Limits...
Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB).

Thiaville JJ, Frelin O, García-Salinas C, Harrison K, Hasnain G, Horenstein NA, Díaz de la Garza RI, Henry CS, Hanson AD, de Crécy-Lagard V - Front Microbiol (2016)

Bottom Line: Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida Gainesville, FL, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Antifolate sensitivity (A) Overnight cultures of E. coli BW25113 and MG1655 carrying pCA24N::panB (ppanB) or pBAD33 (empty) were diluted 100-fold and spread onto M9-glucose agar medium containing chloramphenicol and IPTG (0.1 mM). Twenty microliter of (1) sulfathiazole at 100 μg/ml, (2) sulfathiazole at 50 μg/ml, (3) trimethoprim at 100 μg/ml, (4) trimethoprim at 50 μg/ml, or (N) media with no antibiotic were spotted onto filter disks. (B) Diameter of the halo inhibition were measured for three biological replicates of each and the average and standard deviation of each were calculated.* indicates significant difference from empty vector control (p > 0.05 determined by 2-tailed Student's t-test). (C) Serial dilutions of each strain were spotted onto M9-glucose agar plates with chloramphenicol and IPTG with trimethoprim (0 or 1 μg/ml) or sulfathiazole (0 or 0.1 μg/ml). E. coli overexpressing panB were more sensitive to both folate inhibitors than E. coli with empty vector control.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4814558&req=5

Figure 4: Antifolate sensitivity (A) Overnight cultures of E. coli BW25113 and MG1655 carrying pCA24N::panB (ppanB) or pBAD33 (empty) were diluted 100-fold and spread onto M9-glucose agar medium containing chloramphenicol and IPTG (0.1 mM). Twenty microliter of (1) sulfathiazole at 100 μg/ml, (2) sulfathiazole at 50 μg/ml, (3) trimethoprim at 100 μg/ml, (4) trimethoprim at 50 μg/ml, or (N) media with no antibiotic were spotted onto filter disks. (B) Diameter of the halo inhibition were measured for three biological replicates of each and the average and standard deviation of each were calculated.* indicates significant difference from empty vector control (p > 0.05 determined by 2-tailed Student's t-test). (C) Serial dilutions of each strain were spotted onto M9-glucose agar plates with chloramphenicol and IPTG with trimethoprim (0 or 1 μg/ml) or sulfathiazole (0 or 0.1 μg/ml). E. coli overexpressing panB were more sensitive to both folate inhibitors than E. coli with empty vector control.

Mentions: The effect of overexpressing the panB was tested by transforming either E. coli strain BW25113 or MG1655 with the ASKA clone expressing panB under control of the lac promoter (ppanB) or with an empty vector (pBAD33) control. Sensitivity to trimethoprim and sulfathiazole was assessed for each strain by measuring the halos of inhibition of different concentrations of each drug. The strains overexpressing panB were more sensitive to both trimethoprim and sulfathiazole resulting in larger zones of inhibition (Figures 4A,B). E. coli MG1655 showed a greater increase in sensitivity to sulfathiazole when overexpressing panB than BW25113; conversely, BW25113 overexpressing panB had a greater increase in sensitivity to trimethoprim than MG1655. To confirm the exacerbated sensitivity, ten-fold serial dilutions of late log-phase cultures were spotted onto M9-glucose agar plates containing chloramphenicol, IPTG, and various levels of trimethoprim or sulfathiazole (Figure 4C). Trimethoprim at 1 μg/ml did not affect growth of the empty-vector control cells but reduced growth of the BW25113 panB-overexpressing cells by a factor of 105. Similarly, overexpressing panB made E. coli 102-fold more sensitive to 0.1 μg/ml sulfathiazole. As observed with the inhibition halos, the trimethoprim sensitivity of MG1655 overexpressing panB was less drastic than BW25113, however, the sulfathiazole sensitivity of both panB-overexpressing strains was similar.


Experimental and Metabolic Modeling Evidence for a Folate-Cleaving Side-Activity of Ketopantoate Hydroxymethyltransferase (PanB).

