Conformational interdomain flexibility in a bacterial α-isopropylmalate synthase is necessary for leucine biosynthesis.

α-Isopropylmalate synthase (IPMS) catalyzes the first step in leucine (Leu) biosynthesis and is allosterically regulated by the pathway end product, Leu. IPMS is a dimeric enzyme with each chain consisting of catalytic, accessory, and regulatory domains, with the accessory and regulatory domains of each chain sitting adjacent to the catalytic domain of the other chain. The IPMS crystal structure shows significant asymmetry because of different relative domain conformations in each chain. Owing to the challenges posed by the dynamic and asymmetric structures of IPMS enzymes, the molecular details of their catalytic and allosteric mechanisms are not fully understood. In this study, we have investigated the allosteric feedback mechanism of the IPMS enzyme from the bacterium that causes meningitis, Neisseria meningitidis (NmeIPMS). By combining molecular dynamics simulations with small-angle X-ray scattering, mutagenesis, and heterodimer generation, we demonstrate that Leu-bound NmeIPMS is in a rigid conformational state stabilized by asymmetric interdomain polar interactions. Furthermore, we found removing these polar interactions by mutagenesis impaired the allosteric response without compromising Leu binding. Our results suggest that the allosteric inhibition of NmeIPMS is achieved by restricting the flexibility of the accessory and regulatory domains, demonstrating that significant conformational flexibility is required for catalysis.

Synthesis of 4-methylvaleric acid, a precursor of pogostone, involves a 2-isobutylmalate synthase related to 2-isopropylmalate synthase of leucine biosynthesis.

We show here that the side chain of pogostone, one of the major components of patchouli oil obtained from Pogostemon cablin and possessing a variety of pharmacological activities, is derived from 4-methylvaleric acid. We also show that 4-methylvaleric acid is produced through the one-carbon α-ketoacid elongation pathway with the involvement of the key enzyme 2-isobutylmalate synthase (IBMS), a newly identified enzyme related to isopropylmalate synthase (IPMS) of leucine (Leu) biosynthesis. Site-directed mutagenesis identified Met132 in the N-terminal catalytic region as affecting the substrate specificity of PcIBMS1. Even though PcIBMS1 possesses the C-terminal domain that in IPMS serves to mediate Leu inhibition, it is insensitive to Leu. The observation of the evolution of IBMS from IPMS, as well as previously reported examples of IPMS-related genes involved in making glucosinolates in Brassicaceae, acylsugars in Solanaceae, and flavour compounds in apple, indicate that IPMS genes represent an important pool for the independent evolution of genes for specialised metabolism.

Complete dominant inheritance of intracellular leucine accumulation traits in polyploid yeasts.

The yeast Saccharomyces cerevisiae is widely used for ethanol production. In the production of alcoholic beverages, flavours are affected mainly by yeast metabolism in the fermentation process. To increase the contents of initial scented fruity flavours, such as isoamyl alcohol and isoamyl acetate, leucine accumulation in yeast cells is induced by a decrease of leucine feedback inhibition in the l-leucine synthetic pathway using conventional mutagenesis. Diploid strains are commonly used in sake brewing because of better fermentation performance, such as vitality and endurance, compared with those of haploid strains. Heterozygous mutations are mostly detected in target genes of brewing yeasts generated through mutation breeding. Here we describe that an allele of the LEU4 gene, LEU4G516S , dominantly induced leucine accumulation even in triploid and tetraploid yeasts as with in diploid yeasts. Importantly, we demonstrated that there is no difference in the intracellular amount of branched-chain amino acids between LEU4G516S /LEU4 heterozygous diploids and LEU4G516S /LEU4G516S homozygous diploids. The approach to increase isoamyl alcohol and isoamyl acetate by intracellular leucine accumulation can potentially be applied to a variety of yeast strains, including aneuploid and polyploid yeasts.

Influence of mutation in the regulatory domain of α-isopropylmalate synthase from Saccharomyces cerevisiae on its activity and feedback inhibition.

