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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

Proposed side activity of PanB. PanB initiates normal carbon transfer to α-ketoisovalerate to form the initial adduct which then subsequently undergoes aberrant hydrolytic attack to cleave the glutaminyl-p-ABA group from the pterin system. The resulting adduct can cleave by path A which generates 2-dehydropantoate and H4-HMPterin. In path B, hydrolytic cleavage releases unchanged α-ketoisovalerate and produces the bis-hydroxymethyl intermediate that spontaneously decomposes to formaldehyde and H4-HMPterin. Subsequent redox steps would convert the H4-HMPterin to H2-HMPterin for re-entry into the biosynthetic pathway.
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Figure 3: Proposed side activity of PanB. PanB initiates normal carbon transfer to α-ketoisovalerate to form the initial adduct which then subsequently undergoes aberrant hydrolytic attack to cleave the glutaminyl-p-ABA group from the pterin system. The resulting adduct can cleave by path A which generates 2-dehydropantoate and H4-HMPterin. In path B, hydrolytic cleavage releases unchanged α-ketoisovalerate and produces the bis-hydroxymethyl intermediate that spontaneously decomposes to formaldehyde and H4-HMPterin. Subsequent redox steps would convert the H4-HMPterin to H2-HMPterin for re-entry into the biosynthetic pathway.

Mentions: The panB gene encodes the CH2-THF-dependent enzyme ketopantoate hydroxymethyltransferase (EC 2.1.2.11). This suggested an explanation for the observed association between panB and folK based on the proposed reaction mechanism of PanB. Powers, Snell, and coworkers first purified this enzyme and showed that it is a class II aldolase (i.e., metal requiring) (Powers and Snell, 1976; Teller et al., 1976). In the proposed mechanism, PanB initiates the normal reaction sequence by forming an adduct between α-ketoisovalerate and CH2-THF; the adduct is then resolved by hydrolytic attack to generate THF and the product 2-dehydropantoate. We hypothesized a damage side-reaction in which the adduct first undergoes hydrolytic attack to liberate the pABA-Glu moiety (Figure 1), with concomitant formation of a hydroxymethyl group on the pterin/α-ketoisovalerate adduct (Figure 3). This adduct then undergoes a second hydrolytic attack, with two possible outcomes, both involving production of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-tetrahydropteridine (H4-HMPterin). In path A, the other reaction product is 2-dehydropantoate; in path B, α-ketoisovalerate is regenerated along with an unstable bis-hydroxymethylpterin, which spontaneously loses formaldehyde to give H4-HMPterin. The H4-HMPterin formed by either path could re-enter the folate pathway via FolK after spontaneous oxidation to the dihydro form (Figure 1), or possibly directly. We therefore made genetic tests of the hypothesis that PanB damages CH2-THF and that the resulting pterin moiety is recycled to folate via FolK.


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)

Proposed side activity of PanB. PanB initiates normal carbon transfer to α-ketoisovalerate to form the initial adduct which then subsequently undergoes aberrant hydrolytic attack to cleave the glutaminyl-p-ABA group from the pterin system. The resulting adduct can cleave by path A which generates 2-dehydropantoate and H4-HMPterin. In path B, hydrolytic cleavage releases unchanged α-ketoisovalerate and produces the bis-hydroxymethyl intermediate that spontaneously decomposes to formaldehyde and H4-HMPterin. Subsequent redox steps would convert the H4-HMPterin to H2-HMPterin for re-entry into the biosynthetic pathway.
© Copyright Policy
Related In: Results  -  Collection

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

Figure 3: Proposed side activity of PanB. PanB initiates normal carbon transfer to α-ketoisovalerate to form the initial adduct which then subsequently undergoes aberrant hydrolytic attack to cleave the glutaminyl-p-ABA group from the pterin system. The resulting adduct can cleave by path A which generates 2-dehydropantoate and H4-HMPterin. In path B, hydrolytic cleavage releases unchanged α-ketoisovalerate and produces the bis-hydroxymethyl intermediate that spontaneously decomposes to formaldehyde and H4-HMPterin. Subsequent redox steps would convert the H4-HMPterin to H2-HMPterin for re-entry into the biosynthetic pathway.
Mentions: The panB gene encodes the CH2-THF-dependent enzyme ketopantoate hydroxymethyltransferase (EC 2.1.2.11). This suggested an explanation for the observed association between panB and folK based on the proposed reaction mechanism of PanB. Powers, Snell, and coworkers first purified this enzyme and showed that it is a class II aldolase (i.e., metal requiring) (Powers and Snell, 1976; Teller et al., 1976). In the proposed mechanism, PanB initiates the normal reaction sequence by forming an adduct between α-ketoisovalerate and CH2-THF; the adduct is then resolved by hydrolytic attack to generate THF and the product 2-dehydropantoate. We hypothesized a damage side-reaction in which the adduct first undergoes hydrolytic attack to liberate the pABA-Glu moiety (Figure 1), with concomitant formation of a hydroxymethyl group on the pterin/α-ketoisovalerate adduct (Figure 3). This adduct then undergoes a second hydrolytic attack, with two possible outcomes, both involving production of 2-amino-4-hydroxy-6-hydroxymethyl-7,8-tetrahydropteridine (H4-HMPterin). In path A, the other reaction product is 2-dehydropantoate; in path B, α-ketoisovalerate is regenerated along with an unstable bis-hydroxymethylpterin, which spontaneously loses formaldehyde to give H4-HMPterin. The H4-HMPterin formed by either path could re-enter the folate pathway via FolK after spontaneous oxidation to the dihydro form (Figure 1), or possibly directly. We therefore made genetic tests of the hypothesis that PanB damages CH2-THF and that the resulting pterin moiety is recycled to folate via FolK.

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