Tetrahydrofolat-spezifische Enzyme im Baustoffwechsel von Methanosarcina barkeri sowie die Rolle von Folsäure und p-Aminobenzoesäure als Wachstumsfaktoren in diesem Archaeon
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Date
2005-10-24
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Philipps-Universität Marburg
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Abstract
Tetrahydrofolate (H4F) plays an essential role as a carrier of C1-units in the anabolism of bacteria, eucarya and some archaea, where it is involved in the biosynthesis of purines, thymidylate and methionine. In anabolic reactions of archaea, which seem to lack H4F, the coenzyme is replaced by tetrahydromethanopterin (H4MPT), a structural analog playing a major role as C1-carrier in methanogenesis from CO2 or acetate. In the anabolism of archaea of the order Methanosarcinales H4MPT appears not to be involved in the synthesis of purines, thymidylate and methionine which is concluded from 13C-labelling experiments and raises the question whether these organisms contain both, H4MPT and H4F. Experiments in order to address this question were carried out with Methanosarcina barkeri, which contains tetrahydrosarcinapterin (H4SPT) instead of H4MPT. H4SPT differs from H4MPT by a terminal glutamate residue, which has no impact on the activity of the coenzyme.
In the genome of Methanosarcina barkeri, M. acetivorans, M. thermophila and M. mazei genes were found, which code for putative H4F-specific enzymes as following: serine hydroxymethyltransferase (GlyA), methylene-H4F dehydrogenase/ methenyl-H4F cylcohydrolase (FolD), methylene-H4F reductase (MetF), phosphoribosylglycinamide formyltransferase (PurN) and phosphoribosylglycinaminoimidazolecarboxamide formyltransferase (PurH). These genes - with the exception of glyA - could not be found in the genomes of Methanothermobacter thermoautotrophicus, Methanococcus jannaschii, Methanococcus maripaludis and Methanopyrus kandleri. Two of the genes from M. barkeri, folD and glyA, were heterologously overproduced in Escherichia coli as soluble proteins. The recombinant active enzymes were characterized and polyclonal antibodies were produced in order to detect the proteins in cell extracts of M. barkeri.
The gene folD codes for a bifunctional methylene-H4F dehydrogenase/ methenyl-H4F cyclohydrolase, which catalyzes both, the oxidation of methylene-H4F with NAD+ to methenyl-H4F+ and NADH and the hydrolysis of methenyl-H4F+ to N10-formyl-H4F. Both activites were strictly H4F-specific. The gene glyA codes for serine hydroxymethyltransferase, which catalyzes the aldol-cleavage of L-serine in presence of H4F into glycine and methylene-H4F. In this reaction H4F could be replaced by H4MPT, but the activity counted for less than 1% of that with H4F. The apparent Km values for methylene-H4F (FolD) and (6S-)H4F (GlyA) were below 5 µM. The gene products of folD and glyA could be verified in cell extracts of M. barkeri via western-blot analyses and activity measurements.
These results thus indicated that Methanosarcina should contain H4F, which was further supported by the finding that growth of the archaeon was dependent on the presence of folic acid (200 nM) or p-aminobenzoic acid (20 nM), a biosynthetic precursor of H4F, in the medium. Growth inhibition was observed upon addition of sulfanilamid (>2 mM), an antibiotic known to inhibit H4F biosynthesis specifically. Under p-aminobenzoic acid (pABA) limiting conditions 2 nmol of the vitamin were required to produce 1 g of wet cells from which an intracellular H4F concentration of approximately 5 µM was calculated assuming that p-aminobenzoic acid was completely taken up by the cells and incorporated into H4F.
The intracellular H4SPT concentration of M. barkeri was determined to be 3 mM and found to be independent of the pABA concentration provided in the growth medium. These data indicate that M. barkeri does not use free pABA for the synthesis of H4SPT under pABA limiting growth conditions, which is in contrast to reports found in literature. These results were confirmed by experiments, in which the uptake of [carboxy-14C]-pABA in the cells was monitored. Under the experimental conditions a maximum of 0.4% of H4SPT was synthesized from pABA taken up from the medium, which was followed via the loss of radioactivity according to decarboxylation during the incorporation of pABA into H4SPT.
From these studies it was concluded that Methanosarcina contains two separate C1-pools, in which H4F plays a crucial role in anabolic reactions.
Additionally, substrate specificity of serine hydroxymethyltransferase of Methanococcus jannaschii was tested and found to be H4MPT-specific. The published results can be found in the appendix.
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Created: 2004Issued: 2005-10-24Updated: 2011-08-10
Faculty
Fachbereich Biologie
Language
ger
Data types
DoctoralThesis
Keywords
TetrahydromethanopterinFolatbiosyntheseC1-MetabolismusTetrahydrofolateMethanosarcina barkeriC1-metabolismFolate biosynthesisp-Aminobenzoesäure
DFG-subjects
Methanosarcina barkeriAnabolismusTetrahydrofolsäure
DDC-Numbers
570
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Buchenau, Bärbel: Tetrahydrofolat-spezifische Enzyme im Baustoffwechsel von Methanosarcina barkeri sowie die Rolle von Folsäure und p-Aminobenzoesäure als Wachstumsfaktoren in diesem Archaeon. : Philipps-Universität Marburg 2005-10-24. DOI: https://doi.org/10.17192/z2005.0146.
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