TY - JOUR
T1 - Structure and mechanism of isopropylmalate dehydrogenase from Arabidopsis thaliana
T2 - Insights on leucine and aliphatic glucosinolate biosynthesis
AU - Lee, Soon Goo
AU - Nwumeh, Ronald
AU - Jez, Joseph M.
N1 - Publisher Copyright:
© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.
PY - 2016/6/24
Y1 - 2016/6/24
N2 - 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 Mg2+ 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.
AB - 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 Mg2+ 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.
UR - http://www.scopus.com/inward/record.url?scp=84976430995&partnerID=8YFLogxK
U2 - 10.1074/jbc.M116.730358
DO - 10.1074/jbc.M116.730358
M3 - Article
C2 - 27137927
AN - SCOPUS:84976430995
SN - 0021-9258
VL - 291
SP - 13421
EP - 13430
JO - Journal of Biological Chemistry
JF - Journal of Biological Chemistry
IS - 26
ER -