Isoamyl alcohol (i-AmOH) is produced from α-ketoisocaproate in the l-leucine biosynthetic pathway in yeast and controlled by the negative feedback regulation of α-isopropylmalate synthase (IPMS), which senses the accumulation of l-leucine. It is known that i-AmOH production increases when mutations in the regulatory domain reduce the susceptibility to feedback inhibition. However, the impact of mutations in this domain on the IPMS activity has not been examined. In this study, we obtained 5 IPMS mutants, encoding the LEU4 gene, N515D/S520P/S542F/A551D/A551V, that are tolerant to 5,5,5-trifluoro-dl-leucine. All mutant proteins were purified and examined for both IPMS activity and negative feedback activity by in vitro experiments. The results showed that not only the negative-feedback regulation by l-leucine was almost lost in all mutants, but also the IPMS activity was greatly decreased and the difference in IPMS activity among Leu4 mutants in the presence of l-leucine was significantly correlated with i-AmOH production.

Two alpha isopropylmalate synthase isozymes with similar kinetic properties are extant in the yeast Lachancea kluyveri.

The first committed step in the leucine biosynthetic pathway is catalyzed by α-isopropylmalate synthase (α-IPMS, EC 2.3.3.13), which in the Saccaromycotina subphylum of Ascomycete yeasts is frequently encoded by duplicated genes. Following a gene duplication event, the two copies may be preserved presumably because the encoded proteins diverge in either functional properties and/or cellular localization. The genome of the petite-negative budding yeast Lachancea kluyveri includes two SAKL0E10472 (LkLEU4) and SAKL0F05170 g (LkLEU4BIS) paralogous genes, which are homologous to other yeast α-IPMS sequences. Here, we investigate whether these paralogous genes encode functional α-IPMS isozymes and whether their functions have diverged. Molecular phylogeny suggested that the LkLeu4 isozyme is located in the mitochondria and LkLeu4BIS in the cytosol. Comparison of growth rates, leucine intracellular pools and mRNA levels, indicate that the LkLeu4 isozyme is the predominant α-IPMS enzyme during growth on glucose as carbon source. Determination of the kinetic parameters indicates that the isozymes have similar affinities for the substrates and for the feedback inhibitor leucine. Thus, the diversification of the physiological roles of the genes LkLEU4 and LkLEU4BIS involves preferential transcription of the LkLEU4 gene during growth on glucose and different subcellular localization, although ligand interactions have not diverged.

Biosensor for branched-chain amino acid metabolism in yeast and applications in isobutanol and isopentanol production.

Branched-chain amino acid (BCAA) metabolism fulfills numerous physiological roles and can be harnessed to produce valuable chemicals. However, the lack of eukaryotic biosensors specific for BCAA-derived products has limited the ability to develop high-throughput screens for strain engineering and metabolic studies. Here, we harness the transcriptional regulator Leu3p from Saccharomyces cerevisiae to develop a genetically encoded biosensor for BCAA metabolism. In one configuration, we use the biosensor to monitor yeast production of isobutanol, an alcohol derived from valine degradation. Small modifications allow us to redeploy Leu3p in another biosensor configuration that monitors production of the leucine-derived alcohol, isopentanol. These biosensor configurations are effective at isolating high-producing strains and identifying enzymes with enhanced activity from screens for branched-chain higher alcohol (BCHA) biosynthesis in mitochondria as well as cytosol. Furthermore, this biosensor has the potential to assist in metabolic studies involving BCAA pathways, and offers a blueprint to develop biosensors for other products derived from BCAA metabolism.

Mitochondrial Compartmentalization Confers Specificity to the 2-Ketoacid Recursive Pathway: Increasing Isopentanol Production in Saccharomyces cerevisiae.

