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Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase
Linda Jeanguenin1, Aurora Lara-Núñez, Anne Pribat
1Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611, USA.
Certain bacteria metabolize 5-formyltetrahydrofolate (5-CHO-THF) using glutamate formiminotransferase (FT) instead of the typically assumed 5-CHO-THF cycloligase (5-FCL). This discovery reveals an alternative pathway for folate metabolism in prokaryotes lacking 5-FCL.
Area of Science:
- Biochemistry
- Microbiology
- Enzymology
Background:
- 5-Formyltetrahydrofolate (5-CHO-THF) is a folate metabolite that inhibits folate enzymes and requires metabolism.
- 5-CHO-THF cycloligase (5-FCL) is the enzyme traditionally recognized for metabolizing 5-CHO-THF.
- Comparative genomics suggests some prokaryotes may utilize an alternative enzyme for 5-CHO-THF metabolism.
Purpose of the Study:
- To investigate the potential role of glutamate formiminotransferase (FT) in metabolizing 5-CHO-THF in prokaryotes lacking 5-FCL.
- To develop and utilize a functional assay to study 5-CHO-THF metabolism.
- To confirm the enzymatic activity of FT in 5-CHO-THF metabolism.
Main Methods:
- Developed a functional complementation assay in an Escherichia coli 5-FCL deletion mutant.
- Assessed growth defects and 5-CHO-THF accumulation in the mutant.
- Tested complementation using bacterial and archaeal FT genes.
- Investigated 5-CHO-THF utilization in Streptococcus pyogenes requiring FT expression.
- Characterized recombinant FTs' enzymatic activity towards 5-CHO-THF and glutamate.
Main Results:
- The E. coli 5-FCL deletion mutant accumulated 5-CHO-THF and exhibited a growth defect on glycine, which was complemented by FT genes.
- Streptococcus pyogenes' utilization of 5-CHO-THF depended on its native FT expression.
- Recombinant FTs catalyzed formyl transfer from 5-CHO-THF to glutamate with low k(cat) but favorable K(m) values.
- The kinetic parameters suggest significant in vivo flux through the FT-mediated formyltransferase reaction.
Conclusions:
- Glutamate formiminotransferase (FT) functionally replaces 5-CHO-THF cycloligase (5-FCL) in certain prokaryotes.
- FT plays a crucial role in metabolizing 5-CHO-THF in these organisms.
- This finding expands our understanding of folate metabolism pathways in prokaryotes.
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