Nitrogen assimilation

Nitrogen assimilation is a fundamental biological process that occurs in plants and algae that are incapable of independent nitrogen fixation (see Scheme). The assimilation of nitrogen has marked effects on plant productivity, biomass, and crop yield, and nitrogen deficiency leads to a decrease in structural components. An initial conversion of nitrate to nitrite is followed by a reduction to ammonia by nitrite reductase (also called nitrite oxidoreductase). The ammonia is incorporated into glutamine as an amido nitrogen and is reductively transferred to 2-oxoglutarate to form 2 molecules of glutamate by glutamate synthase. The general steps of assimilation have been known for several years, however, the chemical mechanisms that occur in these processes remain poorly understood in large part due to the lack of detailed structural information concerning the enzymes that catalyze these reactions. Therefore, to understand nitrogen assimilation at a molecular level, we are determining molecular models of these key enzymes based upon the determination of their three dimensional structures and characterizing the functional and structural changes arising from modifications of the substrates and amino acid residues.

In collaboration with David Knaff, two different crystals forms have been obtained of spinach glutamate synthase. The largest are 0.9 mm and the determination of the structure using X-ray diffraction is underway at a resolution limit of 2.7 Å. Structural work is also underway for nitrite reductase. Measurement of the diffraction quality has been performed using the RAXIS IV detector in our protein crystallography facility. Crystals were mounted in capillaries and data was collected at room temperature. The crystals diffract to a resolution limit of 2.7 Å and heavy metal derivatives are being screened.