Biosynthesis of methionine-derived glucosinolates in Arabidopsis thaliana : recombinant expression and characterization of methylthioalkylmalate synthase, the condensing enzyme of the chain-elongation cycle

Abstract

The major class of glucosinolates in Arabidopsis thaliana (L.) Heynh. are biosynthesized from methionine involving a three-step chain-elongation cycle. Each passage through the cycle results in the net addition of a single methylene group, with up to six cycles of elongation occurring in A. thaliana. The first reaction of the cycle is catalyzed by a methylthioalkylmalate synthase (MAMS), which condenses a omega-methylthio-2-oxoalkanoic acid with acetyl-CoA. Here we have demonstrated that MAM1, one of two similar genes in the A. thaliana ecotype Columbia, encodes a MAMS catalyzing the condensing reactions of the first two elongation cycles but not those of further cycles. The Columbia ecotype is dominated by compounds that have undergone only two elongation cycles. The A. thaliana MAM1 protein exhibits basic sequence similarity to other previously described enzymes catalyzing the condensation of 2-oxo acids and acetyl-CoA, such as isopropylmalate synthase (EC 2.3.3.13), an enzyme of leucine biosynthesis, and homocitrate synthase (EC 2.3.3.14). It also shares similar properties with them, including the catalytic requirements for a divalent metal ion and an adenine nucleotide. However, the MAM1 protein does not show activity with the substrates of any of these other enzymes, and was chromatographically separable from isopropylmalate synthase in extracts of A. thaliana. Thus, MAM1 is exclusively an enzyme of secondary metabolism, distinct from primary metabolic enzymes catalyzing similar reactions.

A robust omics-based approach for the identification of glucosinolate biosynthetic genes

“…ATTED-II analysis using a whole dataset showed weak correlation between ESM1 and ESP (Epithiospecifier protein, At1g54040) (data not shown). MAM genes that encode one of the Met side-chain elongation enzymes were also identified and characterized on the basis of the QTL analysis (Field et al 2004;Textor et al 2004;Kroymann et al 2001Kroymann et al , 2003. To my knowledge, however, some other genes that are involved in Met side-chain elongation process, namely, MAM-I (coding for methylthioalkylmalate isomerase) and MAM-D (coding for methylthioalkylmalate dehydrogenase) have not been identified by QTL analysis, presumably because natural variation of these genes does not result in metabolic natural variation.…”

Mathematical modelling of aliphatic glucosinolate chain length distribution in Arabidopsis thaliana leaves

“…Table 1 shows the kinetic parameters and the initial (fixed) concentration of MTOB used in the rate laws. Kinetic values set in Roman type were obtained from Textor et al (2004Textor et al ( , 2007, or, in the case of the CYP79F1/F2 enzymes, from Chen et al (2003). Values set in italics were derived from assumptions given above or chosen from preliminary simulations to optimize correspondence of the model with actual glucosinolate chain-length distributions observed.…”

“…k = 7 * 10 -7 a Parameters given in roman are experimental kinetic data, while those written in italics have been optimized to fit the model. Experimental data for MAM3 was obtained in Textor et al (2007); for MAM1 in Textor et al (2004) and for Cyp79F1 and F2 in Chen et al (2003). Constant concentration of the external metabolite OMTB = 1 lM transamination reactions (which are not yet characterized) by unknown parameters, increasing computational difficulties.…”

Two Arabidopsis Genes (IPMS1 and IPMS2) Encode Isopropylmalate Synthase, the Branchpoint Step in the Biosynthesis of Leucine

“…Kroymann et al, 2001;Junk and Mourad, 2002;Field et al, 2004;Textor et al, 2004). A principal reason for this attention is the potential role of these genes in glucosinolate biosynthesis.…”

“…Each cycle of elongation is initiated by an aldol-type condensation between acetyl-CoA and a v-methylthio-2-oxo acid leading to the formation of a v-methylthioalkylmalate. Both IPMS-like sequences at the Gs-Elong locus of Arabidopsis Col-0 were shown to encode proteins that possess this activity, but each had only very limited IPMS activity (Kroymann et al, 2001;Textor et al, 2004;S. Textor, unpublished data).…”