Analysis of wild-type and mutant plant nitrate reductase expressed in the methylotrophic yeast Pichia pastoris

• Published in: Plant physiology (Bethesda) Vol. 115; no. 3; pp. 1135 - 1143
• Main Authors: Su, W.P, Mertens, J.A, Kanamaru, K, Campbell, W.H, Crawford, N.M
• Format: Journal Article
• Language: English
• Published: Rockville, MD American Society of Plant Physiologists 01-11-1997
• Subjects: ACTIVIDAD ENZIMATICA; ACTIVITE ENZYMATIQUE; ALANINA; ALANINE; Amino acids; Arabidopsis - enzymology; ARABIDOPSIS THALIANA; ATP; Biochemistry and Enzymology; Biological and medical sciences; CISTEINA; CYSTEINE; DELETIONS; Dimers; Enzymes; ENZYMIC ACTIVITY; EXPRESION GENICA; EXPRESSION DES GENES; Fundamental and applied biological sciences. Psychology; Gels; GENE; GENE EXPRESSION; GENES; GENETIC ENGINEERING; GENETIC VARIATION; GENIE GENETIQUE; INGENIERIA GENETICA; KINETICS; Metabolism; MUTACION; MUTAGENESIS; Mutagenesis, Site-Directed; MUTANT; MUTANTES; MUTANTS; MUTATION; NITRATE REDUCTASE; Nitrate Reductases - chemistry; Nitrate Reductases - genetics; Nitrates; NITRATO REDUCTASA; Oxidases; PICHIA; Pichia - genetics; Plant physiology and development; PROTEINAS RECOMBINANTES; PROTEINE RECOMBINANTE; Proteins; PURIFICACION; PURIFICATION; RECOMBINANT PROTEINS; Recombinant Proteins - chemistry; Recombinant Proteins - genetics; SERINA; SERINE; Spectrum Analysis; Spinach; SUBSTRATES; Sulfites; VARIACION GENETICA; VARIATION GENETIQUE; WILD STRAINS; Recombinant microorganism; Nitrate reductase (NADH); Heterologous system; Purification; Enzyme; Spectral properties; Site directed mutagenesis; Gene expression; Structure function relationship; Fungi; Arabidopsis thaliana; Enzymatic activity; Cruciferae; Dicotyledones; Angiospermae; N terminal-Sequence; Ascomycetes; Spermatophyta; Oxidoreductases; Kinetics; Experimental plant; Thallophyta
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.115.3.1135

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