The anabolic pyruvate oxidoreductase from Methanococcus maripaludis

Abstract

In autotrophic methanogens, pyruvate oxidoreductase (POR) plays a key role in the assimilation of CO(2) and the biosynthesis of organic carbon. This enzyme has been purified to homogeneity, and the genes from Methanococcus maripaludis were sequenced. The purified POR contained five polypeptides with molecular masses of 47, 33, 25, 21.5 and 13 kDa. The N-terminal sequences of four of the polypeptides had high similarity to the subunits commonly associated with this enzyme from other archaea. However, the 21.5-kDa polypeptide had not been previously observed in PORs. Nucleotide sequencing of the gene cluster encoding the POR revealed six open reading frames ( porABCDEF). The genes porABCD corresponded to the subunits previously identified in PORs. On the basis of the N-terminal amino acid sequence, porE encoded the 21.5-kDa polypeptide and contained a high cysteinyl residue content and a motif indicative of a [Fe-S] cluster. porF also had a high sequence similarity to porE, a high cysteinyl residue content, and two [Fe-S] cluster motifs. Homologs to porE were also present in the genomic sequences of the autotrophic methanogens Methanocaldococcus jannaschii and Methanothermobacter thermautotrophicus. Based upon these results, it is proposed that PorE and PorF are components of a specialized system required to transfer low-potential electrons for pyruvate biosynthesis. Some biochemical properties of the purified methanococcal POR were also determined. This unstable enzyme was very sensitive to O(2 )and demonstrated high activity with pyruvate, oxaloacetate, and alpha-ketobutyrate. Methyl viologen, rubredoxin, FMN, and FAD were readily reduced. Activity was also observed with spinach and clostridial ferredoxins and cytochrome c. Coenzyme F(420) was not an electron acceptor for the purified enzyme.

Disruption of the Operon Encoding Ehb Hydrogenase Limits Anabolic CO 2 Assimilation in the Archaeon Methanococcus maripaludis

“…Previously, Ehb was proposed to be coupled to POR in methanococci by two small ferredoxins associated with the POR genes (15). Consistent with this hypothesis, mutants with a deletion of the genes encoding these ferredoxins were unable to form methane with pyruvate as an electron donor.…”

“…In addition, genes for two possible electron carriers, named PorE and PorF, are found adjacent to the genes encoding the POR subunits (14,15). One of these proteins copurifies with POR.…”

“…Mutants with deletions in these genes grow poorly in mineral medium and are unable to use pyruvate as an electron donor for methanogenesis (14), similar to the phenotype of the ehbF mutant described here. In addition, a homolog of PorE, named CdhF, is adjacent to the genes encoding the subunits for CODH-ACS (15). Those electron carriers could transfer the electrons from the Ehb system to the POR and CODH-ACS reactions.…”

“…In cell extract, the activity of the carbon monoxide dehydrogenase-acetyl coenzyme A (CoA) synthase (CODH-ACS) was measured similarly as the CO-dependent reduction of methyl viologen (24). Pyruvate oxidoreductase (POR) activity was assayed anaerobically as pyruvate-and CoAdependent methyl viologen reduction (15). Because of the instability of the POR (15), this activity was assayed within 1 day of preparation of the cell extracts.…”

Carbohydrate Metabolism in Archaea: Current Insights into Unusual Enzymes and Pathways and Their Regulation

“…maripaludis is also able to utilize acetate as an alternative carbon source, which is channeled into central metabolic pathways via acetyl-CoA formed by AMPdependent acetyl-CoA synthetase or by the acetate kinase/phosphotransacetylase enzyme couple (571). Pyruvate is formed from acetyl-CoA by the incorporation of CO 2 catalyzed by anabolic/ reversible pyruvate:ferredoxin oxidoreductase (572,573) and either is used for gluconeogenesis via the EMP pathway or enters the reductive branch of the incomplete CAC after conversion to oxaloacetate by pyruvate carboxylase, leading to 2-oxoglutarate (568). Energy/ATP conservation proceeds via an archaeal-type A 1 A o ATP synthase, which utilizes the proton motive force generated during methanogenesis for ATP synthesis (Mca.…”