The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus Has a Novel Structure

Abstract

In Methanothermobacter thermautotrophicus, oxaloacetate synthesis is a major and essential CO 2 -fixation reaction. This methanogenic archaeon possesses two oxaloacetate-synthesizing enzymes, pyruvate carboxylase and phosphoenolpyruvate carboxylase. The phosphoenolpyruvate carboxylase from this organism was purified to homogeneity. The subunit size of this homotetrameric protein was 55 kDa, which is about half that of all known bacterial and eukaryotic phosphoenolpyruvate carboxylases (PPCs). The NH 2 -terminal sequence identified this enzyme as the product of MTH943, an open reading frame with no assigned function in the genome sequence. A BLAST search did not show an obvious sequence similarity between MTH943 and known PPCs, which are generally well conserved. This is the first report of a new type of phosphoenolpyruvate carboxylase that we call PpcA ("A" for "archaeal"). Homologs to PpcA were present in most archaeal genomic sequences, but only in three bacterial (Clostridium perfringens, Oenococcus oeni, and Leuconostoc mesenteroides) and no eukaryotic genomes. PpcA was the only recognizable oxaloacetate-producing enzyme in Methanopyrus kandleri, a hydrothermal vent organism. Each PpcA-containing organism lacked a PPC homolog. The activity of M. thermautotrophicus PpcA was not influenced by acetyl coenzyme A and was about 50 times less sensitive to aspartate than the Escherichia coli PPC. The catalytic core (including His 138 , Arg 587 , and Gly 883 ) of the E. coli PPC was partly conserved in PpcA, but three of four aspartate-binding residues (Lys 773 , Arg 832 , and Asn 881 ) were not. PPCs probably evolved from PpcA through a process that added allosteric sites to the enzyme. The reverse is also equally possible.The synthesis of oxaloacetate (OAA) is a major and essential CO 2 -fixation reaction in the methanarchaea (10,11,15,16,50,52,58,60). These organisms possess an incomplete tricarboxylic acid (TCA) cycle which is used to generate intermediates (OAA and ␣-ketoglutarate [␣-KG]) and a carrier (succinate) for the biosynthesis of amino acids and tetrapyrroles (10,11,15,16,50,52,58,60). The organisms belonging to the orders of Methanococcales, Methanobacteriales, and Methanomicrobiales, which primarily use hydrogen as an energy source (2), employ a reductive sequence starting at OAA and terminating at ␣-KG (10,11,15,16,50,52,60).Methanosarcina species, which predominantly depend on acetotrophic or methylotrophic methanogenesis for energy generation (2), use an oxidative branch of the TCA cycle that initiates with OAA and acetate and terminates at ␣-KG (52, 58). Hence, OAA synthesis is a central anabolic process in methanarchaea. Thus far, pyruvate carboxylase (PYC) (39,41,42,50) and phosphoenolpyruvate carboxylase (PPC) (14,31,47,60) have been found to be capable of fulfilling this requirement, as follows:PYC is ubiquitous in the methanogens (39,41,42,50), and the primary structure, kinetic characteristics, and expression patterns of the methanogen PYCs have been investigated (39,41,42,50). Methanothermob...

Insights into the Autotrophic CO 2 Fixation Pathway of the Archaeon Ignicoccus hospitalis : Comprehensive Analysis of the Central Carbon Metabolism

“…PEP carboxylase is a member of the archaeal-type carboxylase (39). Fructose 1,6-bisphosphate aldolase appears to be different from the known enzyme, since activity was detected but a database search did not yield any hit.…”

Maize Phosphoenolpyruvate Carboxylase

“…In particular, the subunit molecular size of Archaea is very small (about 60 kDa) (8,9) and that of protozoa (malaria pathogen) deduced from its DNA sequence is very large (about 130 kDa) (www.genedb.org/ genedb/malaria/index.jsp, ID: PF14_0246) . Most PEPCs studied previously have been allosteric in nature, but their effectors show a wide variety depending on the species of the organisms (1,10), and kinetic properties modified by effectors often differ even among isoforms in the same organisms (11,12).…”

Genetic, Genomic, and Transcriptomic Studies of Pyruvate Metabolism in Methanosarcina barkeri Fusaro

“…5). Although we were unable to demonstrate Ppc activity in extracts of the mutants, it is known that the archaeal enzyme can be unstable (26). Given that OAA biosynthesis is essential and that the ppc gene is upregulated in the mutants, it is highly likely that Ppc is functionally expressed in the Pyr-1 and Pyr-2 cell lines.…”

“…Many organisms, including some methanogenic archaea, use the enzyme phosphoenolpyruvate carboxylase (Ppc) for this purpose (26). Although previous efforts failed to detect Ppc activity in M. barkeri cell extracts (10), a homolog of the archaeal ppc gene is found in the M. barkeri genome sequence (locus tag Mbar_A2632).…”