PURIFICATION AND CHARACTERIZATION OF ASSIMILATORY NITRITE REDUCTASE FROM CANDIDA-UTILIS

Nitrate assimilation in many plants, algae, yeasts and bacteria is med iated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reduc tase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase fro m an industrially important yeast, Candida utilis, has been purified t o homogeneity. Purified nitrite reductase is a heterodimer and the mol ecular masses of the two subunits are 58 and 66 kDa. The native enzyme exhibits a molecular mass of 126 kDa as analysed by gel filtration. T he identity of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductas e. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrat e-induced C. utilis followed by immunoprecipitation of the in vitro tr anslated products with heterologous nitrite reductase antibody and (b) Northern-blot analysis. The 66 kDa subunit is acidic in nature which is probably due to its phosphorylated status. The enzyme is stable ove r a range of temperatures. Both subunits can catalyse nitrite reductio n, and the reconstituted enzyme, at a higher protein concentration, sh ows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition t o a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic p rocess, a phenomenon not commonly seen for nitrite reductases from oth er systems.