Recursive elongation pathways produce compounds of increasing carbon-chain length with each iterative cycle. Of particular interest are 2-ketoacids derived from recursive elongation, which serve as precursors to a valuable class of advanced biofuels known as branched-chain higher alcohols (BCHAs). Protein engineering has been used to increase the number of iterative elongation cycles completed, yet specific production of longer-chain 2-ketoacids remains difficult to achieve. Here, we show that mitochondrial compartmentalization is an effective strategy to increase specificity of recursive pathways to favor longer-chain products. Using 2-ketoacid elongation as a proof of concept, we show that overexpression of the three elongation enzymes-LEU4, LEU1, and LEU2-in mitochondria of an isobutanol production strain results in a 2.3-fold increase in the isopentanol to isobutanol product ratio relative to overexpressing the same elongation enzymes in the cytosol, and a 31-fold increase relative to wild-type enzyme expression. Reducing the loss of intermediates allows us to further boost isopentanol production to 1.24 ± 0.06 g/L of isopentanol. In this strain, isopentanol accounts for 86% of the total BCHAs produced, while achieving the highest isopentanol titer reported for Saccharomyces cerevisiae. Localizing the elongation enzymes in mitochondria  enables the development of strains in which isopentanol constitutes as much as 93% of BCHA production. This work establishes mitochondrial compartmentalization as a new approach to favor high titers and product specificities of larger products from recursive pathways.

Protein acetylation on 2-isopropylmalate synthase from Thermus thermophilus HB27.

Protein lysine Nε-acetylation is one of the important factors regulating cellular metabolism. We performed a proteomic analysis to identify acetylated proteins in the extremely thermophilic bacterium, Thermus thermophilus HB27. A total of 335 unique acetylated lysine residues, including many metabolic enzymes and ribosomal proteins, were identified in 208 proteins. Enzymes involved in amino acid metabolism were the most abundant among acetylated metabolic proteins. 2-Isopropylmalate synthase (IPMS), which catalyzes the first step in leucine biosynthesis, was acetylated at four lysine residues. Acetylation-mimicking mutations at Lys332 markedly decreased IPMS activity in vitro, suggesting that Lys332, which is located in subdomain II, plays a regulatory role in IPMS activity. We also investigated the acetylation-deacetylation mechanism of IPMS and revealed that it was acetylated non-enzymatically by acetyl-CoA and deacetylated enzymatically by TT_C0104. The present results suggest that leucine biosynthesis is regulated by post-translational protein modifications, in addition to feedback inhibition/repression, and that metabolic enzymes are regulated by protein acetylation in T. thermophilus.

Characterization of two 2-isopropylmalate synthase homologs from Thermus thermophilus HB27.

2-Isopropylmalate synthase (IPMS) catalyzes the first step of leucine biosynthesis and is regulated via feedback inhibition by leucine. The thermophilic bacterium, Thermus thermophilus HB27, has two IPMS homologous genes: TTC0847 and TTC0849, both of which are in the branched-chain amino acid biosynthetic gene cluster. Since enzymes involved in the leucine biosynthetic pathway are evolutionarily related to those in isoleucine biosynthesis, TTC0847 and TTC0849 are expected to function as IPMS or citramalate synthase, which is the first enzyme in the isoleucine biosynthetic pathway from pyruvate. We characterized these proteins in vitro and in vivo, and revealed that TTC0849 plays a key role in the biosynthesis of leucine and isoleucine, whereas TTC0847 is only involved in that of isoleucine.

A Feedback-Insensitive Isopropylmalate Synthase Affects Acylsugar Composition in Cultivated and Wild Tomato.

Acylsugars are insecticidal specialized metabolites produced in the glandular trichomes of plants in the Solanaceae family. In the tomato clade of the Solanum genus, acylsugars consist of aliphatic acids of different chain lengths esterified to sucrose, or less frequently to glucose. Through liquid chromatography-mass spectrometry screening of introgression lines, we previously identified a region of chromosome 8 in the Solanum pennellii LA0716 genome (IL8-1/8-1-1) that causes the cultivated tomato Solanum lycopersicum to shift from producing acylsucroses with abundant 3-methylbutanoic acid acyl chains derived from leucine metabolism to 2-methylpropanoic acid acyl chains derived from valine metabolism. We describe multiple lines of evidence implicating a trichome-expressed gene from this region as playing a role in this shift. S. lycopersicum M82 SlIPMS3 (Solyc08g014230) encodes a functional end product inhibition-insensitive version of the committing enzyme of leucine biosynthesis, isopropylmalate synthase, missing the carboxyl-terminal 160 amino acids. In contrast, the S. pennellii LA0716 IPMS3 allele found in IL8-1/8-1-1 encodes a nonfunctional truncated IPMS protein. M82 transformed with an SlIPMS3 RNA interference construct exhibited an acylsugar profile similar to that of IL8-1-1, whereas the expression of SlIPMS3 in IL8-1-1 partially restored the M82 acylsugar phenotype. These IPMS3 alleles are polymorphic in 14 S. pennellii accessions spread throughout the geographical range of occurrence for this species and are associated with acylsugars containing varying amounts of 2-methylpropanoic acid and 3-methylbutanoic acid acyl chains.

