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N-Ethylglutamate (NEG) was detected in Escherichia coli BL21 cells grown on LB broth, and it was found to occur at a concentration of ~4 mM in these cells under these conditions. The same cells grown on M9 glucose medium contained no detectable amount of NEG. Analysis of the LB broth showed the presence of NEG, a compound never before reported as a natural product. Isotope dilution analysis showed that it occurred at a concentration of 160 [micro]M in LB broth. Analyses of yeast extract and tryptone, the organic components of LB broth, both showed the presence NEG. It was demonstrated that NEG can be generated during the autolysis of the yeast used in the preparation of the yeast extract. Growth of these E. coli cells in LB broth prepared in denterated water showed no incorporation of deuterium into NEG, demonstrating that E. coli cells did not generate the NEG. Cell growth rates were not affected by the addition of 5 mM NEG to either LB or M9 glucose medium. L-[ethyl-[sup.2][H.sub.4]]NEG was found to be readily incorporated into the cells and metabolized by the cells. From these results, it was concluded that all of the NEG present in the cells was taken up from the medium. NEG could serve as the sole nitrogen source for E. coli when grown on M9 glucose medium in the presence of glucose but could not serve as the sole carbon source on M9 medium in the absence of glucose. doi: 10.1128/JB.00721-10

The pathway of lysine biosynthesis in the methanococci has not been identified previously. A variant of the diaminopimelic acid (DAP) pathway uses diaminopimelate aminotransferase (DapL) to catalyze the direct conversion of tetrahydrodipicolinate (THDPA) to LL-DAP. Recently, the enzyme DapL (MTH52) was identified in Methanothermobacter thermautotrophicus and shown to belong to the DapLl group. Although the Methanococcus maripaludis genome lacks a gene that can be unambiguously assigned a DapL function based on sequence similarity, the open reading frame MMP1527 product shares 30% amino acid sequence identity with MTH52. A Ammp1527 deletion mutant was constructed and found to be a lysine auxotroph, suggesting that this DapL homolog in methanococci is required for lysine biosynthesis. In cell extracts of the M. maripaludis wild-type strain, the specific activity of DapL using LL-DAP and [alpha]-ketoglutarate as substrates was 24.3 [+ or -] 2.0 nmol [min.sup.-1] mg of [protein.sup.-1]. The gene encoding the DapL homolog in Methanocaidococcus jannaschii (MJ1391) was cloned and expressed in Escherichia coli, and the protein was purified. The maximum activity of MJ1391 was observed at 70[degrees]C and pH 8.0 to 9.0. The apparent [K.sub.m]s of MJ1391 for LL-DAP and [alpha]-ketoglutarate were 82.8 [+ or -] 10 [micro]M and 0.42 [+ or -] 0.02 mM, respectively. MJ1391 was not able to use succinyl-DAP or acetyl- DAP as a substrate. Phyiogenetic analyses suggested that two lateral gene transfers occurred in the DapL genes, one from the archaea to the bacteria in the DapL2 group and one from the bacteria to the archaea in the DapL1 group. These results demonstrated that the DapL pathway is present in marine methanogens belonging to the Methanococcales. doi: 10.1128/JB.00172-10

The riboflavin kinase in Methanocaldococcus jannaschii has been identified as the product of the M J0056 gene. Recombinant expression of the M J0056 gene in Escherichia coli led to a large increase in the amount of flavin mononucleotide (FMN) in the E. coli cell extract. The unexpected features of the purified recombinant enzyme were its use of CTP as the phosphoryl donor and the absence of a requirement for added metal ion to catalyze the formation of FMN. Identification of this riboflavin kinase fills another gap in the archaeal flavin biosynthetic pathway. Some divalent metals were found to be potent inhibitors of the reaction. The enzyme represents a unique CTP-dependent family of kinases.

