BackgroundMethanomicrobiales is the least studied order of methanogens. While these organisms appear to be more closely related to the Methanosarcinales in ribosomal-based phylogenetic analyses, they are metabolically more similar to Class I methanogens.Methodology/Principal FindingsIn order to improve our understanding of this lineage, we have completely sequenced the genomes of two members of this order, Methanocorpusculum labreanum Z and Methanoculleus marisnigri JR1, and compared them with the genome of a third, Methanospirillum hungatei JF-1. Similar to Class I methanogens, Methanomicrobiales use a partial reductive citric acid cycle for 2-oxoglutarate biosynthesis, and they have the Eha energy-converting hydrogenase. In common with Methanosarcinales, Methanomicrobiales possess the Ech hydrogenase and at least some of them may couple formylmethanofuran formation and heterodisulfide reduction to transmembrane ion gradients. Uniquely, M. labreanum and M. hungatei contain hydrogenases similar to the Pyrococcus furiosus Mbh hydrogenase, and all three Methanomicrobiales have anti-sigma factor and anti-anti-sigma factor regulatory proteins not found in other methanogens. Phylogenetic analysis based on seven core proteins of methanogenesis and cofactor biosynthesis places the Methanomicrobiales equidistant from Class I methanogens and Methanosarcinales.Conclusions/SignificanceOur results indicate that Methanomicrobiales, rather than being similar to Class I methanogens or Methanomicrobiales, share some features of both and have some unique properties. We find that there are three distinct classes of methanogens: the Class I methanogens, the Methanomicrobiales (Class II), and the Methanosarcinales (Class III).

Background: Staphylothermus marinus is an anaerobic, sulfur-reducing peptide fermenter of the archaeal phylum Crenarchaeota. It is the third heterotrophic, obligate sulfur reducing crenarchaeote to be sequenced and provides an opportunity for comparative analysis of the three genomes.

Tyr235 of GTP‐dependent phosphoenolpyruvate (PEP) carboxykinase is a fully invariant residue. The aromatic ring of this residue establishes an energetically favorable weak anion–quadrupole interaction with PEP carboxylate. The role of Tyr235 in catalysis was investigated via kinetic analysis of site‐directed mutagenesis‐derived variants. The Y235F change lowered the apparent Km for PEP by about six‐fold, raised the apparent Km for Mn2+ by about 70‐fold, and decreased oxaloacetate (OAA)‐forming activity by about 10‐fold. These effects were due to an enhanced anion–quadrupole interaction between the aromatic side chain at position 235, which now lacked a hydroxyl group, and PEP carboxylate, which probably increased the distance between PEP and Mn2+ and consequently affected the phosphoryl transfer step and overall catalysis. For the Y235A and Y235S changes, an elimination of the favorable edge‐on interaction increased the apparent Km for PEP by four‐ and six‐fold, respectively, and the apparent Km for Mn2+ by eight‐ and six‐fold, respectively. The pyruvate kinase‐like activity, representing the PEP dephosphorylation step of the OAA‐forming reaction, was affected by the substitutions in a similar way to the complete reaction. These observations indicate that the aromatic ring of Tyr235 helps to position PEP in the active site and the hydroxyl group allows an optimal PEP–Mn2+ distance for efficient phosphoryl transfer and overall catalysis. The Y235A and Y235S changes drastically reduced the PEP‐forming and OAA decarboxylase activities, probably due to the elimination of the stabilizing interaction between Tyr235 and the respective products, PEP and pyruvate.

The crystal structure of an archaeal-type phosphoenolpyruvate carboxylase from Clostridium perfringens has been determined based on X-ray data extending to 3 Ǻ. The asymmetric unit of the structure includes two tetramers (each a dimer-of-dimers) of the enzyme. The precipitant, malonate, employed for the crystallization is itself a weak inhibitor of phosphoenolpyruvate carboxylase and a malonate molecule is seen in the active-site in the crystal structure. The allosteric binding sites for aspartate (an inhibitor) and glucose-6-phosphate (an activator) observed in the Escherichia coli and Zea mays phosphoenolpyruvate carboxylase structures, respectively, are not conserved in the C. perfringens structure. Aspartate inhibits the C. perfringens enzyme competitively with respect to the substrate, Mg ++. phosphoenolpyruvate. A mechanism for inhibition is proposed based on the structure and sequence comparisons with other archaeal-type phosphoenolpyruvate carboxylases with differing sensitivity to inhibition by aspartate.

An archaeal-type phosphoenolpyruvate carboxylase (PepcA) from Clostridium perfringens has been expressed in Escherichia coli in a soluble form with an amino-terminal His tag. The recombinant protein is enzymatically active and two crystal forms have been obtained. Complete diffraction data extending to 3.13 Å resolution have been measured from a crystal soaked in KAu(CN) 2 , using radiation at a wavelength just above the Au L III edge. The asymmetric unit contains two tetramers of PepcA.