title: Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB).
abstract: Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable. While it is clear that chemical oxidation can cleave folates or damage their pterin precursors, very little is known about enzymatic damage to these molecules or about whether the folate biosynthesis pathway responds adaptively to damage to its end-products. The presence of a duplication of the gene encoding the folate biosynthesis enzyme 6-hydroxymethyl-7,8-dihydropterin pyrophosphokinase (FolK) in many sequenced bacterial genomes combined with a strong chromosomal clustering of the folK gene with panB, encoding the 5,10-methylene-THF-dependent enzyme ketopantoate hydroxymethyltransferase, led us to infer that PanB has a side activity that cleaves 5,10-methylene-THF, yielding a pterin product that is recycled by FolK. Genetic and metabolic analyses of Escherichia coli strains showed that overexpression of PanB leads to accumulation of the likely folate cleavage product 6-hydroxymethylpterin and other pterins in cells and medium, and-unexpectedly-to a 46% increase in total folate content. In silico modeling of the folate biosynthesis pathway showed that these observations are consistent with the in vivo cleavage of 5,10-methylene-THF by a side-activity of PanB, with FolK-mediated recycling of the pterin cleavage product, and with regulation of folate biosynthesis by folates or their damage products.
authors.affiliation: Department of Microbiology and Cell Science, University of Florida Gainesville, FL, USA.
authors.forename: Jennifer J
authors.initials: JJ
authors.lastname: Thiaville
authors.affiliation: Horticultural Sciences Department, University of Florida Gainesville, FL, USA.
authors.forename: Océane
authors.initials: O
authors.lastname: Frelin
authors.affiliation: Tecnológico de Monterrey Campus Monterrey, Monterrey, Mexico.
authors.forename: Carolina
authors.initials: C
authors.lastname: García-Salinas
authors.affiliation: Department of Microbiology and Cell Science, University of Florida Gainesville, FL, USA.
authors.forename: Katherine
authors.initials: K
authors.lastname: Harrison
authors.affiliation: Horticultural Sciences Department, University of Florida Gainesville, FL, USA.
authors.forename: Ghulam
authors.initials: G
authors.lastname: Hasnain
authors.affiliation: Department of Chemistry, University of Florida Gainesville, FL, USA.
authors.forename: Nicole A
authors.initials: NA
authors.lastname: Horenstein
authors.affiliation: Tecnológico de Monterrey Campus Monterrey, Monterrey, Mexico.
authors.forename: Rocio I
authors.initials: RI
authors.lastname: Díaz de la Garza
authors.affiliation: Mathematics and Computer Science Division, Argonne National LaboratoryArgonne, IL, USA; Computation Institute, The University of ChicagoChicago, IL, USA.
authors.forename: Christopher S
authors.initials: CS
authors.lastname: Henry
authors.affiliation: Horticultural Sciences Department, University of Florida Gainesville, FL, USA.
authors.forename: Andrew D
authors.initials: AD
authors.lastname: Hanson
authors.affiliation: Department of Microbiology and Cell Science, University of FloridaGainesville, FL, USA; Genetics Institute, University of FloridaGainesville, FL, USA.
authors.forename: Valérie
authors.initials: V
authors.lastname: de Crécy-Lagard
keywords: Acinetobacter baylyi; folate biosynthesis; metabolite repair; paralogs; side-reaction