A novel autophosphorylation mediated regulation of nitrite reductase in Candida utilis

Abstract

The assimilatory nitrite reductase catalyses the conversion of nitrite to ammonia. The enzyme from Candida utilis has been previously purified to homogeneity and shown to be a heterodimer consisting of 58 kDa and 66 kDa subunits. The enzyme has also been shown to be induced by nitrate and repressed by ammonium ions. The levels of nitrite reductase mRNA, its protein and the enzyme activity were modulated together indicating that the primary level of regulation of this enzyme existed at the transcriptional level. Here we report that the 58 kDa and 66 kDa subunits of the enzyme were differentially phosphorylated under the induced and repressed conditions, indicating a second level of regulation. The highly phosphorylated 66 kDa subunit was shown to be dephosphorylated by calf intestinal alkaline phosphatase. The enzymatic activity associated with the native enzyme also decreased due to the dephosphorylation. Each of the subunits could undergo autophosphorylation at serine/threonine residues as demonstrated by thin layer chromatography and recognition by antibodies to phosphoamino acids. The presence of similar phosphorylated subunits under in vivo conditions has also been demonstrated. A model has been proposed to explain the posttranslational regulation of the enzyme.

Assimilation of nitrate by yeasts

“…Dephosphorylation of this enzyme form in vitro by alkaline phosphate correlates with a decrease of nitrite reductase activity. In ammonium-grown cells nitrite reductase activity was 20% of that in cells grown in nitrate and the phosphorylation degree was decreased about 80%, but both subunits were phosphorylated [71,72]. Unfortunately no further information is available either on the phosphorylation sites or on the participants in the reaction.…”

Assimilation of nitrate by yeasts

“…Dephosphorylation of this enzyme form in vitro by alkaline phosphate correlates with a decrease of nitrite reductase activity. In ammonium‐grown cells nitrite reductase activity was 20% of that in cells grown in nitrate and the phosphorylation degree was decreased about 80%, but both subunits were phosphorylated [71,72]. Unfortunately no further information is available either on the phosphorylation sites or on the participants in the reaction.…”

Down-regulation of Eukaryotic Nitrate Transporter by Nitrogen-dependent Ubiquitinylation

“…Ynt1 shows high similarity to NrtA (formerly designated CRNA) and NrtB from Aspergillus nidulans (11), NIT10 from Neurospora crassa (12), and high affinity nitrate transporters from plants and algae (9). Previous studies have shown that nitrate and nitrite reductases are subjected to posttranslational regulation (2,(13)(14)(15)(16). However, there have been no detailed studies exploring posttranslational modifications of nitrate transporters in response to nitrogen sources (17).…”