Two Arabidopsis genes (IPMS1 and IPMS2) encode isopropylmalate synthase, the branchpoint step in the biosynthesis of leucine

Abstract

Heterologous expression of the Arabidopsis (Arabidopsis thaliana) IPMS1 (At1g18500) and IPMS2 (At1g74040) cDNAs in Escherichia coli yields isopropylmalate synthases (IPMSs; EC 2.3.3.13). These enzymes catalyze the first dedicated step in leucine (Leu) biosynthesis, an aldol-type condensation of acetyl-coenzyme A (CoA) and 2-oxoisovalerate yielding isopropylmalate. Most biochemical properties of IPMS1 and IPMS2 are similar: broad pH optimum around pH 8.5, Mg2+ as cofactor, feedback inhibition by Leu, Km for 2-oxoisovalerate of approximately 300 microM, and a Vmax of approximately 2 x 10(3) micromol min(-1) g(-1). However, IPMS1 and IPMS2 differ in their Km for acetyl-CoA (45 microM and 16 microM, respectively) and apparent quaternary structure (dimer and tetramer, respectively). A knockout insertion mutant for IPMS1 showed an increase in valine content but no changes in Leu content; two insertion mutants for IPMS2 did not show any changes in soluble amino acid content. Apparently, in planta each gene can adequately compensate for the absence of the other, consistent with available microarray and reverse transcription-polymerase chain reaction data that show that both genes are expressed in all organs at all developmental stages. Both encoded proteins accept 2-oxo acid substrates in vitro ranging in length from glyoxylate to 2-oxohexanoate, and catalyze at a low rate the condensation of acetyl-CoA and 4-methylthio-2-oxobutyrate, i.e. a reaction involved in glucosinolate chain elongation normally catalyzed by methylthioalkylmalate synthases. The evolutionary relationship between IPMS and methylthioalkylmalate synthase enzymes is discussed in view of their amino acid sequence identity (60%) and overlap in substrate specificity.

Figure 1.

The biosynthesis of Leu and Val from pyruvate. The action of acetohydroxyacid synthase (AHAS), ketoacid reductoismerase (KARI), and dihydroxyacid dehydratase (DHAD) yields 2-oxoisovalerate that is either transaminated to Val or subjected to additional reactions specific for Leu biosynthesis. The dedicated step in Leu biosynthesis is the aldol-type condensation between 2-oxoisovalerate and acetyl-CoA that results in formation of 2-isopropylmalate. Isomerization and oxidative decarboxylation by isopropylmalate isomerase (IPMI) and isopropylmalate dehydrogenase (IPMDH) yield 4-methyl-2-oxovalerate that is transaminated to Leu. The enzymes that catalyze the reactions from pyruvate to 2-oxoisovalerate are also involved in biosynthesis of Ile, using 4-oxobutyrate (product of Thr dehydratase) as an initial substrate, but for simplicity have not been depicted. AHAS and IPMS are subject to feedback inhibition as shown with dashed lines.

Figure 2.

General scheme for the biosynthesis of aliphatic glucosinolates involving Met side-chain elongation (I), backbone synthesis (II), and side-chain modifications (III). The proposed cycle for side-chain elongation of deaminated Met (I) commences with an aldol-type condensation between the respective ω-methylthio-2-oxo acid and acetyl-CoA, a reaction catalyzed by MAM. The methylthioalkylmalate product is converted through subsequent isomerization and oxidative decarboxylation into a ω-methylthio-2-oxo acid that is elongated by one methylene group. The elongated ω-methylthio-2-oxo acid is either transaminated and enters glucosinolate backbone synthesis (II) or undergoes additional cycles of side-chain elongation. Glucosinolate backbone synthesis is represented in a simplified manner without the identified enzymes and cofactors. The depicted end product, 2-methylsulfinylethyl glucosinolate, is a C₂-glucosinolate that has not been side-chain elongated and does not occur naturally in Arabidopsis.

