Biosynthesis of the 7-methylated pterin of methanopterin

2014

Methanopterin (MPT) and its analogs are coenzymes required for methanogenesis and methylotrophy in specialized microorganisms. The methyl groups at C-7 and C-9 of the pterin ring distinguish MPT from all other pterin-containing natural products. However, the enzyme(s) responsible for the addition of these methyl groups has yet to be identified. Here we demonstrate that a putative radical S-adenosyl-L-methionine (SAM) enzyme superfamily member encoded by the MJ0619 gene in the methanogen Methanocaldococcus jannaschii is likely this missing methylase. When MJ0619 was heterologously expressed in Escherichia coli, various methylated pterins were detected, consistent with MJ0619 catalyzing methylation at C-7 and C-9 of 7,8-dihydro-6-hydroxymethylpterin, a common intermediate in both folate and MPT biosynthesis. Site-directed mutagenesis of Cys77 present in the first of two canonical radical SAM CX 3 CX 2 C motifs present in MJ0619 did not inhibit C-7 methylation, while mutation of Cys102, found in the other radical SAM amino acid motif, resulted in the loss of C-7 methylation, suggesting that the first motif could be involved in C-9 methylation, while the second motif is required for C-7 methylation. Further experiments demonstrated that the C-7 methyl group is not derived from methionine and that methylation does not require cobalamin. When E. coli cells expressing MJ0619 were grown with deuterium-labeled acetate as the sole carbon source, the resulting methyl group on the pterin was predominantly labeled with three deuteriums. Based on these results, we propose that this archaeal radical SAM methylase employs a previously uncharacterized mechanism for methylation, using methylenetetrahydrofolate as a methyl group donor.

Section: Resultsmentioning

confidence: 66%

Section: Resultsmentioning

confidence: 89%

Section: Ethanopterin (Mpt) (mentioning

confidence: 99%

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Identification of a Unique Radical S -Adenosylmethionine Methylase Likely Involved in Methanopterin Biosynthesis in Methanocaldococcus jannaschii

Allen

2014

“…Methanopterin was isolated and characterized because of its involvement as a C1 carrier in the reduction of CO2 to CH4 in methanogenic archaebacteria where it functions analogously to folate (21). (The close similarity between the biosynthesis of folate and the biosynthesis of methanopterin further supports the close connection between these C1 carriers [27,29,30,33].) Subsequent work has indicated that this coenzyme completely replaces folate in these organisms which have been found to lack the normal folate coenzymes (14,35).…”

mentioning

confidence: 90%

Distribution of folates and modified folates in extremely thermophilic bacteria

1991

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Analyses were made of the structures and levels of folates and modified folates present in extremely thermophilic bacteria. These procedures involved the chemical analysis of products resulting from the oxidative cleavage of the 6-substituted, folatelike tetrahydropterins present in the cells. Air-oxidized cell extracts of extreme thermophiles from two members of the archaebacterial order Thermococcales, Thermococcus celer and Pyrococcusfuriosus, contained only 7-methylpterin, indicating that these cells contain a modified folate with a methylated pterin. Cell extracts also contained 6-acetyl-7-methyl-7,8-dihydropterin, another product derived from the oxidative cleavage of a dimethylated folate, demonstrating that both the C-7 and C-9 carbons of the pterin were methylated. Extracts, however, contained neither p-aminobenzoylpolyglutamates nor methaniline, the oxidative cleavage products of folates and methanopterin, respectively, indicating that they contain a previously undescribed C1 carrier(s). On the basis of the level of the 7-methylpterin isolated, the levels of modified folate were 2 to 10 times higher than those typically found in mesophilic bacteria and 10 to 100 times less than the levels of methanopterin found in the methanogenic bacteria. Oxidized cell extracts of Sulfolobus spp. of the archaebacterial order Sulfolobaks contained only pterin, and, like members of the order Thermococcales, they contained neither p-aminobenzoylpolyglutamates nor methaniline. Oxidized cell extracts of the extreme thermophiles Pyrobaculum sp. strain H10 and Pyrodictium occultum, from the archaebacterial orders Thermoproteales and Pyrodictiales, respectively, and Thermotoga maritima, from the eubacterial order Thermotogales, contained pterin and p-aminobenzoylpolyglutamates, indicating that these cells contained unmodified folates. The levels of p-aminobenzoylpolyglutamates in these archaebacterial cell extracts indicate that the folates were present in the cells at levels 4 to 10 times higher than generally found in those mesophilic eubacteria which do not use folates in energy metabolism. The levels and chain lengths of the of p-aminobenzoylpolyglutamates present in Thermotoga maritima were typical of those found in mesophilic eubacteria.Folic acid and its polyglutamyl derivatives represent one of the central coenzymes found in biological systems where folic acid functions as a C1 carrier. The core of the folate coenzyme structures was established in 1946 (1), and no modifications of this original structure were found until 1980 when the first structurally modified folate, methanopterin, was characterized (25,26). Methanopterin was isolated and characterized because of its involvement as a C1 carrier in the reduction of CO2 to CH4 in methanogenic archaebacteria where it functions analogously to folate (21). (The close similarity between the biosynthesis of folate and the biosynthesis of methanopterin further supports the close connection between these C1 carriers [27,29,30,33].) Subsequent work has indicated that thi...