Archaea have been shown to produce isoprenoids from mevalonate; however, genome analysis has failed to identify several genes in the mevalonate pathway on the basis of sequence similarity. A predicted archaeal kinase, coded for by the MJ0044 gene, was associated with other mevalonate pathway genes in the archaea and was predicted to be the 'missing' phosphomevalonate kinase. The MJ0044-derived protein was tested for phosphomevalonate kinase activity and was found not to catalyze this reaction. The MJ0044 gene product was found to phosphorylate isopentenyl phosphate, generating isopentenyl diphosphate. Unlike other known kinases associated with isoprene biosynthesis, Methanocaldococcus jannaschii isopentenyl phosphate kinase is predicted to be a member of the aspartokinase superfamily.

One of the early steps in the blosynthesis of coenzyme [F.sub.420] in Methanocaldococcus jannaschii requires generation of 2-phospho-L-lactate, which is formed by the phosphorylation of L-lactate. Preliminary studies had shown that L-lactate in M. jannaschii is not derived from pyruvate, and thus an alternate pathway(s) for its formation was examined. Here we report that L-lactate is formed by the NA[D.sup.+]-dependent oxidation of L-lactaldehyde by the M JI411 gene product. The lactaldehyde, in tnrn, was found to be generated either by the NAD(P)H reduction of methylglyoxal or by the aldol cleavage of fuculose-l-phosphate by fuculose-l-phosphate aldolase, the MJ1418 gene product.

Recent work has raised a question as to the involvement of erythrose-4-phosphate, a product of the pentose phosphate pathway, in the metabolism of the methanogenic archaea (R. H. White, Biochemistry. 43:7618-7627, 2004). To address the possible absence of erythrose-4-phosphate in Methanocaldococcus jannaschii, we have assayed cell extracts of this methanogen for the presence of this and other intermediates in the pentose phosphate pathway and have determined and compared the labeling patterns of sugar phosphates derived metabolically from [6,6-[sup.2][H.sub.2]]- and [U-[sup.13]C]-labeled glucose-6-phosphate incubated with cell extracts. The results of this work have established the absence of pentose phosphate pathway intermediates erythrose-4-phosphate, xylose-5-phosphate, and sedoheptulose-7-phosphate in these cells and the presence of D-arabino-3-hexulose-6-phosphate, an intermediate in the ribulose monophosphate pathway. The labeling of the D-arabino-3-hexulose-6-phosphate, as well as the other sugar-Ps, indicates that this hexose-6-phosphate was the precursor to ribulose-5-phosphate that in turn was converted into ribose-5-phosphate by ribose-5-phosphate isomerase. Additional work has demonstrated that ribulose-5-phosphate is derived by the loss of formaldehyde from D-arabino-3-hexulose-6-phosphate, catalyzed by the protein product of the MJ1447 gene.

Proteins in the ATP-grasp superfamily of amide bond-forming ligases have evolved to function in a number of unrelated biosynthetic pathways. Previously identified homologs encoding glutathione synthetase, D-alanine:D-alanine ligase and the bacterial ribosomal protein S6:glutamate ligase have been vertically inherited within certain organismal lineages. Although members of this specificity-diverse superfamily share a common reaction mechanism, the nonoverlapping set of amino acid and peptide substrates recognized by each family provided few clues as to their evolutionary history. Two members of this family have been identified in the hyperthermophilic marine archaeon Methanococcus jannaschii and shown to catalyze the final reactions in two coenzyme biosynthetic pathways. The MJ0620 (mptN) locus encodes a tetrahydromethanopterin:[alpha]-L-glutamate ligase that forms tetrahydrosarcinapterin, a single carbon-carrying coenzyme. The MJ1001 (cofF) locus encodes a [gamma]-[F.sub.420]-2:[alpha]L-glutamate ligase, which caps the [gamma]-glutamyl tail of the hydride carrier coenzyme [F.sub.420]. These two genes share a common ancestor with the ribosomal protein S6:glutamate ligase and a putative [alpha]-aminoadipate ligase, defining the first group of ATP-grasp enzymes with a shared amino acid substrate specificity. As in glutathione biosynthesis, two unrelated amino acid ligases catalyze sequential reactions in coenzyme [F.sub.420] polyglutamate formation: a [gamma]-glutamyl ligase adds 1-3 L-glutamate residues and the ATP-grasp-type ligase described here caps the chain with a single [alpha]-linked L-glutamate residue. The analogous pathways for glutathione, [F.sub.420], folate, and murein peptide biosyntheses illustrate convergent evolution of nonribosomal peptide biosynthesis through the recruitment of single-step amino acid ligases.

