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Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p -aminobenzoic acid

Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this... Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H 4 SPT) rather than tetrahydrofolate (H 4 F) as a pterin C 1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN , folD , glyA and metF , that are predicted to encode for H 4 F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N 5 , N 10 -methylene-H 4 F dehydrogenase/ N 5 , N 10 -methenyl-H 4 F cyclohydrolase) and GlyA (serine:H 4 F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli , purified and found to be specific for methylene-H 4 F and H 4 F, respectively (apparent K m below 5 μM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri . The results thus indicate that M. barkeri should contain H 4 F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p -aminobenzoic acid, a biosynthetic precursor of H 4 F. From the p -aminobenzoic acid requirement, an intracellular H 4 F concentration of approximately 5 μM was estimated. Evidence is presented that the p -aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H 4 SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H 4 SPT and H 4 F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C 1 pools in these methanogens. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Archives of Microbiology Springer Journals

Tetrahydrofolate-specific enzymes in Methanosarcina barkeri and growth dependence of this methanogenic archaeon on folic acid or p -aminobenzoic acid

Buchenau, Bärbel; Thauer, Rudolf
Archives of Microbiology , Volume 182 (4) – Oct 1, 2004
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References (69)

Publisher
Springer Journals
Copyright
Copyright © 2004 by Springer-Verlag
Subject
LifeSciences
ISSN
0302-8933
eISSN
1432-072X
DOI
10.1007/s00203-004-0714-0
pmid
15349715
Publisher site
See Article on Publisher Site

Abstract

Methanogenic archaea are generally thought to use tetrahydromethanopterin or tetrahydrosarcinapterin (H 4 SPT) rather than tetrahydrofolate (H 4 F) as a pterin C 1 carrier. However, the genome sequence of Methanosarcina species recently revealed a cluster of genes, purN , folD , glyA and metF , that are predicted to encode for H 4 F-specific enzymes. We show here for folD and glyA from M. barkeri that this prediction is correct: FolD (bifunctional N 5 , N 10 -methylene-H 4 F dehydrogenase/ N 5 , N 10 -methenyl-H 4 F cyclohydrolase) and GlyA (serine:H 4 F hydroxymethyltransferase) were heterologously overproduced in Escherichia coli , purified and found to be specific for methylene-H 4 F and H 4 F, respectively (apparent K m below 5 μM). Western blot analyses and enzyme activity measurements revealed that both enzymes were synthesized in M. barkeri . The results thus indicate that M. barkeri should contain H 4 F, which was supported by the finding that growth of M. barkeri was dependent on folic acid and that the vitamin could be substituted by p -aminobenzoic acid, a biosynthetic precursor of H 4 F. From the p -aminobenzoic acid requirement, an intracellular H 4 F concentration of approximately 5 μM was estimated. Evidence is presented that the p -aminobenzoic acid taken up by the growing cells was not required for the biosynthesis of H 4 SPT, which was found to be present in the cells at a concentration above 3 mM. The presence of both H 4 SPT and H 4 F in M. barkeri is in agreement with earlier isotope labeling studies indicating that there are two separate C 1 pools in these methanogens.

Journal

Archives of Microbiology – Springer Journals

Published: Oct 1, 2004

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