Figure 3.

Radio-HPLC analyses of the biochemical assay for IPMS2. Results for IPMS1 had a similar pattern. A, Incubation of IPMS2 with 500 μm [¹⁴C]acetyl-CoA and 3 mm 2-oxoisovalerate shows [¹⁴C]-2-isopropylmalate (IPM) as the only radioactive labeled product. B, In the absence of 2-oxo acid substrate, a small amount of [¹⁴C]acetate (Ac) is formed, whereas most of the [¹⁴C]acetyl-CoA (Ac-CoA) remains intact and is hardly retained in the HPLC column. C, UV trace (230 nm) of the HPLC showing the elution pattern of a standard solution containing 10 mm acetic acid (Ac) and 5 mm 2-isopropylmalate (IPM), and the injection peak at 15 min. D, Incubation of the IPMS2 gene product with 4-methylthio-2-oxobutyrate and [¹⁴C]acetyl-CoA yields a small amount of [¹⁴C]2-(2′-methylthio)ethylmalate (MTEM), whereas most of the [¹⁴C]acetyl-CoA (Ac-CoA) remains.

Figure 4.

Properties of Arabidopsis IPMSs. A, Effect of MgCl₂ concentration on IPMS2 activity; 0 mm corresponds to a desalted enzyme preparation without addition of MgCl₂. A similar graph was obtained for IPMS1. B, pH Curves for enzyme activity of IPMS1 and IPMS2 using MES (▴), BisTris-propane (▪), and 2-amino-2-methyl-1-propanol (♦). Each data point corresponds to a duplicate enzyme activity measurement with DTNB that is corrected for chemical hydrolysis of acetyl-CoA at each particular pH value. C, Determination of the molecular mass of IPMS1 (□) and IPMS2 (○) by calibrated gel-filtration chromatography. The column was calibrated by measuring the elution volume (♦) of β-amylase (200 kD), alcohol dehydrogenase (150 kD), bovine serum albumin (66 kD), carbonic anhydrase (29 kD), and cytochrome C (12.4 kD). The void volume of the column determined with Blue Dextran (2.0 × 10³ kD) was 44 mL.

Figure 5.

Leu inhibition of the heterologously expressed IPMS1 and IPMS2 from Arabidopsis.

Figure 6.

Semiquantitative RT-PCR analyses of IPMS transcript levels in leaf tissues of Salk T-DNA insertion lines, homozygous (mm) for the insertion or lacking the insertions (ww), and Col-0 wild type (WT). A, Results for the IPMS1 mutant line S_101771 compared to the IPMS2 mutant line S_000074; B, results for the IPMS2 mutant lines S_000074 and S_051060.

Figure 7.

Analyses of the free amino acid content of homozygous T-DNA insertion lines for IPMS1 (A; Salk_101771 [mm]) and IPMS2 (B; Salk_051060 [mm] and Salk_000074 [mm]). The IMPS1 mutant shows a significant increase in Val content in comparison with the corresponding outsegregants (ww) and Col-0 wild type. Error bars indicate sd.

Figure 8.

Alignment of deduced amino acid sequences for IPMS1, IPMS2, MAM1, and MAM3 with IPMS sequences from wild tomato (GenBank accession nos. AAB61598 and AAB61599), E. coli (Swiss-Prot: P09151), and M. tuberculosis whose protein structure has been elucidated (PDB: 1SR9). Black shading indicates individual amino acids that are conserved within all sequences, dark gray shading individual amino acids that are identical in at least six out of eight sequences, and light gray shading amino acids that are identical in at least five out of eight sequences. The ChloroP-predicted cleavage sites are marked with an underscore. Amino acid residues mentioned in the text are represented below the alignment, likewise amino acid residues of the Leu binding site that are marked with an “L” and the conserved motifs GxGERXG and HxH(D/N)D. Amino acid positions are numbered relative to 1SR9.