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 - Funding Information:
This work was supported in part by National Science Foundation Grant MCB-0904215. Portions of this research were carried out at the Argonne National Laboratory Structural Biology Center of the Advanced Photon Source, a national user facility operated by the University of Chicago for the Department of Energy Office of Biological and Environmental Research Grant DE-AC02-06CH11357. The authors declare that they have no conflicts of interest with the contents of this article. Supported in part by the Washington University Summer Scholars Program in Biology and Biomedicine and the Washington University uSTAR Summer Scholars Program.
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 -