Oxaloacetate Synthesis in the Methanarchaeon Methanosarcina barkeri : Pyruvate Carboxylase Genes and a Putative Escherichia coli -Type Bifunctional Biotin Protein Ligase Gene ( bpl/birA ) Exhibit a Unique Organization

Abstract

Evidence is presented that, in Methanosarcina barkeri oxaloacetate synthesis, an essential and major CO 2 fixation reaction is catalyzed by an apparent ␣ 4 ␤ 4 -type acetyl coenzyme A-independent pyruvate carboxylase (PYC), composed of 64.2-kDa biotinylated and 52.9-kDa ATP-binding subunits. The purified enzyme was most active at 70°C, insensitive to aspartate and glutamate, mildly inhibited by ␣-ketoglutarate, and severely inhibited by ATP, ADP, and excess Mg 2؉ . It showed negative cooperativity towards bicarbonate at 70°C but not at 37°C. The organism expressed holo-PYC without an external supply of biotin and, thus, synthesized biotin. pycA, pycB, and a putative bpl gene formed a novel operon-like arrangement. Unlike other archaeal homologs, the putative biotin protein ligases (BPLs) of M. barkeri and the closely related euryarchaeon Archaeoglobus fulgidus appeared to be of the Escherichia coli-type (bifunctional, with two activities: BirA or a repressor of the biotin operon and BPL). We found the element Tyr(Phe)ProX 5 Phe(Tyr) to be fully conserved in biotindependent enzymes; it might function as the hinge for their "swinging arms."The oxaloacetate (OAA) synthesis step is an essential physiological component and a major CO 2 fixation site in a methanarchaeon (38), for it primes therein an incomplete tricarboxylic acid cycle reaction sequence that generates intermediates for the synthesis of amino acids (via OAA, ␣-ketoglutarate [␣-KG], and succinate) and tetrapyrroles (via ␣-KG) (38). Methanococcus jannaschii and Methanococcus maripaludis use a pyruvate carboxylase (PYC) for OAA synthesis (28, 37). Methanobacterium thermoautotrophicum possesses two OAA-generating enzymes: PYC and phosphoenolpyruvate carboxylase (PPC) (18,22,31). The PYC and PPC reactions take the following forms:The avenue for OAA biosynthesis in Methanosarcina species is unknown. Weimer and Zeikus (42) showed that Methanosarcina barkeri is devoid of PPC activity. We describe below PYC as an OAA-synthesizing activity in M. barkeri and present a biochemical and molecular genetic characterization of the enzyme. (Fig. 1A). Hence, the organism was capable of synthesizing biotin and of biotinylating a candidate polypeptide without any external source of biotin. The intense band at ϳ65 kDa was typical of the biotin-carrying subunit (PYCB) of an arcaheal PYC (28, 31). Thus, we purified and characterized the corresponding protein. As shown below, it possessed PYC activity and the above-mentioned avidinreacting ϳ65-kDa band indeed corresponded to the PYCB subunit of the enzyme (Fig. 1B and C). For the cells grown with a supply of biotin, the relative intensity of the ϳ65-kDa band increased. Thus, M. barkeri exhibited a less stringent version of the control on PYC synthesis seen in Methanobacterium thermoautotrophicum (31); the latter requires exogenously supplied biotin to express holo-PYC (31), although it makes biotin (33), and a similar phenomenon exists in several bacteria (reviewed in reference 31). Whether other bands in the blot (Fig. 1A) r...

The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus Has a Novel Structure

“…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). Methanothermobacter thermautotrophicus (formerly known as Methanobacterium thermoautotrophicum strain ⌬H) (2, 57, 61) and Methanothermus sociabilis also possess phosphoenolpyruvate carboxylase (14,31,47,60), but very little of such information is available on these enzymes.…”

“…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). Methanothermobacter thermautotrophicus (formerly known as Methanobacterium thermoautotrophicum strain ⌬H) (2, 57, 61) and Methanothermus sociabilis also possess phosphoenolpyruvate carboxylase (14,31,47,60), but very little of such information is available on these enzymes.…”

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

“…Pyruvate carboxylase was assayed from cell extracts by coupling the formation of oxaloacetate to malate dehydrogenase (11). NADH oxidation by malate dehydrogenase was measured spectrophotometrically (ε 340 ϭ 6.22 mM Ϫ1 cm Ϫ1 ) at 37°C.…”

“…Pyruvate then enters the gluconeogenic pathway via phosphoenolpyruvate (PEP) and the branched tricarboxylic cycle via oxaloacetate (OAA). The former is produced via the enzyme PEP synthase (10), whereas the latter is produced via the biotin-dependent enzyme pyruvate carboxylase (Pyc [11]). …”