Thiaville JJ, Frelin O, García-Salinas C, Harrison K, Hasnain G, Horenstein NA, Díaz de la Garza RI, Henry CS, Hanson AD, de Crécy-Lagard V - Front Microbiol (2016)

Antifolate sensitivity (A) Overnight cultures of E. coli BW25113 and MG1655 carrying pCA24N::panB (ppanB) or pBAD33 (empty) were diluted 100-fold and spread onto M9-glucose agar medium containing chloramphenicol and IPTG (0.1 mM). Twenty microliter of (1) sulfathiazole at 100 μg/ml, (2) sulfathiazole at 50 μg/ml, (3) trimethoprim at 100 μg/ml, (4) trimethoprim at 50 μg/ml, or (N) media with no antibiotic were spotted onto filter disks. (B) Diameter of the halo inhibition were measured for three biological replicates of each and the average and standard deviation of each were calculated.* indicates significant difference from empty vector control (p > 0.05 determined by 2-tailed Student's t-test). (C) Serial dilutions of each strain were spotted onto M9-glucose agar plates with chloramphenicol and IPTG with trimethoprim (0 or 1 μg/ml) or sulfathiazole (0 or 0.1 μg/ml). E. coli overexpressing panB were more sensitive to both folate inhibitors than E. coli with empty vector control.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814558&req=5

Figure 4: Antifolate sensitivity (A) Overnight cultures of E. coli BW25113 and MG1655 carrying pCA24N::panB (ppanB) or pBAD33 (empty) were diluted 100-fold and spread onto M9-glucose agar medium containing chloramphenicol and IPTG (0.1 mM). Twenty microliter of (1) sulfathiazole at 100 μg/ml, (2) sulfathiazole at 50 μg/ml, (3) trimethoprim at 100 μg/ml, (4) trimethoprim at 50 μg/ml, or (N) media with no antibiotic were spotted onto filter disks. (B) Diameter of the halo inhibition were measured for three biological replicates of each and the average and standard deviation of each were calculated.* indicates significant difference from empty vector control (p > 0.05 determined by 2-tailed Student's t-test). (C) Serial dilutions of each strain were spotted onto M9-glucose agar plates with chloramphenicol and IPTG with trimethoprim (0 or 1 μg/ml) or sulfathiazole (0 or 0.1 μg/ml). E. coli overexpressing panB were more sensitive to both folate inhibitors than E. coli with empty vector control.
Mentions: The effect of overexpressing the panB was tested by transforming either E. coli strain BW25113 or MG1655 with the ASKA clone expressing panB under control of the lac promoter (ppanB) or with an empty vector (pBAD33) control. Sensitivity to trimethoprim and sulfathiazole was assessed for each strain by measuring the halos of inhibition of different concentrations of each drug. The strains overexpressing panB were more sensitive to both trimethoprim and sulfathiazole resulting in larger zones of inhibition (Figures 4A,B). E. coli MG1655 showed a greater increase in sensitivity to sulfathiazole when overexpressing panB than BW25113; conversely, BW25113 overexpressing panB had a greater increase in sensitivity to trimethoprim than MG1655. To confirm the exacerbated sensitivity, ten-fold serial dilutions of late log-phase cultures were spotted onto M9-glucose agar plates containing chloramphenicol, IPTG, and various levels of trimethoprim or sulfathiazole (Figure 4C). Trimethoprim at 1 μg/ml did not affect growth of the empty-vector control cells but reduced growth of the BW25113 panB-overexpressing cells by a factor of 105. Similarly, overexpressing panB made E. coli 102-fold more sensitive to 0.1 μg/ml sulfathiazole. As observed with the inhibition halos, the trimethoprim sensitivity of MG1655 overexpressing panB was less drastic than BW25113, however, the sulfathiazole sensitivity of both panB-overexpressing strains was similar.

Bottom Line: Tetrahydrofolate (THF) and its one-carbon derivatives, collectively termed folates, are essential cofactors, but are inherently unstable.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology and Cell Science, University of Florida Gainesville, FL, USA.

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.

No MeSH data available.


Related in: MedlinePlus