Purification and biochemical characterization of simplified eukaryotic nitrate reductase expressed in Pichia pastoris

Abstract

NAD(P)H:nitrate reductase (NaR, EC 1.7.1.1-3) is a useful enzyme in biotechnological applications, but it is very complex in structure and contains three cofactors—flavin adenine dinucleotide, heme–Fe, and molybdenum–molybdopterin (Mo–MPT). A simplified nitrate reductase (S-NaR1) consisting of Mo–MPT-binding site and nitrate-reducing active site was engineered from yeast Pichia angusta NaR cDNA (YNaR1). S-NaR1 was cytosolically expressed in high-density fermenter culture of methylotrophic yeast Pichia pastoris. Total amount of S-NaR1 protein produced was ∼0.5 g per 10 L fermenter run, and methanol phase productivity was 5 μg protein/g wet cell weight/h. Gene copy number in genomic DNA of different clones showed direct correlation with the expression level. S-NaR1 was purified to homogeneity in one step by immobilized metal affinity chromatography (IMAC) and total amount of purified protein per run of fermentation was ∼180 mg. Polypeptide size was ∼55 kDa from electrophoretic analysis, and S-NaR1 was mainly homo-tetrameric in its active form, as shown by gel filtration. S-NaR1 accepted electrons efficiently from reduced bromphenol blue ( k cat=2081 s −1) and less so from reduced methyl viologen ( k cat=159 s −1). The nitrate K M for S-NaR1 was 30 ± 3 μM, which is very similar to YNaR1. S-NaR1 is capable of specific nitrate reduction, and direct electric current, as shown by catalytic nitrate reduction using protein film cyclic voltammetry, can drive this reaction. Thus, S-NaR1 is an ideal form of this enzyme for commercial applications, such as an enzymatic nitrate biosensor formulated with S-NaR1 interfaced to an electrode system.