Characterization of the folate salvage enzyme p-aminobenzoylglutamate hydrolase in plants.
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.
Bozzo
Gale G
GG
Horticultural Sciences Department, University of Florida, Gainesville, FL 32611, United States.
Basset
Gilles J C
GJ
Naponelli
Valeria
V
Noiriel
Alexandre
A
Gregory
Jesse F
JF
Hanson
Andrew D
AD
D017360:Arabidopsis / Q000201:enzymology; D002850:Chromatography, Gel; D005492:Folic Acid / Q000378:metabolism*; D005971:Glutamates / Q000378:metabolism*; D006867:Hydrolases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000302:isolation & purification / Q000378:metabolism*; D006868:Hydrolysis; D007700:Kinetics; D018532:Pisum sativum / Q000201:enzymology; D018515:Plant Leaves / Q000201:enzymology; D018517:Plant Roots / Q000201:enzymology; D010944:Plants / Q000201:enzymology*; D011623:gamma-Glutamyl Hydrolase / Q000378:metabolism
D005971:Glutamates; D005492:Folic Acid; C011398:4-aminobenzoylglutamic acid; D006867:Hydrolases; D011623:gamma-Glutamyl Hydrolase