Diversification of Paralogous α-Isopropylmalate Synthases by Modulation of Feedback Control and Hetero-Oligomerization in Saccharomyces cerevisiae.

Production of α-isopropylmalate (α-IPM) is critical for leucine biosynthesis and for the global control of metabolism. The budding yeast Saccharomyces cerevisiae has two paralogous genes, LEU4 and LEU9, that encode α-IPM synthase (α-IPMS) isozymes. Little is known about the biochemical differences between these two α-IPMS isoenzymes. Here, we show that the Leu4 homodimer is a leucine-sensitive isoform, while the Leu9 homodimer is resistant to such feedback inhibition. The leu4Δ mutant, which expresses only the feedback-resistant Leu9 homodimer, grows slowly with either glucose or ethanol and accumulates elevated pools of leucine; this phenotype is alleviated by the addition of leucine. Transformation of the leu4Δ mutant with a centromeric plasmid carrying LEU4 restored the wild-type phenotype. Bimolecular fluorescent complementation analysis showed that Leu4-Leu9 heterodimeric isozymes are formed in vivo. Purification and kinetic analysis showed that the hetero-oligomeric isozyme has a distinct leucine sensitivity behavior. Determination of α-IPMS activity in ethanol-grown cultures showed that α-IPM biosynthesis and growth under these respiratory conditions depend on the feedback-sensitive Leu4 homodimer. We conclude that retention and further diversification of two yeast α-IPMSs have resulted in a specific regulatory system that controls the leucine-α-IPM biosynthetic pathway by selective feedback sensitivity of homomeric and heterodimeric isoforms.

Isolation and characterization of awamori yeast mutants with L-leucine accumulation that overproduce isoamyl alcohol.

Awamori shochu is a traditional distilled alcoholic beverage made from steamed rice in Okinawa, Japan. Although it has a unique aroma that is distinguishable from that of other types of shochu, no studies have been reported on the breeding of awamori yeasts. In yeast, isoamyl alcohol (i-AmOH), known as the key flavor of bread, is mainly produced from α-ketoisocaproate in the pathway of L-leucine biosynthesis, which is regulated by end-product inhibition of α-isopropylmalate synthase (IPMS). Here, we isolated mutants resistant to the L-leucine analog 5,5,5-trifluoro-DL-leucine (TFL) derived from diploid awamori yeast of Saccharomyces cerevisiae. Some of the mutants accumulated a greater amount of intracellular L-leucine, and among them, one mutant overproduced i-AmOH in awamori brewing. This mutant carried an allele of the LEU4 gene encoding the Ser542Phe/Ala551Val variant IPMS, which is less sensitive to feedback inhibition by L-leucine. Interestingly, we found that either of the constituent mutations (LEU4(S542F) and LEU4(A551V)) resulted in the TFL tolerance of yeast cells and desensitization to L-leucine feedback inhibition of IPMS, leading to intracellular L-leucine accumulation. Homology modeling also suggested that L-leucine binding was drastically inhibited in the Ser542Phe, Ala551Val, and Ser542Phe/Ala551Val variants due to steric hindrance in the cavity of IPMS. As we expected, awamori yeast cells expressing LEU4(S542F), LEU4(A551V), and LEU4(S542F/A551V) showed a prominent increase in extracellular i-AmOH production, compared with that of cells carrying the vector only. The approach described here could be a practical method for the breeding of novel awamori yeasts to expand the diversity of awamori taste and flavor.

Subdomain II of α-isopropylmalate synthase is essential for activity: inferring a mechanism of feedback inhibition.

