Plant Physiol, June 2000, Vol. 123, pp. 743-756

Recombinant Expression of Molybdenum Reductase Fragments of Plant Nitrate Reductase at High Levels in Pichia pastoris¹

Department of Biological Sciences and Phytotechnology Research Center, Michigan Technological University, 1400 Townsend Drive, Houghton, Michigan 49931

Mo reductase (MoR; formerly cytochrome c reductase) fragments of NADH:NO₃ 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 L¹, corn MoR at 32 mg L¹, and corn MoR plus with putative NR interface domain N terminus (MoR+) at 17 mg L¹. Constitutively expressed MoR+ was structurally stable while it was degraded when expressed by methanol induction, which suggests methanol growth produces more proteinase. Methanol-induced 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.