A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii

Mukhopadhyay, Biswarup; Patel, Vipool J.; Wolfe, R. S.

doi:10.1007/s002030000225

Cited by 27 publications

(30 citation statements)

References 35 publications

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“…The identity of a polypeptide in a gel band was determined by in-gel trypsin digestion, MALDI-TOF 2 mass spectrometry, and data base searches as described previously (13,14). Protein was assayed according to Bradford (15).…”

Section: Methodsmentioning

confidence: 99%

“…2C). Mass spectrometric analyses (13,14) showed that each of these bands corresponded to MJ0870 (data not shown). Therefore, we concluded that the 70-kDa band in Fig.…”

Section: Growth Of M Jannaschii With Sulfite As the Sulfur Source Anmentioning

confidence: 96%

“…The assay was initiated by the addition of enzyme. For pH studies, the potassium phosphate buffer was replaced with constant ionic strength buffers (14). H 2 F 420 was generated by chemical reduction of F 420 , which was purified from M. thermautotrophicus (17,19), in water with NaBH 4 (20).…”

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confidence: 99%

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A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii

Johnson

Mukhopadhyay

2005

Journal of Biological Chemistry

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197

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Methanocaldococcus jannaschii is a hypertheromphilic, strictly hydrogenotrophic, methanogenic archaeon of ancient lineage isolated from a deep-sea hydrothermal vent. It requires sulfide for growth. Sulfite is inhibitory to the methanogens. Yet, we observed that M. jannaschii grows and produces methane with sulfite as the sole sulfur source. We found that in this organism sulfite induces a novel, highly active, coenzyme F 420 -dependent sulfite reductase (Fsr) with a cell extract specific activity of 0. Methanogenesis by the methanogenic archaea is inhibited by sulfite (1). This oxyanion is a strong nucleophile and is known to be toxic to cells of all types due to its reactivity toward proteins and sulfhydryl groups (2). Methanogens perhaps have an additional reason for sulfite sensitivity. In vitro sulfite reacts with and inactivates purified methylcoenzyme M reductase (3, 4), an essential enzyme for methanogenesis (5). Yet two methanogens, Methanothermococcus thermolithotrophicus and Methanothermobacter thermautotrophicus, have been reported to tolerate and even use sulfite as a sole sulfur source (6, 7). Also, as shown in this report, Methanocaldococcus jannaschii, a deeply rooted hyperthermophilic methanogenic archaeon isolated from a deep-sea hydrothermal vent (8), grows with sulfite. However, the genomes of M. thermautotrophicus and M. jannaschii do not carry a clear homolog of a sulfite reductase (9, 10); the genome sequence of M. thermolithotrophicus is yet to be determined. With the goal of identifying the sulfite detoxification and assimilation mechanisms of these organisms, we have studied sulfite metabolism of M. jannaschii. As shown below, this work has led to the discovery of a new type of sulfite reductase. MATERIALS AND METHODSGrowth of M. jannaschii-The organism was grown on H 2 plus CO 2 (80:20, v/v; 3 ϫ 10 5 Pa) in a mineral salts medium in sealed 500-ml serum bottles, as described previously (8, 11), with either sodium sulfite (1 mM) or sulfide (1 mM) as the sole sulfur source and medium reductant. The cells were harvested by centrifugation at 9600 ϫ g and 4°C anaerobically under an N 2 plus CO 2 atmosphere (80:20, v/v).Methane Measurement-Methane was assayed by use of a HewlettPackard model HP5890 gas chromatograph (Agilent Technologies, Inc., Palo Alto, CA) fitted with a flame ionization detector and a 0.5-mm ϫ 30-m HP-PLOT (aluminum oxide, 15 m) column. The column, detector, and injector were maintained at 100, 150, and 150°C, respectively. The carrier gas (N 2 ) flow rate was 1 ml/min. A methane standard (Matheson Tri-Gas, Montgomeryville, PA) was used for calibration.Protein Analysis-SDS-PAGE was performed according to Laemmli (12), and the same method but employing buffers without SDS and omitting the sample denaturation and reduction step was used for nondenaturing gel electrophoresis. The identity of a polypeptide in a gel band was determined by in-gel trypsin digestion, MALDI-TOF 2 mass spectrometry, and data base searches as described previously (13,14). Protein was assayed according...

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Section: Methodsmentioning

confidence: 99%

“…2C). Mass spectrometric analyses (13,14) showed that each of these bands corresponded to MJ0870 (data not shown). Therefore, we concluded that the 70-kDa band in Fig.…”

Section: Growth Of M Jannaschii With Sulfite As the Sulfur Source Anmentioning

confidence: 96%

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A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii

Johnson

Mukhopadhyay

2005

Journal of Biological Chemistry

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197

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“…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.…”

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confidence: 99%

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confidence: 99%

The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus Has a Novel Structure

Patel

Kraszewski²,

Mukhopadhyay

2004

J Bacteriol

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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...

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“Pyruvate Carboxylase, Structure and Function”

Valle

2017

Subcellular Biochemistry

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Pyruvate carboxylase is a metabolic enzyme that fuels the tricarboxylic acid cycle with one of its intermediates and also participates in the first step of gluconeogenesis. This large enzyme is multifunctional, and each subunit contains two active sites that catalyze two consecutive reactions that lead to the carboxylation of pyruvate into oxaloacetate, and a binding site for acetyl-CoA, an allosteric regulator of the enzyme. Pyruvate carboxylase oligomers arrange in tetramers and covalently attached biotins mediate the transfer of carboxyl groups between distant active sites. In this chapter, some of the recent findings on pyruvate carboxylase functioning are presented, with special focus on the structural studies of the full length enzyme. The emerging picture reveals large movements of domains that even change the overall quaternary organization of pyruvate carboxylase tetramers during catalysis.

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A stable archaeal pyruvate carboxylase from the hyperthermophile Methanococcus jannaschii

Cited by 27 publications

References 35 publications

A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii

A New Type of Sulfite Reductase, a Novel Coenzyme F420-dependent Enzyme, from the Methanarchaeon Methanocaldococcus jannaschii

The Phosphoenolpyruvate Carboxylase from Methanothermobacter thermautotrophicus Has a Novel Structure

“Pyruvate Carboxylase, Structure and Function”

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