Molecular cloning and characterization of nitrate reductase from Ricinus communis L. heterologously expressed in Pichia pastoris.
In a previous paper, we showed that nitrate reductase (NR; EC 1.6.6.1) from leaves of Ricinus communis L. differed from most other higher-plant NRs by an unusually strong Mg2+-sensitivity, a different pH-activity profile and only little ATP-dependent inactivation [A. Kandlbinder et al. (2000) J Exp Bot 51:1099-1105]. In order to elucidate these deviating properties in more detail, the NR gene from R. communis was cloned, expressed heterologously and characterized. The deduced protein sequence showed that Ricinus NR has a serine phosphorylation site and a 14-3-3 binding motif, a common characteristic of NRs. Functional Ricinus NR protein was expressed in the yeast Pichia pastoris and compared with the features of Arabidopsis thaliana NR2 synthesized by the same expression system (AtNR2). The recombinant Ricinus NR (RcNR) itself was not inactivated by incubation with MgATP. As yeast extracts might lack factors required for NR regulation, desalted leaf extracts containing NR kinases and 14-3-3 proteins were prepared from 4-day-darkened (and therefore NR-free) leaves of Ricinus, and added to the assay of RcNR to check for ATP-dependent inactivation and Mg2+-sensitivity. When RcNR was combined with the NR-free extracts described above, its unusually high Mg2+-sensitivity was restored, but it remained unresponsive to ATP. In contrast, AtNR2 became inactive when incubated with the protein mixture and ATP. Thus, insensitivity to ATP appears to be an inherent property of Ricinus NR, whereas the high Mg2+-sensitivity depends on one or several factors in Ricinus leaves. This as yet unknown factor(s) was boiling-sensitive and appeared to interact specifically with recombinant Ricinus NR to provide the Mg2+-sensitivity of the authentic leaf enzyme.
Tsai
Chyn-Bey
CB
Lehrstuhl für Molekulare Pflanzenphysiologie und Biophysik, Julius-von-Sachs-Institut für Biowissenschaften, Julius-von-Sachs-Platz 2, 97082 Würzburg, Germany.
Kaiser
Werner M
WM
Kaldenhoff
Ralf
R
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000595:Amino Acid Sequence; D003001:Cloning, Molecular / Q000379:methods; D018744:DNA, Plant / Q000235:genetics / Q000302:isolation & purification; D007700:Kinetics; D008969:Molecular Sequence Data; D050901:Nitrate Reductase; D009565:Nitrate Reductases / Q000235:genetics* / Q000378:metabolism; D010843:Pichia / Q000201:enzymology / Q000235:genetics*; D018749:RNA, Plant / Q000235:genetics / Q000302:isolation & purification; D011994:Recombinant Proteins / Q000378:metabolism; D012278:Ricinus / Q000201:enzymology*; D016415:Sequence Alignment; D017386:Sequence Homology, Amino Acid
D018744:DNA, Plant; D018749:RNA, Plant; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate; D009565:Nitrate Reductases; D050901:Nitrate Reductase