A deficiency in imidazolonepropionase (IPase), an enzyme in the pathway catabolizing histidine to glutamate, has been previously shown to cause an oxygen-dependent growth reduction in Escherichia coli cells (Kabeer, 2000). The growth reduction is thought to be caused by an inhibition of aspartate aminotransferase (AAT) as a result of oxidative breakdown of the accumulated substrate for IPase, imidazolone propionate (IoPA). In order to investigate this potential causation of growth reduction, a recombinant 6-His-tagged version of urocanase, the enzyme that synthesizes IoPA, was established. Optimization of the expression and subsequent purification of this urocanase provided sufficient yields of pure enzyme to study the specificity of urocanase for its tightly bound nicotinamide adenine dinucleotide (NAD+) cofactor. In addition, the His-tagged urocanase allowed enzymatic synthesis of IoPA and one of its breakdown products, α-ketoglutaramate (α-KGM), through immobilization of the enzyme on Ni+2-binding beads. Following purification of each synthesis product, assays of AAT activity from E. coli crude extract in the presence of these compounds showed a decrease in AAT specific activity corresponding to increased amounts of α-KGM. Through the use of recombinant urocanase enzyme, the effect of IoPA and α-KGM on AAT have been characterized that point to a model of inhibition that ultimately causes a reduction in E. coli growth.
