Identification and purification of hydroxyisourate hydrolase, a novel ureide-metabolizing enzyme

We report the identification and purification of a novel enzyme from soybea n root nodules that catalyzes the hydrolysis of 5-hydroxyisourate, which is the true product of the urate oxidase reaction. The product of this reacti on is 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline, and the new enzyme is designated 5-hydroxyisourate hydrolase, The enzyme was purified from crude extracts of soybean root nodules similar to 100-fold to apparent homogeneit y with a final specific activity of 10 mu mol/min/mg, The enzyme exhibited a native molecular mass of similar to 68 kDa by gel filtration chromatograp hy and migrated as a single band on SDS-polyacrylamide gel electrophoresis with a subunit molecular mass of 68 +/- 2 kDa. The purified enzyme obeyed n ormal Michaelis-Menten kinetics, and the K-m for 5-hydroxyisourate was dete rmined to be 15 mu M The amino terminal end of the purified protein was seq uenced, and the resulting sequence was not found in any available data base s, confirming the novelty of the protein. These data suggest the existence of a hitherto unrecognized enzymatic pathway for the formation of allantoin .