Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris.

摘要: In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed most other higher-plant NRs by an unusually strong Mg2+-sensitivity, different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099–1105]. order to elucidate these deviating properties in more detail, the NR gene R. was cloned, expressed heterologously characterized. The deduced protein sequence has serine phosphorylation site 14-3-3 binding motif, common characteristic NRs. Functional yeast Pichia pastoris compared with features Arabidopsis thaliana NR2 synthesized same expression system (AtNR2). recombinant (RcNR) itself not inactivated incubation MgATP. As extracts might lack factors required for regulation, desalted leaf containing kinases proteins were prepared 4-day-darkened (and therefore NR-free) Ricinus, added assay RcNR check Mg2+-sensitivity. When combined NR-free described above, its high Mg2+-sensitivity restored, but it remained unresponsive ATP. contrast, AtNR2 became inactive when incubated mixture Thus, insensitivity ATP appears be inherent property NR, whereas depends on one or several leaves. This as yet unknown factor(s) boiling-sensitive appeared interact specifically provide authentic enzyme.