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...

Pyruvate carboxylase (PYC) is an ecologically, medically, and industrially important enzyme. It is widespread in all three domains of life, the archaea, bacteria, and eukarya. PYC catalyzes ATP-dependent carboxylation of pyruvate to oxaloacetate. Detailed structure-function studies of this enzyme have been hampered due to the unavailability of a facile recombinant overexpression system. Except for the ␣ 4 enzyme from a thermophilic Bacillus species, Escherichia coli has been unsuitable for overexpression of PYCs. We show that a Pseudomonas aeruginosa strain carrying the T7 polymerase gene can serve as a host for the overexpression of Mycobacterium smegmatis ␣ 4 PYC and Pseudomonas aeruginosa ␣ 4 ␤ 4 PYC under the control of the T7 promoter from a broad-host-range conjugative plasmid. Overexpression occurred both in aerobic (LB medium) and nitrate-respiring anaerobic (LB medium plus glucose and nitrate) cultures. The latter system presented a simpler option because it involved room temperature cultures in stationary screw-cap bottles. We also developed a P. aeruginosa ⌬pyc strain that allowed the expression of recombinant PYCs in the absence of the native enzyme. Since P. aeruginosa can be transformed genetically and lysed for cell extract preparation rather easily, our system will facilitate site-directed mutagenesis, kinetics, X-ray crystallographic, and nuclear magnetic resonance-based structure-function analysis of PYCs. During this work we also determined that, contrary to a previous report (C. K. Stover et al., Nature 406:959-964, 2000), the open reading frame (ORF) PA1400 does not encode a PYC in P. aeruginosa. The ␣ 4 ␤ 4 PYC of this organism was encoded by the ORFs PA5436 and PA5435.

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.