Characterization of Aryloxyalkanoate Dioxygenase-12, a Nonheme Fe(II)/α-Ketoglutarate-Dependent Dioxygenase, Expressed in Transgenic Soybean and <i>Pseudomonas fluorescens</i>

Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium <i>Delftia acidovorans</i> MC1 and is a nonheme Fe­(II)/α-ketoglutarate-dependent dioxygenase, which can impart herbicide tolerance to transgenic plants by catalyzing the degradation of certain phenoxyacetate, pyridyloxyacetate, and aryloxyphenoxypropionate herbicides. The development of commercial herbicide-tolerant crops, in particular AAD-12-containing soybean, has prompted the need for large quantities of the enzyme for safety testing. To accomplish this, the enzyme was produced in <i>Pseudomonas fluorescens</i> (<i>Pf</i>) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and <i>in vitro</i> activity analyses demonstrated to be equivalent to the <i>Pf</i>-generated enzyme. Furthermore, results from <i>in vitro</i> kinetic analyses using a variety of plant endogenous compounds revealed activity with <i>trans</i>-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (<i>k</i>_cat/<i>K</i>_m) of AAD-12 using <i>trans</i>-cinnamate (51.5 M^–1 s^–1) and IAA (8.2 M^–1 s^–1) as substrates were very poor when compared to the efficiencies of plant endogenous enzymes. The results suggest that the presence of AAD-12 in transgenic soybean would not likely have an impact on major plant metabolic pathways