Molecular Basis of the Evolution of Methylthioalkylmalate Synthase and the Diversity of Methionine-Derived Glucosinolates

Abstract

The globally cultivated Brassica species possess diverse aliphatic glucosinolates, which are important for plant defense and animal nutrition. The committed step in the side chain elongation of methionine-derived aliphatic glucosinolates is catalyzed by methylthioalkylmalate synthase, which likely evolved from the isopropylmalate synthases of leucine biosynthesis. However, the molecular basis for the evolution of methylthioalkylmalate synthase and its generation of natural product diversity in Brassica is poorly understood. Here, we show that Brassica genomes encode multiple methylthioalkylmalate synthases that have differences in expression profiles and 2-oxo substrate preferences, which account for the diversity of aliphatic glucosinolates across Brassica accessions. Analysis of the 2.1 Å resolution x-ray crystal structure of Brassica juncea methylthioalkylmalate synthase identified key active site residues responsible for controlling the specificity for different 2-oxo substrates and the determinants of side chain length in aliphatic glucosinolates. Overall, these results provide the evolutionary and biochemical foundation for the diversification of glucosinolate profiles across globally cultivated Brassica species, which could be used with ongoing breeding strategies toward the manipulation of beneficial glucosinolate compounds for animal health and plant protection.

An integrative approach to investigate natural variation in the accumulation of aliphatic glucosinolates in Arabidopsis thaliana

“…To get a clearer understanding of the effects of the localisation of polymorphic amino acid residues in the active sites of the metabolic enzymes, we extracted the information about the active sites from the NCBI’s conserved domain database (Marchler-Bauer et al, 2015). For example, the amino acid positions 93, 94, 97, 124, 162, 164, 186, 227, 229, 231, 257, 259, 260, 261, 262, 290, 292, and 294 are known to be key for activity of MAM synthases, based on the database and a crystal structure (Kumar et al, 2019; Marchler-Bauer et al, 2015; Petersen et al, 2019). We refer to the amino acid positions from 93 to 294 as active region of the enzyme.…”

“…The amino acid residues at position 98, 99, 132, 138, 139, 147, 165, 173, 177, 187, 228, 245, 257, 258, 271, 289 and 290 of the MAM1 and positions 156, 231, 241 and 242 of MAM3 accumulate polymorphic residues across the 72 Arabidopsis ecotypes. Polymorphisms in the active sites of an enzyme, in principle, can change the catalytic properties of the enzyme (Kroymann et al, 2001; Kumar et al, 2019; Petersen et al, 2019). However, the quantitative effect on the enzymatic properties cannot be explained due to unavailability of enzyme abundances in these 72 ecotypes.…”

“…In A. thaliana , the enzyme isoforms MAM1 and MAM2 catalyse the formation of short-chain ( 3C and 4C ) aliphatic GSLs, whereas the isoform MAM3 catalyses the formation of both short-chain and long-chain ( 5C - 8C ) GSLs (Halkier2006). Indeed, orthologues of MAM1 and MAM2 are also responsible for diversity of aliphatic GSLs across a range of Brassica species (Kumar et al, 2019). The distinct function of the two genes was confirmed by complementation of Arabidopsis mam1 mutant, when MAM1 from B. juncea restored wild type GSL profile but MAM2 did not (Kumar et al, 2019).…”

“…Indeed, orthologues of MAM1 and MAM2 are also responsible for diversity of aliphatic GSLs across a range of Brassica species (Kumar et al, 2019). The distinct function of the two genes was confirmed by complementation of Arabidopsis mam1 mutant, when MAM1 from B. juncea restored wild type GSL profile but MAM2 did not (Kumar et al, 2019). Our investigation of genotypic and phenotypic distances between different Arabidopsis ecotypes showed that some ecotypes have identical metabolic genotype but exhibit high diversity in their associated metabolic phenotype, and vice-versa (cf.…”

Growth under high light and elevated temperature affects metabolic responses and accumulation of health-promoting metabolites in kale varieties

“…The biosynthetic machineries behind aliphatic GSL biosynthesis are drawing increasing 545 research interest, as understanding the molecular machineries responsible for the enormous 546 GSL diversity may pave the way for traditional breeding or biotechnological manipulation of 547 GSL content and their pungent metabolites in Brassica crops (Petersen, Wang, Crocoll & 548 Halkier 2018;Kumar et al 2019). In this study, we provide evidence indicating that besides 549 the evolutionary and biochemical foundations of GSL metabolism (Kumar et al 2019), 550 optimized light conditions can be applied to modulate the GSL profiles to increase the contents 551 of beneficial GSL compounds while decreasing those with deleterious effects. 552 553…”