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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

Model-predicted changes in the concentrations of folates and their precursors in response to overexpression of PanB. (A) The response when only the Vmax of the PanB folate-cleaving reaction is increased, leaving other model parameters the same. (B) The response when both the PanB Vmax and the external flux into 6-hydroxymethyldihydropterin are increased at the same time.
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Figure 7: Model-predicted changes in the concentrations of folates and their precursors in response to overexpression of PanB. (A) The response when only the Vmax of the PanB folate-cleaving reaction is increased, leaving other model parameters the same. (B) The response when both the PanB Vmax and the external flux into 6-hydroxymethyldihydropterin are increased at the same time.

Mentions: Next, we simulated the overexpression of PanB by increasing only the kinetic constant of the mass-action kinetic equation for the PanB folate-cleaving reaction (which integrates the enzyme concentration of PanB), leaving all other model parameters the same. In this simulation, all metabolite concentrations started at the steady-state values predicted from the wild-type model (Figure 7A). Immediately, the CH2-THF (and THF) concentration began to drop due to the larger flux through the PanB side reaction. This led to a rise in the concentrations of H2-HMPt (and its pyrophosphate, the PanK reaction product), which in turn led to a rise in the flux through the THF synthesis pathway, leading to the slow recovery of the THF concentration back to—but not exceeding—the wild-type values. Thus, this simulation successfully replicates the rise in H2-HMPt concentration that accompanied the overexpression of PanB in our experiments, but it fails to replicate the rise in THF concentrations, meaning the overexpression of PanB alone cannot explain all experimental observations.


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)

Model-predicted changes in the concentrations of folates and their precursors in response to overexpression of PanB. (A) The response when only the Vmax of the PanB folate-cleaving reaction is increased, leaving other model parameters the same. (B) The response when both the PanB Vmax and the external flux into 6-hydroxymethyldihydropterin are increased at the same time.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 7: Model-predicted changes in the concentrations of folates and their precursors in response to overexpression of PanB. (A) The response when only the Vmax of the PanB folate-cleaving reaction is increased, leaving other model parameters the same. (B) The response when both the PanB Vmax and the external flux into 6-hydroxymethyldihydropterin are increased at the same time.
Mentions: Next, we simulated the overexpression of PanB by increasing only the kinetic constant of the mass-action kinetic equation for the PanB folate-cleaving reaction (which integrates the enzyme concentration of PanB), leaving all other model parameters the same. In this simulation, all metabolite concentrations started at the steady-state values predicted from the wild-type model (Figure 7A). Immediately, the CH2-THF (and THF) concentration began to drop due to the larger flux through the PanB side reaction. This led to a rise in the concentrations of H2-HMPt (and its pyrophosphate, the PanK reaction product), which in turn led to a rise in the flux through the THF synthesis pathway, leading to the slow recovery of the THF concentration back to—but not exceeding—the wild-type values. Thus, this simulation successfully replicates the rise in H2-HMPt concentration that accompanied the overexpression of PanB in our experiments, but it fails to replicate the rise in THF concentrations, meaning the overexpression of PanB alone cannot explain all experimental observations.

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