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 Å 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 ∼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.
authors.affiliation: Department of Biology, Genetics Institute, Plant Molecular, and Cellular Biology Program, University of Florida, Gainesville, Florida 32610 and.
authors.forename: Yan
authors.initials: Y
authors.lastname: He
authors.affiliation: Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Ashley
authors.initials: A
authors.lastname: Galant
authors.affiliation: Department of Biology, Genetics Institute, Plant Molecular, and Cellular Biology Program, University of Florida, Gainesville, Florida 32610 and.
authors.forename: Qiuying
authors.initials: Q
authors.lastname: Pang
authors.affiliation: Department of Biology, Genetics Institute, Plant Molecular, and Cellular Biology Program, University of Florida, Gainesville, Florida 32610 and.
authors.forename: Johanna M
authors.initials: JM
authors.lastname: Strul
authors.affiliation: Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Sherifat F
authors.initials: SF
authors.lastname: Balogun
authors.affiliation: Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
authors.affiliation: Department of Biology, Genetics Institute, Plant Molecular, and Cellular Biology Program, University of Florida, Gainesville, Florida 32610 and. Electronic address: schen@ufl.edu.
authors.forename: Sixue
authors.initials: S
authors.lastname: Chen
mesh_terms: D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D001665:Binding Sites; D002384:Catalysis; D018360:Crystallography, X-Ray; D019143:Evolution, Molecular; D005961:Glucosinolates / Q000235:genetics / Q000378:metabolism*; D007930:Leucine / Q000235:genetics / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D010088:Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D005961:Glucosinolates; D010088:Oxidoreductases; D007930:Leucine