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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

Clustering and phylogeny of folK. (A) Physical clustering of folK homologs with panB or folB in specific genomes. (B) A phylogenetic tree of FolB-clustered FolK (red), PanB-clustered FolK (blue), and FolB and PanB-clustered FolK (green). The sequences were aligned with MUSCLE and the tree was calculated with MEGA6 using the maximum likelihood method.
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Figure 2: Clustering and phylogeny of folK. (A) Physical clustering of folK homologs with panB or folB in specific genomes. (B) A phylogenetic tree of FolB-clustered FolK (red), PanB-clustered FolK (blue), and FolB and PanB-clustered FolK (green). The sequences were aligned with MUSCLE and the tree was calculated with MEGA6 using the maximum likelihood method.

Mentions: Physical clustering analysis showed that in ~70% of the genomes with two copies of folK, each folK gene occurs in a particular genomic context. One copy clusters with folB, which encodes the preceding step in folate synthesis (Figure 1), and the other clusters with a pantothenate biosynthesis gene, panB, as seen in Acinetobacter baylyi ADP1 (Figure 2A). These clusters also reflect the gene organization in genomes that have only one folK gene. Thus, 25% of the organisms in SEED harbor a single folK gene that clusters with folB (e.g., Salmonella enterica) and 18% harbor a single folK gene that clusters with panB (e.g., Bacillus subtilis) (Figure 2A). Furthermore in certain genomes (e.g., Desulforudis audaxviator) a single folK gene clusters with both panB and folB (Figure 2A). To eliminate any bias introduced by overrepresentation of certain species, the analysis was repeated on a set of 981 organisms chosen for their diversity (Niehaus et al., 2015). Very similar results were obtained: 7% of the genomes contained two folK genes with one next to panB and the other next to folB; 20% of the genomes had a unique folK next to folB; and 11% had a unique folK next to panB. Consistent with the functional link between the folK and panB genes implied by their clustering in bacteria, the FolK and PanB enzymes localize to the same subcellular compartment (the mitochondrial matrix) in plants and yeast (Güldener et al., 2004; Ottenhof et al., 2004; Perocchi et al., 2006; Gerdes et al., 2012). To explore structural, and potential functional, differences between the products of folK genes clustered with panB or folB genes, we aligned ~270 FolK sequences that were chosen from a diverse set of prokaryotes that included organisms with folK duplications. In order to produce a higher quality alignment, proteins were kept only if their lengths were within 0.25 standard deviations of the average length of the set, or if there was another FolK encoded in the same genome. Sequence logo comparisons showed differences in enrichments for specific amino acids at given positions between the FolK proteins whose genes physically cluster with panB genes and those that cluster with folB (Figure S1). These amino acid differences between the two groups were confirmed by phylogenetic analyses on the same set of sequences as that for the majority of FolK proteins; the phylogenetic clustering broadly matched the physical clustering (Figure 2B). The separation is not perfect, however, so these signatures do not necessarily point to a functional divergence between the two FolK groups.


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)

Clustering and phylogeny of folK. (A) Physical clustering of folK homologs with panB or folB in specific genomes. (B) A phylogenetic tree of FolB-clustered FolK (red), PanB-clustered FolK (blue), and FolB and PanB-clustered FolK (green). The sequences were aligned with MUSCLE and the tree was calculated with MEGA6 using the maximum likelihood method.
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Related In: Results  -  Collection

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Figure 2: Clustering and phylogeny of folK. (A) Physical clustering of folK homologs with panB or folB in specific genomes. (B) A phylogenetic tree of FolB-clustered FolK (red), PanB-clustered FolK (blue), and FolB and PanB-clustered FolK (green). The sequences were aligned with MUSCLE and the tree was calculated with MEGA6 using the maximum likelihood method.
Mentions: Physical clustering analysis showed that in ~70% of the genomes with two copies of folK, each folK gene occurs in a particular genomic context. One copy clusters with folB, which encodes the preceding step in folate synthesis (Figure 1), and the other clusters with a pantothenate biosynthesis gene, panB, as seen in Acinetobacter baylyi ADP1 (Figure 2A). These clusters also reflect the gene organization in genomes that have only one folK gene. Thus, 25% of the organisms in SEED harbor a single folK gene that clusters with folB (e.g., Salmonella enterica) and 18% harbor a single folK gene that clusters with panB (e.g., Bacillus subtilis) (Figure 2A). Furthermore in certain genomes (e.g., Desulforudis audaxviator) a single folK gene clusters with both panB and folB (Figure 2A). To eliminate any bias introduced by overrepresentation of certain species, the analysis was repeated on a set of 981 organisms chosen for their diversity (Niehaus et al., 2015). Very similar results were obtained: 7% of the genomes contained two folK genes with one next to panB and the other next to folB; 20% of the genomes had a unique folK next to folB; and 11% had a unique folK next to panB. Consistent with the functional link between the folK and panB genes implied by their clustering in bacteria, the FolK and PanB enzymes localize to the same subcellular compartment (the mitochondrial matrix) in plants and yeast (Güldener et al., 2004; Ottenhof et al., 2004; Perocchi et al., 2006; Gerdes et al., 2012). To explore structural, and potential functional, differences between the products of folK genes clustered with panB or folB genes, we aligned ~270 FolK sequences that were chosen from a diverse set of prokaryotes that included organisms with folK duplications. In order to produce a higher quality alignment, proteins were kept only if their lengths were within 0.25 standard deviations of the average length of the set, or if there was another FolK encoded in the same genome. Sequence logo comparisons showed differences in enrichments for specific amino acids at given positions between the FolK proteins whose genes physically cluster with panB genes and those that cluster with folB (Figure S1). These amino acid differences between the two groups were confirmed by phylogenetic analyses on the same set of sequences as that for the majority of FolK proteins; the phylogenetic clustering broadly matched the physical clustering (Figure 2B). The separation is not perfect, however, so these signatures do not necessarily point to a functional divergence between the two FolK groups.

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