Analyses of the [F.sub.420]s present in Methanococcus jannaschii have shown that these cells contain a series of [gamma]-glutamyl-linked [F.sub.420]s capped with a single, terminal [alpha]-linked L-glutamate. The predominant form of [F.sub.420] was designated as [alpha]-[F.sub.420]-3 and represented 86% of the [F.sub.420]s in these cells. Analyses of Methanosarcina thermophila, Methanosarcina barkeri, Methanobacterium thermoautotrophicum, Archaeoglobus fulgidus, and Mycobacterium smegmatis showed that they contained only [gamma]-glutamyl-linked [F.sub.420]s.

Results demonstrate that the early steps of riboflavin biosynthesis in Methanococcus jannaschii and M. thermophilia are very similar to those in yeasts and fungi. The biosynthesis is mediated by a pyrimidine nucleotide reductase.

Research demonstrates that two genes of Methanococcus jannaschii encode enzymes catalyzing NAD-dependent decarboxylation of 7-mercaptoheptanoic acid intermediates and an isomer of DL-threo-isopropylmalate leading to coenzyme B and leucine synthesis. Data suggest that the enzymes are heat-sensitive.

Research describes the two genes involved in the decarboxylation of alpha-ketoacids in Methanococcus jannaschii. Data indicate that the subunits together code for sulfopyruvate decarboxylase which catalyzes the biosynthesis of coenzyme M.

Research has been conducted on the dehydrogenase genes from Methanococcus jannaschii and Methanothermus fervidus. The cloning and overexpression of these genes and testing of the gene products for their catalytic ability have been carried out.

Research was conducted to study the purification and characterization of serine hydroxymthyltransferase (SHMT) from the thermophilic organism Sulfolobus solfataricus. SHMT activity was determined by monitoring the rate of exchange of the alpha-hydrogen of (2-(super 3)H)glycine while a Superdex 200 column was utilized to chromatograph the enzyme from several purification steps. Results showed similarities in the size and properties of the S. solfataricus SHMT relative to procaryotic and eucaryotic SHMT.

An analysis of the purification of the Azotobacter vinelandii nifV-encoded homocitrate synthase was conducted. The nifV gene was expressed at high levels in Escherichia coli and was later on purified. Results of the study revealed that nifV is capable of catalyzing the condensation of acetyl-CoA and other keto acids. Furthermore, E. coli cells produces homocitrate into a growth medium.

GTP cychlohydrolase I is a catalyzing enzyme involved in the biosynthesis of the pterin ring of folic acid, sepiapterin and biopterin. The reaction leads to the removal of C-8 carbon as formate, with the resulting intermediated converted to 7,8-dihy-dro-D-erythro-neopterin (H2neopterin). The absence of an Escherichia coli-like GTP cyclohydrolase I in the archae methanoccus thermophila and methanobacterium thermoautotrophicum Delta H has been demonstrated. GTP is changed into H2neopterin 2:3-cyclic phosphtate during the process.

The autonomy of purine biosynthesis from the presence of folates and modified folates was determined through Sephadex G-25 chromatographic analysis of 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) injected cell extracts. In archaea domain, the role of C1 carrier coenzyme was mimicked by methanopterin (MPT) in place of folate. Results have shown that the organism in the same domain were able to metabolize purine in the absence of folate.

The biosynthesis 5-aminoimidazole-4-carboxamide ribonucleotide (ZMP) and N-(beta-D-ribofuranosyl)formamide 5'-phosphate (FAR-P) by Methanobacterium thermoautotrophicum deltaH was analyzed in cell cultures. Analysis of cell extracts from Methanobacterium thermoautotrophicum deltaH indicated the presence of pro-phosphoribosyl formimino-5-aminoimidazole-4-carboxamide ribonucleotide (5'-proFAR). Furthermore, synthesis of ZMP and FAR-P from 5'-proFAR involved a three-step procedure characterized by the hydrolysis of the intermediate metabolite.

