Structure and Mechanism of Isopropylmalate Dehydrogenase from Arabidopsis thaliana

Abstract

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 The evolution of specialized metabolic pathways from primary metabolism provides plants with the ability to generate molecules that contribute to their survival (1). The classic cycle of gene duplication and divergence of sequence that leads to new substrate specificities is at the core of how plants diversify metabolism for new purposes. One example of this process is the evolution of enzymes from leucine biosynthesis into variants for the production of sulfur-containing glucosinolates in plants of the order Brassicales (2-4). In the biosynthesis of methionine-derived glucosinolates, the sequential addition of methylene groups that leads to elongated aliphatic glucosinolates mimics the reactions in leucine biosynthesis (2).In the leucine biosynthesis pathway of plants and microbes, the NAD ϩ -dependent enzyme isopropylmalate dehydrogenase (IPMDH) 3 catalyzes the oxidation and decarboxylation of 3-isopropyl-L-malate (IPM) to 4-methyl-2-oxovalerate ( Fig. 1) (2). Subsequent transamination of 4-methyl-2-oxovalerate produces leucine. In the synthesis of aliphatic glucosinolate biosynthesis, the corresponding 3-malate derivative (i.e. 3-(2Ј-methylthio)ethylmalate) is produced from methionine. Branched-chain aminotransferases catalyze the deamination of methionine to 4-methythio-2-oxobutanoic acid (5, 6). 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).In plants, complementation of yeast with a Leu2 mutation by genes from canola, potato, and Arabidopsis thaliana identified IPMDH in the leucine biosynthesis pathway (11-13). 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. The loss of glucosinolate synthesis could be complemented by expression of AtIPMDH1 but not by expression of either AtIPMDH2 or AtIPMDH3 (10). T-DNA mutants of AtIPMDH2 and AtIPMDH3 reduced leucine levels but did not significantly alter glucosinolate production in Arabidopsis (14). Moreover, the Arabidopsis AtIPMDH2/AtIPMDH3 double mutant had defects in...

Molecular basis for branched steviol glucoside biosynthesis

“…Protein Expression and Purification. The general protein expression and purification protocol for UGT76G1 uses a combination of affinity and sizeexclusion chromatographies based on a previously published protocol (32). For production of SeMet-substituted protein, the pET-28a-UGT76G1 construct was transformed into E. coli BL21 (DE3) cells, which were grown to A 600 ∼ 0.8 at 37°C in M9 minimal media supplemented with SeMet containing 50 μg·mL −1 kanamycin (33).…”

flasher, a novel mutation in a glucosinolate modifying enzyme, conditions changes in plant architecture and hormone homeostasis

“…Similarly, other GSL biosynthetic enzymes can utilize substrates derived from both methionine (the entry molecule for the aliphatic pathway) and tryptophan (the entry point for the indolic pathway), and include UGT74B1 (Grubb et al, 2004), SUR1 (Mikkelsen et al, 2004) and SOT16 (Klein and Papenbrock, 2009). Analyses of mutations in the isopropylmalate dehydrogenases and methylthioalkylmalate synthases, enzymes that are responsible for methionine chain elongations that precede the GSL core enzymes, revealed that single amino acid changes in the active sites can dramatically alter the substrate specificity of the enzymes (He et al, 2011;Lee et al, 2016;Kumar et al, 2019). Thus, although it has been demonstrated that FSH/GS-OX5 has a preference for longchain GSLs, catalyzing the conversion of methylthioalkyl GSLs to methylsulfinylalkyl GSLs (Li et al, 2008), the enzyme may also recognize unknown substrates, generating the product(s) that negatively influences growth and development.…”

Promiscuity, impersonation and accommodation: evolution of plant specialized metabolism

“…The AtIPMDH2 Leu biosynthetic enzyme crystal structure with 3-isopropylmalate (3-IPM) revealed several binding interactions with the polar portion of the substrate [12]. The structure revealed that residues interacting with the polar groups of 3-IPM are conserved between all IPMDH enzymes (Figure 2B) [13].…”

“…This, combined with the similarity of the polar groups of 3-IPM and (3-(2′-methylthio)-ethylmalate, suggested that recognition of the side chain is responsible for substrate discrimination. There are no specific substrate-enzyme interactions between the 3-IPM aliphatic isopropyl side chain and the residues in the largely hydrophobic pocket in the active site (Figure 2B) [12,13]. Thus, the differences between the glucosinolate biosynthetic enzyme AtIPMDH1 and Leu IPMDH enzymes presumably are responsible for the difference in substrate specificity.…”

“…3-IPM for AtIPMDH2 and AtIPMDH3 and 3-(2′-methylthio)-ethylmalate for AtIPMDH1– and an increase with the non-native substrate [13]. Because the chemical structures of 3-(2′-methylthio)-ethylmalate and 3-IPM differ only by the length and structure of the side chain, this result demonstrates that the substitution of Leu by Phe is sufficient to facilitate the accommodation of the 3-(2′-methylthio)-ethylmalate side chain in the enzyme [12]. …”