title: Characterization of aryloxyalkanoate dioxygenase-12, a nonheme Fe(II)/α-ketoglutarate-dependent dioxygenase, expressed in transgenic soybean and Pseudomonas fluorescens.
abstract: Aryloxyalkanoate dioxygenase-12 (AAD-12) was discovered from the soil bacterium Delftia acidovorans 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. (1) 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 Pseudomonas fluorescens (Pf) and purified to near homogeneity. A small amount of AAD-12 was partially purified from transgenic soybean and through various analytical, biochemical, and in vitro activity analyses demonstrated to be equivalent to the Pf-generated enzyme. Furthermore, results from in vitro kinetic analyses using a variety of plant endogenous compounds revealed activity with trans-cinnamate and indole-3-acetic acid (IAA). The catalytic efficiencies (kcat/Km) of AAD-12 using trans-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.
authors.affiliation: Dow AgroSciences LLC , 9330 Zionsville Road, Indianapolis, Indiana 46268, United States.
authors.forename: Samantha L
authors.initials: SL
authors.lastname: Griffin
authors.forename: Jeffrie A
authors.initials: JA
authors.lastname: Godbey
authors.forename: Trent J
authors.initials: TJ
authors.lastname: Oman
authors.forename: Shawna K
authors.initials: SK
authors.lastname: Embrey
authors.forename: Anton
authors.initials: A
authors.lastname: Karnoup
authors.forename: Krishna
authors.initials: K
authors.lastname: Kuppannan
authors.forename: Brian W
authors.initials: BW
authors.lastname: Barnett
authors.forename: Gaofeng
authors.initials: G
authors.lastname: Lin
authors.forename: Nicholas V J
authors.initials: NV
authors.lastname: Harpham
authors.forename: Amber N
authors.initials: AN
authors.lastname: Juba
authors.forename: Barry W
authors.initials: BW
authors.lastname: Schafer
authors.forename: Robert M
authors.initials: RM
authors.lastname: Cicchillo
mesh_terms: D001426:Bacterial Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D049308:Dioxygenases / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D053209:Herbicide Resistance; D006540:Herbicides / Q000378:metabolism* / Q000494:pharmacology; D007501:Iron / Q000378:metabolism; D007656:Ketoglutaric Acids / Q000378:metabolism; D007700:Kinetics; D030821:Plants, Genetically Modified / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D011551:Pseudomonas fluorescens / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D013025:Glycine max / Q000737:chemistry / Q000187:drug effects / Q000235:genetics / Q000378:metabolism*; D013379:Substrate Specificity
chemical_list: D001426:Bacterial Proteins; D006540:Herbicides; D007656:Ketoglutaric Acids; D007501:Iron; D049308:Dioxygenases