One of the key units of the small-molecule pool in Methanobacterium thermoautotrophicum delta H under optimal conditions of cell growth, is a unique compound 1,3,4,6-hexanetetracarboxylic acid. This pool cannot function as a metabolic intermediate in cell biosynthesis. Free tetracarboxylate is a conservative constituent of the mechanism that preserves high positive internal osmotic pressure in this bacterium.

Abstract Background The ability to perform de novo biosynthesis of purines is present in organisms in all three domains of life, reflecting the essentiality of these molecules to life. Although the pathway is quite similar in eukaryotes and bacteria, the archaeal pathway is more variable. A careful manual curation of genes in this pathway demonstrates the value of manual curation in archaea, even in pathways that have been well-studied in other domains. Results We searched the Integrated Microbial Genome system (IMG) for the 17 distinct genes involved in the 11 steps of de novo purine biosynthesis in 65 sequenced archaea, finding 738 predicted proteins with sequence similarity to known purine biosynthesis enzymes. Each sequence was manually inspected for the presence of active site residues and other residues known or suspected to be required for function. Many apparently purine-biosynthesizing archaea lack evidence for a single enzyme, either glycinamide ribonucleotide formyltransferase or inosine monophosphate cyclohydrolase, suggesting that there are at least two more gene variants in the purine biosynthetic pathway to discover. Variations in domain arrangement of formylglycinamidine ribonucleotide synthetase and substantial problems in aminoimidazole carboxamide ribonucleotide formyltransferase and inosine monophosphate cyclohydrolase assignments were also identified. Manual curation revealed some overly specific annotations in the IMG gene product name, with predicted proteins without essential active site residues assigned product names implying enzymatic activity (21 proteins, 2.8% of proteins inspected) or Enzyme Commission (E. C.) numbers (57 proteins, 7.7%). There were also 57 proteins (7.7%) assigned overly generic names and 78 proteins (10.6%) without E.C. numbers as part of the assigned name when a specific enzyme name and E. C. number were well-justified. Conclusions The patchy distribution of purine biosynthetic genes in archaea is consistent with a pathway that has been shaped by horizontal gene transfer, duplication, and gene loss. Our results indicate that manual curation can improve upon automated annotation for a small number of automatically-annotated proteins and can reveal a need to identify further pathway components even in well-studied pathways. Reviewers This article was reviewed by Dr. Céline Brochier-Armanet, Dr Kira S Makarova (nominated by Dr. Eugene Koonin), and Dr. Michael Galperin.

Research has been conducted on the enzyme which causes IMP cyclohydrolase activity in Methanococcus jannaschii. The purification and sequencing of this enzyme have been carried out and the results indicate that its genetic locus corresponds to gene MJ0626.

Research has been conducted on the cell extracts of Methanococcus jannaschii which convert L-ornithine to L-proline. Results indicate that this cyclization reaction proceeds with the C-2 nitrogen loss.

The cloning and overexpression of the Methanococcus jannaschii gene MJ1392 were conducted to gain an insight into the identity of genes in the methanogens that were possibly involved in the biosynthesis of coenzymes. Another objective was to identify the reaction catalyzed by the encoded enzyme. Results provide strong evidence that the gene product is (R)-citramalate synthase.

dTMP is required for DNA synthesis in Archaea. It is catalyzed by the enzyme thymidylate synthase. Studies involving two archaean bacteria, Methanosarcina thermophila and Sulfolobus solfataricus, revealed that biosynthesis of dTMP require 5,10-methylenetetrahydro derivatives of modified folates to serve as the C1 donors. Except for this characteristic, archaean bacteria carry out the same basic reaction during dTMP biosynthesis that are analogous to other organisms.

The modified folates present in Thermococcus litoralis were structurally characterized by high performance liquid chromatography of partially digested samples. The results showed that the core structure and the linked side chains are N-acetylglucosamine homologs of the modified folates found in Pyrococcus furiosus. These results demonstrate the close taxonomic relationship between the Thermococcales and P. furiosus.