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Title: Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana

Abstract

The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 {angstrom} resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2'-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2'-methylthio)ethylmalate {approx}100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results inmore » functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.« less

Authors:
; ; ; ; ; ;  [1];  [2]
  1. (WU)
  2. (
Publication Date:
Research Org.:
Argonne National Lab. (ANL), Argonne, IL (United States). Advanced Photon Source (APS)
Sponsoring Org.:
National Science Foundation (NSF)
OSTI Identifier:
1046238
Resource Type:
Journal Article
Journal Name:
J. Biol. Chem.
Additional Journal Information:
Journal Volume: 286; Journal Issue: (33) ; 08, 2011; Journal ID: ISSN 0021-9258
Country of Publication:
United States
Language:
ENGLISH
Subject:
59 BASIC BIOLOGICAL SCIENCES; 60 APPLIED LIFE SCIENCES; AMINO ACIDS; ARABIDOPSIS; BIOSYNTHESIS; CRYSTAL STRUCTURE; IN VIVO; LEUCINE

Citation Formats

He, Yan, Galant, Ashley, Pang, Qiuying, Strul, Johanna M., Balogun, Sherifat F., Jez, Joseph M., Chen, Sixue, and Florida). Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana. United States: N. p., 2012. Web. doi:10.1074/jbc.M111.262519.
He, Yan, Galant, Ashley, Pang, Qiuying, Strul, Johanna M., Balogun, Sherifat F., Jez, Joseph M., Chen, Sixue, & Florida). Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana. United States. doi:10.1074/jbc.M111.262519.
He, Yan, Galant, Ashley, Pang, Qiuying, Strul, Johanna M., Balogun, Sherifat F., Jez, Joseph M., Chen, Sixue, and Florida). Wed . "Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana". United States. doi:10.1074/jbc.M111.262519.
@article{osti_1046238,
title = {Structural and functional evolution of isopropylmalate dehydrogenases in the leucine and glucosinolate pathways of Arabidopsis thaliana},
author = {He, Yan and Galant, Ashley and Pang, Qiuying and Strul, Johanna M. and Balogun, Sherifat F. and Jez, Joseph M. and Chen, Sixue and Florida)},
abstractNote = {The methionine chain-elongation pathway is required for aliphatic glucosinolate biosynthesis in plants and evolved from leucine biosynthesis. In Arabidopsis thaliana, three 3-isopropylmalate dehydrogenases (AtIPMDHs) play key roles in methionine chain-elongation for the synthesis of aliphatic glucosinolates (e.g. AtIPMDH1) and leucine (e.g. AtIPMDH2 and AtIPMDH3). Here we elucidate the molecular basis underlying the metabolic specialization of these enzymes. The 2.25 {angstrom} resolution crystal structure of AtIPMDH2 was solved to provide the first detailed molecular architecture of a plant IPMDH. Modeling of 3-isopropylmalate binding in the AtIPMDH2 active site and sequence comparisons of prokaryotic and eukaryotic IPMDH suggest that substitution of one active site residue may lead to altered substrate specificity and metabolic function. Site-directed mutagenesis of Phe-137 to a leucine in AtIPMDH1 (AtIPMDH1-F137L) reduced activity toward 3-(2'-methylthio)ethylmalate by 200-fold, but enhanced catalytic efficiency with 3-isopropylmalate to levels observed with AtIPMDH2 and AtIPMDH3. Conversely, the AtIPMDH2-L134F and AtIPMDH3-L133F mutants enhanced catalytic efficiency with 3-(2'-methylthio)ethylmalate {approx}100-fold and reduced activity for 3-isopropylmalate. Furthermore, the altered in vivo glucosinolate profile of an Arabidopsis ipmdh1 T-DNA knock-out mutant could be restored to wild-type levels by constructs expressing AtIPMDH1, AtIPMDH2-L134F, or AtIPMDH3-L133F, but not by AtIPMDH1-F137L. These results indicate that a single amino acid substitution results in functional divergence of IPMDH in planta to affect substrate specificity and contributes to the evolution of specialized glucosinolate biosynthesis from the ancestral leucine pathway.},
doi = {10.1074/jbc.M111.262519},
journal = {J. Biol. Chem.},
issn = {0021-9258},
number = (33) ; 08, 2011,
volume = 286,
place = {United States},
year = {2012},
month = {10}
}