The trace element molybdenum is essential for nearly all organisms and forms the catalytic centre of a large variety of enzymes such as nitrogenase, nitrate reductases, sulphite oxidase and xanthine oxidoreductases. Nature has developed two scaffolds holding molybdenum in place, the iron-molybdenum cofactor and pterin-based molybdenum cofactors. Despite the different structures and functions of molybdenum-dependent enzymes, there are important similarities, which we highlight here. The biosynthetic pathways leading to both types of cofactor have common mechanistic aspects relating to scaffold formation, metal activation and cofactor insertion into apoenzymes, and have served as an evolutionary 'toolbox' to mediate additional cellular functions in eukaryotic metabolism.

Molybdenum cofactors, enzymes and pathways

Cell biology of molybdenum

The molybdenum cofactor (Moco) forms the active site of all eukaryotic molybdenum (Mo) enzymes. Moco consists of molybdenum covalently bound to two sulfur atoms of a unique tricyclic pterin moiety referred to as molybdopterin. Moco is synthesized from GTP by an ancient and conserved biosynthetic pathway that can be divided into four steps involving the biosynthetic intermediates cyclic pyranopterin monophosphate, molybdopterin, and adenylated molybdopterin. In a fifth step, sulfuration or bond formation between Mo and a protein cysteine result in two different catalytic Mo centers. There are four Mo enzymes in plants: (1) nitrate reductase catalyzes the first and rate-limiting step in nitrate assimilation and is structurally similar to the recently identified, (2) peroxisomal sulfite oxidase that detoxifies excessive sulfite. (3) Aldehyde oxidase catalyzes the last step of abscisic acid biosynthesis, and (4) xanthine dehydrogenase is essential for purine degradation and stress response.

Molybdenum cofactor biosynthesis and molybdenum enzymes

Nitrate Reductase Regulates Plant Nitric Oxide Homeostasis.

The nitrate anion is a simple, abundant and relatively stable species, yet plays a significant role in global cycling of nitrogen, global climate change, and human health. Although it has been known for quite some time that nitrate is an important species environmentally, recent studies have identified potential medical applications. In this respect the nitrate anion remains an enigmatic species that promises to offer exciting science in years to come. Many bacteria readily reduce nitrate to nitrite via nitrate reductases. Classified into three distinct types – periplasmic nitrate reductase (Nap), respiratory nitrate reductase (Nar) and assimilatory nitrate reductase (Nas), they are defined by their cellular location, operon organization and active site structure. Of these, Nap proteins are the focus of this review. Despite similarities in the catalytic and spectroscopic properties Nap from different Proteobacteria are phylogenetically distinct. This review has two major sections: in the first section, nitrate in the nitrogen cycle and human health, taxonomy of nitrate reductases, assimilatory and dissimilatory nitrate reduction, cellular locations of nitrate reductases, structural and redox chemistry are discussed. The second section focuses on the features of periplasmic nitrate reductase where the catalytic subunit of the Nap and its kinetic properties, auxiliary Nap proteins, operon structure and phylogenetic relationships are discussed.

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Nitrate and periplasmic nitrate reductases

Nitrate assimilation in autotrophs provides most of the reduced nitrogen on earth. In eukaryotes, reduction of nitrate to nitrite is catalyzed by the molybdenum-containing NAD(P)H:nitrate reductase (NR; EC 1.7.1.1-3). In addition to the molybdenum center, NR contains iron-heme and flavin adenine dinucleotide as redox cofactors involved in an internal electron transport chain from NAD(P)H to nitrate. Recombinant, catalytically active Pichia angusta nitrate-reducing, molybdenum-containing fragment (NR-Mo) was expressed in P. pastoris and purified. Crystal structures for NR-Mo were determined at 1.7 and 2.6 A. These structures revealed a unique slot for binding nitrate in the active site and identified key Arg and Trp residues potentially involved in nitrate binding. Dimeric NR-Mo is similar in overall structure to sulfite oxidases, with significant differences in the active site. Sulfate bound in the active site caused conformational changes, as compared with the unbound enzyme. Four ordered water molecules located in close proximity to Mo define a nitrate binding site, a penta-coordinated reaction intermediate, and product release. Because yeast NAD(P)H:NR is representative of the family of eukaryotic NR, we propose a general mechanism for nitrate reduction catalysis.

Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site.

The invention provides modification to the polynucleotide coding sequence for Pichia angusta NAD (P)H: nitrate reductase [YNaR1; GenBank accession number Z49110], which has Enzyme Commission number 1.7.1.2 (formerly EC 1.6.6.2), yielding the polynucleotide coding sequence for simplified eukaryotic nitrate reductase (S-NaR1). The invention also provides a method for recombinant expression of said polynucleotide code in the cells of the methylotrophic yeast Pichia pastoris to produce the polypeptide for S-NaR1, which binds the host-produced molybdenum-molybdopterin cofactor and intracellularly forms catalytically active, nitrate-reducing enzyme as small and stable multimeric proteins. The invention also provides a method for rapid and high-yielding purification of S-NaR1 by utilizing the hexa-histidine sequence at the carboxyl-terminus of said polypeptide for immobilized metal affinity chromatography.

Simplified eukaryotic nitrate reductase

Nitrate analysis in water is one of the most frequently applied methods in environmental chemistry. Current methods for nitrate are generally based on toxic substances. Here, we show that a viable alternative method is to use the enzyme nitrate reductase. The key to applying this Green Chemistry solution for nitrate analysis is plentiful, inexpensive, analytical grade enzyme. We demonstrate that recombinant Arabidopsis nitrate reductase, expressed in the methylotrophic yeast Pichia pastoris, is a highly effective catalyst for nitrate analysis at 37°C. Recombinant production of enzyme ensures consistent quality and provides means to meet the needs of environmental chemistry.

https://www3.nd.edu/~aseriann/Nitrate_Assay.pdf

Guillaume G. Barbier

Papers

Structural basis of eukaryotic nitrate reduction: crystal structures of the nitrate reductase active site.

Simplified eukaryotic nitrate reductase

Nitrate reductase for nitrate analysis in water

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

Viscosity effects on eukaryotic nitrate reductase activity.