title: Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana: INSIGHTS ON LEUCINE AND ALIPHATIC GLUCOSINOLATE BIOSYNTHESIS.
abstract: Isopropylmalate dehydrogenase (IPMDH) and 3-(2'-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different β-hydroxyacids in the leucine- and methionine-derived glucosinolate biosynthesis pathways, respectively, in plants. Evolution of the glucosinolate biosynthetic enzyme from IPMDH results from a single amino acid substitution that alters substrate specificity. Here, we present the x-ray crystal structures of Arabidopsis thaliana IPMDH2 (AtIPMDH2) in complex with either isopropylmalate and Mg(2+) or NAD(+) These structures reveal conformational changes that occur upon ligand binding and provide insight on the active site of the enzyme. The x-ray structures and kinetic analysis of site-directed mutants are consistent with a chemical mechanism in which Lys-232 activates a water molecule for catalysis. Structural analysis of the AtIPMDH2 K232M mutant and isothermal titration calorimetry supports a key role of Lys-232 in the reaction mechanism. This study suggests that IPMDH-like enzymes in both leucine and glucosinolate biosynthesis pathways use a common mechanism and that members of the β-hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Soon Goo
authors.initials: SG
authors.lastname: Lee
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130.
authors.forename: Ronald
authors.initials: R
authors.lastname: Nwumeh
authors.affiliation: From the Department of Biology, Washington University, St. Louis, Missouri 63130 jjez@wustl.edu.
authors.forename: Joseph M
authors.initials: JM
authors.lastname: Jez
mesh_terms: D050539:3-Isopropylmalate Dehydrogenase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D017360:Arabidopsis / Q000201:enzymology* / Q000235:genetics; D029681:Arabidopsis Proteins / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism; D020134:Catalytic Domain; D018360:Crystallography, X-Ray; D005961:Glucosinolates / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D007930:Leucine / Q000096:biosynthesis* / Q000737:chemistry / Q000235:genetics; D013329:Structure-Activity Relationship
chemical_list: D029681:Arabidopsis Proteins; D005961:Glucosinolates; D050539:3-Isopropylmalate Dehydrogenase; D007930:Leucine
keywords: enzyme; plant biochemistry; protein evolution; protein structure; reaction mechanism; x-ray crystallography