Abstract
Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of omega-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in the Arabidopsis ecotype Columbia: MAM1 and MAM3 (formerly known as MAM-L). Here, we describe the biochemical properties of the MAM3 enzyme, which is able to catalyze all six condensation reactions of Met chain elongation that occur in Arabidopsis. Underlining its broad substrate specificity, MAM3 also accepts a range of non-Met-derived 2-oxoacids, e.g. converting pyruvate to citramalate and 2-oxoisovalerate to isopropylmalate, a step in leucine biosynthesis. To investigate its role in vivo, we identified plant lines with mutations in MAM3 that resulted in a complete lack or greatly reduced levels of long-chain glucosinolates. This phenotype could be complemented by reintroduction of a MAM3 expression construct. Analysis of MAM3 mutants demonstrated that MAM3 catalyzes the formation of all glucosinolate chain lengths in vivo as well as in vitro, making this enzyme the major generator of glucosinolate chain length diversity in the plant. The localization of MAM3 in the chloroplast suggests that this organelle is the site of Met chain elongation.
Highlights
Chain elongated, methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana)
We propose that MAM-L be renamed MAM3 based on the biochemical properties of the encoded enzyme demonstrated here, the existing rules for Arabidopsis gene nomenclature, and the prior naming of MAM1 and MAM2 (Kroymann et al, 2003)
Purification of the recombinant polyhistidine-containing protein by application to a nickel-nitrilotriacetic acid agarose (Ni-NTA) affinity chromatography resin and elution with L-His resulted in an active enzyme fraction that was more than 90% pure as judged by SDS-PAGE with Coomassie staining
Summary
Methionine (Met)-derived glucosinolates are a major class of secondary metabolites in Arabidopsis (Arabidopsis thaliana). Isomerization and oxidation-decarboxylation reactions yield a 2-oxo acid that is extended by one methylene group This product can undergo additional cycles of elongation or it can be transaminated to form an elongated amino acid, which enters the pathway for formation of the core glucosinolate structure. We have focused on the elongation cycle enzymes at this important branching point, the MAM synthases, which catalyze the condensation of 2-oxo acid derivatives of Met with acetyl-CoA. The MAM synthases belong to a large family of enzymes that condense various 2-oxo acids with an acyl-CoA ester (Textor et al, 2004) One member of this group is isopropylmalate synthase (IPMS; EC 2.3.3.13), which condenses 2-oxo-isovalerate with acetyl-CoA, the chain elongation reaction in Leu biosynthesis
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