoB yields a heterodisulfide with CoM (CoM-S-S-CoB), which is reduced to regenerate the thiols by heterodisulfide reductase (Hdr) [3]. Without coenzyme M being present to complete the biosynthesis of methane, the organism is unable to produce the necessary energy for growth. The biosynthetic pathway of coenzyme M (CoM) in Methanocaldococcus jannaschii, an organism closely related to M. maripaludis, is proposed to proceed in five steps. Four enzymes involved in the biosynthesis of CoM have been biochemically characterized [7–10]. In addition, the genes encoding these enzymes have been identified in diverse methanogens, including M. maripaludis. The proposed pathway for the biosynthesis of CoM starts with the sulfonation of PEP by a phosphoenolpyruvate sulfotransferase (ComA). Then, a phosphosulfolactate phosphatase (ComB) hydrolyses phosphosulfolactate, and a dehydrogenase (ComC) oxidizes the (R)-sulfolactate intermediate to form sulfopyruvate. In the fourth step, a sulfopyruvate decarboxylase (ComDE) catalyzes the decarboxylation of sulfopyruvate to form
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