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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.

The article describes the newly discovered [Fe.sup.2}-dependent archael-specific GTP cyclohydrolase, MptA, which is obtained from Methanocaldococcus jannaschii and catalyzes the first step in the biosynthesis of methanopterin. The analysis reveals that the mutation of conserved histidine residues, expected to be involved in such binding, reduces the enzymatic activity, but not the amount of bond iron.

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.

Studies done to demonstrate that 6-deoxy-5-ketofructose 1-phosphate and L-aspartate semialdehyde are precursors to DHQ are reported. It was observed that the true sugar precursor is not 6-deoxy-5-ketofructose 1-phosphate but its C-2 reduced material, 6-deoxy-5-ketomannitol 1-P and this precursor has the advantage that the other product of the reaction would be D-glyceraldehyde 3-P, which would be readily metabolized by the cells.

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.

Research has been conducted on guanosine 5'-triphosphate (GTP) cyclohydrolase from Methanococcus jannaschii. The identification and characterization of this cyclohydrolase producing 2-amino-5-formylamino-6-ribofuranosylamino-4(3H)-pyrimidinone monophosphate and inorganic phosphate have been carried out and discussed, and the details are reported.

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.

The Methanococcus jannaschii MJ256 gene, termed cofD, encodes CofD enzyme that catalyzes the fourth step in the biosynthesis of coenzyme F(sub)420. On the basis of the reactions catalyzed by CofD, the enzyme is called lactyl(2) diphospho-(5')guanosine:Fo 2-phospho-L-lactate transferase. The enzyme requires magnesium for its activity.

Research has been conducted on the coenzyme F (sub)420 in the methanoarchaea. The biochemical route for the phosphodiester bond formation in this coenzyme has been established and described.

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.

The reaction catalyzed by the enzyme 4-(beta-D-ribofuranosyl)aminobenzene 5'-phosphate synthase (beta-RFA-P synthase) from Methanosarcina thermophila was found to be analogous to other phosphoribosyltranferase reactions. The similarity is that pyrophosphate is released as a product of the reaction, which is dependent upon magnesium ions. The molecular weight of the enzyme was calculated to be 65,000 using gel filtration chromatography.

Experiments were performed to identify the biochemistry of the steps involved in the conversion of alpha-ketoglutarate and acetylCoA to alpha-ketosuberate in Methanosarcina thermophila. Trans-homoaconitrate was formed with the condensation of alpha-ketoglutarate and acetylCoA. Two consecutive elongation reactions involving alpha-ketoadipate that was formed from the dehydration and rehydration of cis-homoaconitate resulted to alpha-ketosuberate. A protein product from the Methanococcus gene served as a catalyst in one of the process.

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.