期刊
JOURNAL OF BIOLOGICAL CHEMISTRY
卷 291, 期 26, 页码 13421-13430出版社
AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC
DOI: 10.1074/jbc.M116.730358
关键词
enzyme; plant biochemistry; protein evolution; protein structure; x-ray crystallography; reaction mechanism
资金
- National Science Foundation [MCB-0904215]
- Department of Energy Office of Biological and Environmental Research [DE-AC02-06CH11357]
- Washington University Summer Scholars Program in Biology and Biomedicine
- Washington University uSTAR Summer Scholars Program
Isopropylmalate dehydrogenase (IPMDH) and 3-(2-methylthio)ethylmalate dehydrogenase catalyze the oxidative decarboxylation of different -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 -hydroxyacid reductive decarboxylase family employ different active site features for similar reactions.
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