“…On the basis of its structural similarity to folic acid and on the basis of biochemical evidence that it can serve as a single carbon carrier at the oxidation level of methenyl (6,24), methylene (7,14), and methyl (8)(9)(10), there is little doubt that methanopterin functions in a manner analogous to that of folate in these cells. In addition, existing biosynthetic evidence indicates that methanopterin is the first example of a structurally modified folate (25)(26)(27). It also appears that folates are absent or at very low levels in these bacteria (5,14,17).…”

mentioning

confidence: 99%

Analysis and characterization of the folates in the nonmethanogenic archaebacteria

1988

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A detailed analysis of the folate coenzymes in the nonmethanogenic archaebacteria has been performed. By using the Lactobacillus casei microbiological assay for folates, the levels of folates in Sulfolobus solfataricus and Sulfolobus acidocaldarius were found to be 3.7 and 8.3 ng/g (dry weight) of cells, respectively, compared with 88,000 and 28,000 ng/g (dry weight) of cells in Halobacterium halobium and Halobacterium strain GN-1, respectively. The levels of folates found in the Sulfolobus spp. were -100 times less than those found in the typical eubacterium, whereas the levels in the halobacteria were -10 times higher. The folate in Sulfolobus solfataricus was shown to consist of only 5-formyltetrahydropteroylglutamate, and the folate in Halobacterium strain GN-1 was shown to consist of only pteroyldiglutamate. The low folate levels in the Sulfolobus spp. are the same as those found in the methanogenic bacteria, suggesting that another C1 carrier may function in these cells.Folate and its polyglutamates are well-established coenzymes involved in C1 metabolism in both eubacteria and eucaryotes (1), and until recently, they were the only recognized C1 carriers known to function in nature. However, during work on the nature of the coenzymes involved in the reduction of carbon dioxide to methane in the methanogenic archaebacteria, a new pterin-containing coenzyme, methanopterin, was discovered and chemically characterized (22,23). On the basis of its structural similarity to folic acid and on the basis of biochemical evidence that it can serve as a single carbon carrier at the oxidation level of methenyl (6, 24), methylene (7,14), and methyl (8-10), there is little doubt that methanopterin functions in a manner analogous to that of folate in these cells. In addition, existing biosynthetic evidence indicates that methanopterin is the first example of a structurally modified folate (25)(26)(27). It also appears that folates are absent or at very low levels in these bacteria (5,14,17). The occurrence of this unique pterin-containing coenzyme in the methanogenic archaebacteria suggests that other structurally modified folates may be present in the other two groups of archaebacteria, i.e., the extremely thermophilic bacteria and the extremely halophilic bacteria. To address this possibility, the levels and the structures of compounds possessing folate bioactivity in these organisms have been investigated. MATERIALS AND METHODSChemicals and supplies. Nitrobenzoyl chloride was obtained from Aldrich Chemical Co., Inc., Milwaukee, Wis. and Halobacterium strain GN-1, supplied by Barbara J. Javor of the Scripps Institution of Oceanography, La Jolla, Calif., were grown for 3 to 4 days at 39°C on the complex medium described by Javor (16).To determine the effect of the growth medium on the cellular content of folate, cells were grown in 2.8-liter Fernbach flasks containing 1.5 liters of medium, aerated by shaking at 150 rpm. The large quantity of cells needed for the isolation and characterization of folates was grown in 20-...