Manganese is essential for activity of allantoate amidinohydrolase from Chlamydomonas reinhardtii

Allantoicase (allantoate amidinohydrolase, EC 3.5.3.4) from Chlamydomonas reinhardtii catalyses the degradation of allantoate to (−)ureidoglycolate and of (+)ureidoglycolate to glyoxylate, in both cases with urea as the other product. Allantoicase activity purified in buffers without any cations increased after manganese addition to the assay mixture reaching a saturation maximun with 0.25 mM. The allantoicase activity was strongly inhibited after EDTA treatment. The activity of the EDTA-treated enzyme was restored after incubation with Mn 2+, whereas the incubation with Cu 2+ resulted in a fully inactivated preparation. Others cations tested (Ca 2+, Fe 2+, Mg 2+, Zn 2+, Co 2+) had no effect on activity. The same results were obtained independently of the substrate used in all the experiments performed. A manganese-depleted allantoicase, obtained by purification with manganese free buffers, bound radioactive manganese in vitro. This binding is strong since enzyme retained the radioactive cation after gel chromatography and SDS-PAGE. Results obtained are the first clear demonstration of manganese binding in vitro to any allantoate-degrading enzyme from a photosynthetic organism and that manganese is essential for allantoicase activity.