Moonlighting glutamate formiminotransferases can functionally replace 5-formyltetrahydrofolate cycloligase.
5-Formyltetrahydrofolate (5-CHO-THF) is formed by a side reaction of serine hydroxymethyltransferase. Unlike other folates, it is not a one-carbon donor but a potent inhibitor of folate enzymes and must therefore be metabolized. Only 5-CHO-THF cycloligase (5-FCL) is generally considered to do this. However, comparative genomic analysis indicated (i) that certain prokaryotes lack 5-FCL, implying that they have an alternative 5-CHO-THF-metabolizing enzyme, and (ii) that the histidine breakdown enzyme glutamate formiminotransferase (FT) might moonlight in this role. A functional complementation assay for 5-CHO-THF metabolism was developed in Escherichia coli, based on deleting the gene encoding 5-FCL (ygfA). The deletion mutant accumulated 5-CHO-THF and, with glycine as sole nitrogen source, showed a growth defect; both phenotypes were complemented by bacterial or archaeal genes encoding FT. Furthermore, utilization of supplied 5-CHO-THF by Streptococcus pyogenes was shown to require expression of the native FT. Recombinant bacterial and archaeal FTs catalyzed formyl transfer from 5-CHO-THF to glutamate, with k(cat) values of 0.1-1.2 min(-1) and K(m) values for 5-CHO-THF and glutamate of 0.4-5 μM and 0.03-1 mM, respectively. Although the formyltransferase activities of these proteins were far lower than their formiminotransferase activities, the K(m) values for both substrates relative to their intracellular levels in prokaryotes are consistent with significant in vivo flux through the formyltransferase reaction. Collectively, these data indicate that FTs functionally replace 5-FCL in certain prokaryotes.
Jeanguenin
Linda
L
Department of Horticultural Sciences, University of Florida, Gainesville, Florida 32611, USA.
Lara-Núñez
Aurora
A
Pribat
Anne
A
Mageroy
Melissa Hamner
MH
Gregory
Jesse F
JF
Rice
Kelly C
KC
de Crécy-Lagard
Valérie
V
Hanson
Andrew D
AD
D000818:Animals; D001105:Archaea / Q000378:metabolism; D019731:Carbon-Nitrogen Ligases / Q000737:chemistry*; D004926:Escherichia coli / Q000201:enzymology / Q000378:metabolism; D005492:Folic Acid / Q000737:chemistry; D005816:Genetic Complementation Test; D023281:Genomics; D051042:Glutamate Formimidoyltransferase / Q000378:metabolism*; D018698:Glutamic Acid / Q000737:chemistry; D006639:Histidine / Q000737:chemistry; D007700:Kinetics; D008957:Models, Genetic; D009154:Mutation; D010641:Phenotype; D011994:Recombinant Proteins / Q000737:chemistry; D013297:Streptococcus pyogenes / Q000378:metabolism; D013552:Swine
D011994:Recombinant Proteins; D018698:Glutamic Acid; D006639:Histidine; D005492:Folic Acid; D051042:Glutamate Formimidoyltransferase; D019731:Carbon-Nitrogen Ligases; C021557:5,10-methenyltetrahydrofolate synthetase