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PLANT PHYSIOLOGY , Vol 115, Issue 3 1135-1143, Copyright © 1997 by American Society of Plant Biologists
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BIOCHEMISTRY AND ENZYMOLOGY |
Analysis of Wild-Type and Mutant Plant Nitrate Reductase Expressed in the Methylotrophic Yeast Pichia pastoris
W. Su, J. A. Mertens, K. Kanamaru, W. H. Campbell and N. M. Crawford
Department of Biology, University of California, San Diego, La Jolla, California 92093-0116 (W.S., K.K., N.M.C.)
Recombinant Arabidopsis thaliana NADH:nitrate reductase (NR; EC 1.6.6.1)
was produced in the methylotrophic yeast Pichia pastoris and purified to
near-electrophoretic homogeneity. Purified enzyme had the spectral and
kinetic properties typical of highly purified NR from natural plant
sources. Site-directed mutagenesis altering several key residues and
regions was carried out, and the mutant enzyme forms were expressed in P.
pastoris. When the invariant cysteine residue, cysteine-191, in the
molybdo-pterin region of the A. thaliana NIA2 protein was replaced with
serine or alanine, the NR protein was still produced but was inactive,
showing that this residue is essential for enzyme activity. Deletions or
substitutions of the conserved N terminus of NR retained activity and the
ability to be inactivated in vitro when incubated with ATP. Enzyme with a
histidine sequence appended to the N terminus was still active and was
easily purified using metal-chelate affinity chromatography. These results
demonstrate that P. pastoris is a useful and reliable system for producing
recombinant holo-NR from plants.
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