Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastoris

• Published in: Plant physiology (Bethesda) 2000-06, Vol.123 (2), p.743-756
• Main Authors: Jeffrey A. Mertens, Shiraishi, Naomasa, Campbell, Wilbur H.
• Format: Article
• Language: English
• Subjects: Agronomy. Soil science and plant productions; Aldehyde Oxidoreductases - genetics; Aldehyde Oxidoreductases - isolation & purification; Aldehyde Oxidoreductases - metabolism; Biochemical Processes and Macromolecular Structures; Biological and medical sciences; Cell lines; Dimers; Economic plant physiology; Enzymes; Fundamental and applied biological sciences. Psychology; Gels; Kinetics; Metabolism; Mops; Multienzyme Complexes - genetics; Multienzyme Complexes - isolation & purification; Multienzyme Complexes - metabolism; Nitrate Reductase; Nitrate Reductases - genetics; Nitrates; Nitrogen metabolism; Nitrogen metabolism and other ones (excepting carbon metabolism); Nutrition. Photosynthesis. Respiration. Metabolism; Oxidation-Reduction; Pichia; Pichia - enzymology; Pichia - genetics; Plant physiology and development; Proteins; Recombinant Proteins - genetics; Recombinant Proteins - isolation & purification; Recombinant Proteins - metabolism; Spinach; Spinacia oleracea - enzymology
• ISSN: 0032-0889; 1532-2548
• DOI: 10.1104/pp.123.2.743

Mo reductase (MoR; formerly cytochrome c reductase) fragments of $\text{NADH:NO}_{3}$ reductase (NR; EC1.6.6.1) were cytosolically expressed in Pichia pastoris, a methylotrophic yeast, using spinach (Spinacia oleracea) and corn (Zea maize) cDNAs. In fermenter cultures, spinach MoR was expressed at 420 mg L-1, corn MoR at 32 mg L-1, and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L-1. Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanolinduced expression yielded more target protein. All three MoR were purified to homogeneity and their polypeptides were approximately 41 (MoR) and approximately 66 (MoR+) kD. MoR was monomeric and MoR+ dimeric, confirming the predicted role for dimer interface domain of NR. MoR+, although differing in quaternary structure from MoR, has similar kinetic properties for ferricyanide and cytochrome c reductase activities and visible spectra, which were like NR. Redox potentials of MoR and MoR+ were similar for flavin, whereas MoR+ had a more negative potential for heme-iron. Reaction schemes for MoR catalyzed reactions were proposed based on fast-reaction rapid-scan stopped-flow kinetic analysis of MoR. P. pastoris is an excellent system for producing the large amounts of NR fragments needed for detailed biochemical studies.