|Daga, Pankaj -|
|Lee, Ryan -|
|Tranel, Patrick -|
|Doerksen, Robert -|
Submitted to: Biochimica et Biophysica Acta
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: April 9, 2010
Publication Date: April 23, 2010
Citation: Dayan, F.E., Daga, P.R., Duke, S.O., Lee, R.M., Tranel, P.J., Doerksen, R.J. 2010. Biochemical and Structural Consequences of a Glycine Deletion in the a-8 Helix of Protoporphyrinogen Oxidase. Biochimica et Biophysica Acta. 1804:1548-1556. Interpretive Summary: Herbicide-resistant biotypes of the weed Amaranthus tuberculatus have been discovered. Resistance to inhibitors of the enzyme protoporphyrinogen oxidase occurred through the selection of an unusual amino acid deletion. This report studies the consequences that the deletion of this glycine has the structure and activity of the enzyme, both in terms of the enzyme’s affinity for its substrate (protoporphyrinogen) and for selected inhibitors. Our results indicate that the rather innocuous deletion of a glycine has a dramatic effect on the active site because this amino acid happens to be involved in capping alpha helix-8, which is responsible for the positioning of the substrate with respect to the FAD ring involved in the catalysis.
Technical Abstract: An unusual mutation, the deletion of Gly210, in the mitochondrial protoporphyrinogen oxidase (PPO) of Amaranthus tuberculatus has been reported in herbicide-resistant biotypes of this weed. However, a mechanistic understanding of the consequences of this Gly210 deletion is lacking. According to our comparative protein models, based on an X-ray co-crystal structure of Nicotiana tabacum PPO and molecular dynamics simulations, deletion of Gly210 changed the architecture of the substrate binding domain significantly. Specifically, Gly210 caps the a-8 helix of PPO, and its deletion resulted in the unraveling of the last turn of that helix, which increased the volume of the active site cavity by ~25% and caused a ~3 Å shift of the carbonyl oxygen of Gly207 away from the isoalloxazine ring of flavin adenine dinucleotide (FAD). Kinetic studies of both wild-type and resistant isoforms revealed that this alteration did not affect the binding of protoporphyrinogen IX (Protogen) (similar Km) nor the FAD content, but it had a deleterious effect on the catalytic efficiency (lower kcat) of the oxidation of Protogen by the cofactor. This loss of catalytic efficiency is compensated by a change in the binding mode of the inhibitors (from competitive to mixed-type inhibition) and a significant decrease in the binding affinity of the inhibitors (higher Ki), which enables the survival of these biotypes under the selection pressure caused by PPO-inhibiting herbicides. The architectural and mechanistic consequences of the Gly210 deletion suggest that this mutation may be superior to a single amino acid substitution for achieving resistance to PPO inhibitors.