A redox‐active isopropylmalate dehydrogenase functions in the biosynthesis of glucosinolates and leucine in Arabidopsis

Abstract

SUMMARYWe report a detailed functional characterization of an Arabidopsis isopropylmalate dehydrogenase (AtI-PMDH1) that is involved in both glucosinolate biosynthesis and leucine biosynthesis. AtIPMDH1 shares high homology with enzymes from bacteria and yeast that are known to function in leucine biosynthesis. In plants, AtIPMDH1 is co-expressed with nearly all the genes known to be involved in aliphatic glucosinolate biosynthesis. Mutation of AtIPMDH1 leads to a significant reduction in the levels of free leucine and of glucosinolates with side chains of four or more carbons. Complementation of the mutant phenotype by ectopic expression of AtIPMDH1, together with the enzyme's substrate specificity, implicates AtIPMDH1 in both glucosinolate and leucine biosynthesis. This functional assignment is substantiated by subcellular localization of the protein in the chloroplast stroma, and the gene expression patterns in various tissues. Interestingly, AtIPMDH1 activity is regulated by a thiol-based redox modification. This work characterized an enzyme in plants that catalyzes the oxidative decarboxylation step in both leucine biosynthesis (primary metabolism) and methionine chain elongation of glucosinolates (specialized metabolism). It provides evidence for the hypothesis that the two pathways share a common origin, and suggests a role for redox regulation of glucosinolate and leucine synthesis in plants.

The ZmASR1 Protein Influences Branched-Chain Amino Acid Biosynthesis and Maintains Kernel Yield in Maize under Water-Limited Conditions

“…Distribution data for ZmAHAS1, ZmBCAT2, and ZmBCAT4 are as yet unavailable, although it is noteworthy that ZmBCAT1 and ZmBCAT3 showed enriched expression in mesophyll cells . Additionally, it may be noted that Arabidopsis Trx-m1 is the most effective Trx on AtIPMDH1 in comparison with Arabidopsis Trx-f1 or bacterial Trx (He et al, 2009). Its maize ortholog (Trx-m4) also showed enriched expression in mesophyll cells (Majeran et al, 2005;Friso et al, 2010).…”

“…Just recently, a wheat (Triticum aestivum) group 2 LEA protein named DHN-5, which is closely related to the maize LEA protein RAB17 and involved in salt and osmotic tolerance (Brini et al, 2007), has been shown to enhance the thermostability and activity of b-D-glucosidase (Brini et al, 2010), a target protein of ZmASR1. It is striking that b-D-glucosidase and 12 other ZmASR1 target genes belong to the list of established and potential thioredoxin (Trx) targets (He et al, 2009;Montrichard et al, 2009). Closer analysis shows that seven additional ZmASR1 target genes may contain Cys residues involved in disulfide bridges, making a total of 20 ZmASR1 target genes potentially linked to Trx.…”

Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana

“…Structural and functional studies revealed the point mutation responsible for functional divergence of the IPMDH for either leucine or aliphatic glucosinolate synthesis pathways in Arabidopsis (9). Substitution of an active site leucine in AtIPMDH2 and AtIPMDH3 for a phenylalanine in AtIPMDH1 altered substrate preference from IPM (AtIPMDH2 and AtIPMDH3) to 3-(2Ј-methylthio)ethylmalate (AtIPMDH1) (9).…”

“…Later studies of the three IPMDH isoforms in Arabidopsis (AtIPMDH1-3) revealed differences in the biochemical properties and metabolic contributions of each protein (9,10). Steady-state kinetic analysis of AtIPMDH1-3 showed that each enzyme catalyzed the conversion of 3-isopropylmalate to 4-methyl-2-oxovalerate; however, the catalytic efficiency of AtIPMDH1 was up to 40-fold lower than the two other isoforms (9,10). Analysis of Arabidopsis T-DNA insertion mutants that disrupted AtIPMDH1 showed decreased levels of C4 -C8 aliphatic glucosinolates and leucine.…”

“…Subsequent steps performed by methylthioalkylmalate synthase and an isopropylmalate isomerase homolog generate 3-(2Ј-methylthio)ethylmalate) (7,8), which undergoes oxidation and decarboxylation to yield 5-methylthiol-2-oxopentoate ( Fig. 1) (9,10). This product can then be transaminated for further elongation of the aliphatic moiety to yield C4 to C8 aliphatic glucosinolates (2).…”