ANALYSIS OF WILD-TYPE AND MUTANT PLANT NITRATE REDUCTASE EXPRESSED INTHE METHYLOTROPHIC YEAST PICHIA-PASTORIS

Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6. 1) was produced in the methylotrophic yeast Pichia pastoris and purifi ed to near-electrophoretic homogeneity. purified enzyme had the spectr al and kinetic properties typical of highly purified NR from natural p lant sources. Site-directed mutagenesis altering several key residues and regions was carried out, and the mutant enzyme forms were expresse d in P. pastoris. When the invariant cysteine residue, cysteine-191, i n the molybdo-pterin region of the A. thaliana NIA2 protein was replac ed with serine or alanine, the NR protein was still produced but was i nactive, showing that this residue is essential for enzyme activity. D eletions or substitutions of the conserved N terminus of NR retained a ctivity and the ability to be inactivated in vitro when incubated with ATE. Enzyme with a histidine sequence appended to the N terminus was still active and was easily purified using metal-chelate affinity chro matography. These results demonstrate that P. pastoris is a useful and reliable system for producing recombinant holo-NR from plants.