Purification and characterization of assimilatory nitrite reductase from Candida utilis.
Nitrate assimilation in many plants, algae, yeasts and bacteria is mediated by two enzymes, nitrate reductase (EC 1.6.6.2) and nitrite reductase (EC 1.7.7.1). They catalyse the stepwise reduction of nitrate to nitrite and nitrite to ammonia respectively. The nitrite reductase from an industrially important yeast, Candida utilis, has been purified to homogeneity. Purified nitrite reductase is a heterodimer and the molecular 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. The identify of the two subunits of nitrite reductase was confirmed by immunoblotting using antibody for Cucurbita pepo leaf nitrite reductase. The presence of two different sized transcripts coding for the two subunits was confirmed by (a) in vitro translation of mRNA from nitrate-induced C. utilis followed by immunoprecipitation of the in vitro translated 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 over a range of temperatures. Both subunits can catalyse nitrite reduction, and the reconstituted enzyme, at a higher protein concentration, shows an activity similar to that of the purified enzyme. Each of these subunits has been shown to contain a few unique peptides in addition to a large number of common peptides. Reduced Methyl Viologen has been found to be as effective an electron donor as NADPH in the catalytic process, a phenomenon not commonly seen for nitrite reductases from other systems.
Sengupta
S
S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore, India.
Shaila
M S
MS
Rao
G R
GR
D002175:Candida / Q000201:enzymology*; D004591:Electrophoresis, Polyacrylamide Gel; D015151:Immunoblotting; D009572:Nitrite Reductases / Q000302:isolation & purification* / Q000378:metabolism; D010449:Peptide Mapping; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011489:Protein Denaturation; D012333:RNA, Messenger
D012333:RNA, Messenger; D009572:Nitrite Reductases