Purification and Properties of a Folate-catabolizing Enzyme

Abstract

We have identified and purified to homogeneity an enzyme from rat liver that catalyzes the oxidative catabolism of 5-formyltetrahydrofolate to p-aminobenzoylglutamate and a pterin derivative. Purification of the enzyme utilized six column matrices, including a pterin-6-carboxylic acid affinity column. Treatment of crude rat liver extracts with EDTA or heat decreased the specific activity of the enzyme by up to 85%. Peptides generated from the purified protein were sequenced and found to be identical to primary sequences present within rat light chain or heavy chain ferritin. Commercial rat ferritin did not display catabolic activity, but activity could be acquired with iron loading. The purified enzyme contained 2000 atoms of iron/ferritin 24-mer and displayed similar electrophoretic properties as commercial rat liver ferritin. The ferritin-catalyzed reaction displayed burst kinetics, and the enzyme catalyzed only a single turnover in vitro. Expression of rat heavy chain ferritin cDNA resulted in increased rates of folate turnover in cultured Chinese hamster ovary cells and human mammary carcinoma cells and reduced intracellular folate concentrations in Chinese hamster ovary cells. These results indicate that ferritin catalyzes folate turnover in vitro and in vivo and may be an important factor in regulating intracellular folate concentrations.

Methenyltetrahydrofolate Synthetase Regulates Folate Turnover and Accumulation

“…Therefore, alterations in the relative distribution of the folate one-carbon forms may influence total folate accumulation if an individual folate onecarbon derivative accumulates beyond the capacity of cellular proteins to sequester it. Recently, we identified heavy chain ferritin (HCF) as a protein that catalyzes folate catabolism in vitro and in cell cultures (27). By altering the distribution of folate cofactors, MTHFS may make certain folate derivatives more susceptible to undergo degradation by HCF-mediated catabolism.…”

“…The cellular forms of folate also differ in their susceptibility to oxidative degradation, with 5-substituted forms of reduced folate being the most stable (26,27). 5-Formyl-THF, which is used clinically (Leucovorin TM ) to elevate cellular folate levels, is the most stable naturally occurring form of reduced folate.…”

“…Increased rates of folate catabolism and turnover can deplete cellular folate concentrations (27), and may account for differences in folate turnover rates and total folate content that exist among tissues (28 -30). Localized cellular folate deficiency can occur in the absence of whole body folate deficiency, and this phenomenon may result from accelerated rates of folate catabolism (9,15,31,32).…”

“…Folic acid, a synthetic form of folate found in fortified food and vitamin supplements, is also a stable form of folate. Unsubstituted forms of folate, which include THF and dihydrofolate, and the 10-substituted folates, are chemically labile and susceptible to rapid oxidative degradation in vitro (26,27). There are two pathways for folate oxidation: one resulting in the formation of dihydro forms of the cofactor (dihydrofolate, 5-methyldihydrofolate); the other resulting in the oxidative cleavage of the C9 -N10 bond of dihydrofolates and tetrahydrofolates resulting in the formation of the pterin derivative and para-aminobenzoyl(poly)glutamate (pABG) (15).…”

Folate pathway gene expression differs in subtypes of acute lymphoblastic leukemia and influences methotrexate pharmacodynamics

“…P values are from pairwise comparisons using the Wilcoxon rank sum test after adjustment for multiple testing. (27), and GTP cyclohydrolase 1 (GCH1), which catalyzes sequential reactions in folate biosynthesis. The expression patterns among ALL subtypes of the 3 MTX transporters and FPGS are depicted in Figure 3; the expression patterns of 4 additional subtype-discriminating folate/antifolate pathway genes (ATIC, GART, MTHFD1, and MTHFD2) are provided in Supplemental Figure 4.…”