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

Abstract Folates break down in vivo to give pterin and p -aminobenzoylglutamate ( p ABAGlu) fragments, the latter usually having a polyglutamyl tail. Pilot studies have shown that plants can hydrolyze p ABAGlu and its polyglutamates to p -aminobenzoate, a folate biosynthesis precursor. The enzymatic basis of this hydrolysis was further investigated. p ABAGlu 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 ∼200 kDa. Partially purified preparations showed a pH optimum near 7.5, a K m value for p ABAGlu of 370 μM, 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 Mn 2+ , 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 p ABAGlu. The sole significant matches were auxin conjugate hydrolase family members and the At4g17830 protein. None was found to have significant p ABAGlu 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.