Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase

Franck E. Dayan, Pankaj R. Daga, Stephen O. Duke, Ryan M. Lee, Patrick J Tranel, Robert J. Doerksen

Research output: Contribution to journalArticle

Abstract

A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼ 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.

Original languageEnglish (US)
Pages (from-to)1548-1556
Number of pages9
JournalBiochimica et Biophysica Acta - Proteins and Proteomics
Volume1804
Issue number7
DOIs
StatePublished - Jul 1 2010

Fingerprint

Protoporphyrinogen Oxidase
Glycine
Herbicides
Herbicide Resistance
Catalytic Domain
Amaranthus
Flavin-Adenine Dinucleotide
Molecular Dynamics Simulation
Tobacco
Molecular dynamics
Proteins
Switches
X-Rays
X rays
Mutation
Kinetics

Keywords

  • Binding kinetics
  • Codon deletion
  • Evolution of resistance
  • Helix capping
  • Herbicide resistance
  • Mechanism of resistance
  • Molecular dynamics simulation

ASJC Scopus subject areas

  • Analytical Chemistry
  • Biophysics
  • Biochemistry
  • Molecular Biology

Cite this

Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase. / Dayan, Franck E.; Daga, Pankaj R.; Duke, Stephen O.; Lee, Ryan M.; Tranel, Patrick J; Doerksen, Robert J.

In: Biochimica et Biophysica Acta - Proteins and Proteomics, Vol. 1804, No. 7, 01.07.2010, p. 1548-1556.

Research output: Contribution to journalArticle

Dayan, Franck E. ; Daga, Pankaj R. ; Duke, Stephen O. ; Lee, Ryan M. ; Tranel, Patrick J ; Doerksen, Robert J. / Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase. In: Biochimica et Biophysica Acta - Proteins and Proteomics. 2010 ; Vol. 1804, No. 7. pp. 1548-1556.
@article{3684bff899fa4f79848d708a4e9097f7,
title = "Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase",
abstract = "A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼ 50{\%}. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.",
keywords = "Binding kinetics, Codon deletion, Evolution of resistance, Helix capping, Herbicide resistance, Mechanism of resistance, Molecular dynamics simulation",
author = "Dayan, {Franck E.} and Daga, {Pankaj R.} and Duke, {Stephen O.} and Lee, {Ryan M.} and Tranel, {Patrick J} and Doerksen, {Robert J.}",
year = "2010",
month = "7",
day = "1",
doi = "10.1016/j.bbapap.2010.04.004",
language = "English (US)",
volume = "1804",
pages = "1548--1556",
journal = "Biochimica et Biophysica Acta - Proteins and Proteomics",
issn = "1570-9639",
publisher = "Elsevier",
number = "7",

}

TY - JOUR

T1 - Biochemical and structural consequences of a glycine deletion in the α-8 helix of protoporphyrinogen oxidase

AU - Dayan, Franck E.

AU - Daga, Pankaj R.

AU - Duke, Stephen O.

AU - Lee, Ryan M.

AU - Tranel, Patrick J

AU - Doerksen, Robert J.

PY - 2010/7/1

Y1 - 2010/7/1

N2 - A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼ 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.

AB - A rare Gly210 deletion in protoporphyrinogen oxidase (PPO) was recently discovered in herbicide-resistant Amaranthus tuberculatus. According to the published X-ray structure of Nicotiana tabacum PPO, Gly210 is adjacent to, not in, the PPO active site, so it is a matter of interest to determine why its deletion imparts resistance to herbicides. In our kinetic experiments, this deletion did not affect the affinity of protoporphyrinogen IX nor the FAD content, but decreased the catalytic efficiency of the enzyme. The suboptimal Kcat was compensated by a significant increase in the Kis for inhibitors and a switch in their interactions from competitive to mixed-type inhibition. In our protein modeling studies on herbicide-susceptible PPO and resistant PPO, we show that Gly210 plays a key role in the αL helix-capping motif at the C-terminus of the α-8 helix which helps to stabilize the helix. In molecular dynamics simulations, the deletion had significant architecture consequences, destabilizing the α-8 helix-capping region and unraveling the last turn of the helix, leading to enlargement of the active site cavity by ∼ 50%. This seemingly innocuous deletion of Gly210 of the mitochondrial PPO imparts herbicide resistance to this dual-targeted protein without severely affecting its normal physiological function, which may explain why this unusual mutation was the favored evolutionary path for achieving resistance to PPO inhibitors.

KW - Binding kinetics

KW - Codon deletion

KW - Evolution of resistance

KW - Helix capping

KW - Herbicide resistance

KW - Mechanism of resistance

KW - Molecular dynamics simulation

UR - http://www.scopus.com/inward/record.url?scp=77952419950&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=77952419950&partnerID=8YFLogxK

U2 - 10.1016/j.bbapap.2010.04.004

DO - 10.1016/j.bbapap.2010.04.004

M3 - Article

C2 - 20399914

AN - SCOPUS:77952419950

VL - 1804

SP - 1548

EP - 1556

JO - Biochimica et Biophysica Acta - Proteins and Proteomics

JF - Biochimica et Biophysica Acta - Proteins and Proteomics

SN - 1570-9639

IS - 7

ER -