Soybean Genes Involved in Urease Activation

University Of Missouri-Columbia, Columbia MO

Investigators

Abstract

Enzymes are protein catalysts, substances which speed up biological reactions. Enzymes are essential for the complex chemistry of life processes. While enzymes consist of protein, most require the participation of a cation (positively charged metal ion) in catalysis. Nickle (Ni2+) is a rare component of enzymes. It is not known to be required by any of the 50 to 100,000 human enzymes. However, it is of extreme global importance as a component of four enzymes essential for inter-conversions of the one-carbon compounds, urea, carbon dioxide, carbon monoxide and methane. Urease is one of the four Ni metalloenyzmes. Urease is produced by a wide variety of bacteria, fungi and algae, and is extremely important in recycling urea from industrial and biological sources: Greater than 6 x 1010 pounds of urea nitrogen are applied annually to the world's cropland. In terrestrial mammals, urea is the major "jettisoned" form of excess metabolic nitrogen; adults excrete the protein equivalent of about 1,000 soybean seeds daily as urea. Urease is inactive without other protein factors to insert Ni into its catalytic (active) site. Soybean produces two ureases which are inactive without the function of two genes, Eu2 and Eu3. The primary objective of this research project is to determine whether these genes code Ni insertion proteins. Eu3 codes a Ni-binding protein (Eu3). Antibodies to Eu3 inhibit urease activation in anin vitro (test tube) assay we established for urease activation by Ni. Eu3 is absent in one mutant lacking all urease activity and altered in another mutant with trace amounts of activity. We have three goals for further elucidation of urease activation: (1) Structure-function analysis of Eu3 in vitro and in transgenic tissues. This involves directed alteration of the sequence of the Eu3 component amino acids and examining the effect on Eu3 function. (2) Identification and characterization of the Eu2 protein. (3) Charactization of a new gene, AJ6. This gene is unusual in being essential for the activation of only one of soybean's two ureases. The pursuit of these goals is aided by a convenient radioactive Ni tracer, 63Ni. Successfully addressing these objectives will expand our understanding of plant urease activation, a complex process which has already shown some differences from bacterial urease activation. These insights may also help us understand how plants assimilate minerals.