Purification and partial characterization of nitrate reductase from halotolerant cyanobacterium Aphanothece halophytica

Abstract

Nitrate is a major nitrogen source for the growth of cyanobacteria. Nitrate reductase (NR) catalyzes the conversion of nitrate to nitrite. The enzyme is important in nitrate metabolism regulation. In this work, we found that ammonium, the intermediate of nitrogen metabolism, reduced nitrate reductase in Aphanothece halophytica. Glutamine also decreased nitrate reductase activity. Uner salt stress condition, nitrate reductase was lower than normal condition due to repression of growth. Nitrate reductase from A. halophytica was localized in cytoplasmic fraction, suggesting its possible involvement in the nitrate assimilation. Purification of nitrate reductase was done by 4 steps including: ultracentrifugation, 20 – 60 % ammonium sulfate precipitation, DEAE-Toyopearl chromatography and Bio-Gel hydroxyapatite chromatography. The enzyme was purified to homogeneity with 15.7 % yield and 406 purification folds. The Km value for nitrate was 465 µM and Vmax value was 32 nmol/min/mg protein using methyl viologen assay. The purified nitrate reductase used ferredoxin as physiological electron donor with specific activity was 14.6 nmol/ min/ mg protein. Inhibition studies revealed that p-chloromercuribenzoate, iodoacetamide, N-ethylmaleimide, potassium cyanide and chlorate could strongly inhibit enzyme activity whereas azide and nitrite had little or no effect on the enzyme activity.