Characterization of the folate salvage enzyme p-aminobenzoylglutamate hydrolase in plants

Abstract

Folates break down in vivo to give pterin and p-aminobenzoylglutamate (pABAGlu) fragments, the latter usually having a polyglutamyl tail. Pilot studies have shown that plants can hydrolyze pABAGlu and its polyglutamates to p-aminobenzoate, a folate biosynthesis precursor. The enzymatic basis of this hydrolysis was further investigated. pABAGlu hydrolase activity was found in all species and organs tested; activity levels implied that the proteins responsible are very rare. The activity was located in cytosol/vacuole and mitochondrial fractions of pea (Pisum sativum L.) leaves, and column chromatography of the activity from Arabidopsis tissues indicated at least three peaks. A major activity peak from Arabidopsis roots was purified 86-fold by a three-column procedure; activity loss during purification exceeded 95%. Size exclusion chromatography gave a molecular mass of approximately 200 kDa. Partially purified preparations showed a pH optimum near 7.5, a Km value for pABAGlu of 370 microM, and activity against folic acid. Activity was relatively insensitive to thiol and serine reagents, but was strongly inhibited by 8-hydroxyquinoline-5-sulfonic acid and stimulated by Mn2+, pointing to a metalloenzyme. The Arabidopsis genome was searched for proteins similar to Pseudomonas carboxypeptidase G, which contains zinc and is the only enzyme yet confirmed to attack pABAGlu. The sole significant matches were auxin conjugate hydrolase family members and the At4g17830 protein. None was found to have significant pABAGlu hydrolase activity, suggesting that this activity resides in hitherto unrecognized enzymes. The finding that Arabidopsis has folate-hydrolyzing activity points to an enzymatic component of folate degradation in plants.

Folates in Plants: Research Advances and Progress in Crop Biofortification

“…Although plant PGH genes have not been identified yet, PGH activities were found in Arabidopsis, pea and tomato (Orsomando et al, 2006). Studies on Arabidopsis and pea detected PGH activity in mitochondria, cytosol and vacuoles and suggested existence of at least two isoforms of the enzyme (Bozzo et al, 2008), which is in agreement with the finding of two isoforms of the enzyme in E. coli (Hussein et al, 1998). …”

Metabolism of the Folate Precursor p-Aminobenzoate in Plants

“…After the incubation, protoplasts were prepared from the [ 14 C]pABA-fed leaflets together with unlabeled pea leaflets (24 g) following a 5-h digestion as described (20) except that the centrifugation step used to harvest protoplasts included a cushion of 50% (v/v) Percoll in 10 mM MESNaOH, pH 7.0, 0.5 M sorbitol, 5 mM CaCl 2 , 0.5 mg/ml polyvinylpyrrolidone-40. The protoplast layers were adjusted to 13% Percoll in the same buffer and purified on a three-step sucrosesorbitol gradient as described (21). Vacuoles were prepared from lysed protoplasts (20) after adding to the lysate unlabeled folic acid to a final concentration of 50 M and 2-25 Ci (0.05-0.58 nmol) of [ 3 H]folic acid.…”

Purification and Characterization of the Folate Catabolic Enzyme p -Aminobenzoyl-Glutamate Hydrolase from Escherichia coli

“…Plants have high rates of folate catabolism, degrading approximately 10% of their total folates per day, and there is evidence that plants may salvage these breakdown products (3,(16)(17)(18)(19). Bozzo et al identified a PGH activity in pea (Pisum sativum L.) leaves and in Arabidopsis roots that has some similarities to the E. coli enzyme: it hydrolytically cleaves PABA-GLU and is stimulated by manganese (3).…”