The committed step of leucine biosynthesis, converting acetyl-CoA and α-ketoisovalerate into α-isopropylmalate, is catalyzed by α-isopropylmalate synthase (IPMS), an allosteric enzyme subjected to feedback inhibition by the end product L-leucine. We characterized the short form IPMS from Leptospira biflexa (LbIPMS2), which exhibits a catalytic activity comparable with that of the long form IPMS (LbIPMS1) and has a similar N-terminal domain followed by subdomain I and subdomain II but lacks the whole C-terminal regulatory domain. We found that partial deletion of the regulatory domain of LbIPMS1 resulted in a loss of about 50% of the catalytic activity; however, when the regulatory domain was deleted up to Arg-385, producing a protein that is almost equivalent to the intact LbIPMS2, about 90% of the activity was maintained. Moreover, in LbIPMS2 or LbIPMS1, further deletion of several residues from the C terminus of subdomain II significantly impaired or completely abolished the catalytic activity, respectively. These results define a complete and independently functional catalytic module of IPMS consisting of both the N-terminal domain and the two subdomains. Structural comparison of LbIPMS2 and the Mycobacterium tuberculosis IPMS revealed two different conformations of subdomain II that likely represent two substrate-binding states related to cooperative catalysis. The biochemical and structural analyses together with the previously published hydrogen-deuterium exchange data led us to propose a conformation transition mechanism for feedback inhibition mediated by subdomains I and II that might associated with alteration of the binding affinity toward acetyl-CoA.

Mechanistic and bioinformatic investigation of a conserved active site helix in α-isopropylmalate synthase from Mycobacterium tuberculosis, a member of the DRE-TIM metallolyase superfamily.

The characterization of functionally diverse enzyme superfamilies provides the opportunity to identify evolutionarily conserved catalytic strategies, as well as amino acid substitutions responsible for the evolution of new functions or specificities. Isopropylmalate synthase (IPMS) belongs to the DRE-TIM metallolyase superfamily. Members of this superfamily share common active site elements, including a conserved active site helix and an HXH divalent metal binding motif, associated with stabilization of a common enolate anion intermediate. These common elements are overlaid by variations in active site architecture resulting in the evolution of a diverse set of reactions that include condensation, lyase/aldolase, and carboxyl transfer activities. Here, using IPMS, an integrated biochemical and bioinformatics approach has been utilized to investigate the catalytic role of residues on an active site helix that is conserved across the superfamily. The construction of a sequence similarity network for the DRE-TIM metallolyase superfamily allows for the biochemical results obtained with IPMS variants to be compared across superfamily members and within other condensation-catalyzing enzymes related to IPMS. A comparison of our results with previous biochemical data indicates an active site arginine residue (R80 in IPMS) is strictly required for activity across the superfamily, suggesting that it plays a key role in catalysis, most likely through enolate stabilization. In contrast, differential results obtained from substitution of the C-terminal residue of the helix (Q84 in IPMS) suggest that this residue plays a role in reaction specificity within the superfamily.

V-type allosteric inhibition is described by a shift in the rate-determining step for α-isopropylmalate synthase from Mycobacterium tuberculosis.

The kinetic parameters affected by allosteric mechanisms contain collections of rate constants that vary based on differences in the relative rates of individual steps in the reaction. Thus, it may not be useful to compare enzymes with similar allosteric mechanisms unless the point of regulation has been identified. Rapid reaction kinetics and kinetic isotope effects provide a detailed description of V-type feedback allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis, an evolutionarily conserved model allosteric system. Results are consistent with a shift in the rate-determining step from product release to the hydrolytic step in catalysis in the presence of the effector.

Amino-acid substitutions at the domain interface affect substrate and allosteric inhibitor binding in α-isopropylmalate synthase from Mycobacterium tuberculosis.

α-Isopropylmalate synthase (α-IPMS) is a multi-domain protein catalysing the condensation of α-ketoisovalerate (α-KIV) and acetyl coenzyme A (AcCoA) to form α-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and α-IPMS is allosterically regulated by this amino acid. Existing crystal structures of α-IPMS from Mycobacterium tuberculosis (MtuIPMS) indicate that this enzyme has a strikingly different domain arrangement in each monomer of the homodimeric protein. This asymmetry results in two distinct interfaces between the N-terminal catalytic domains and the C-terminal regulatory domains in the dimer. In this study, residues Arg97 and Asp444 across one of these unequal domain interfaces were substituted to evaluate the importance of protein asymmetry and salt bridge formation between this pair of residues. Analysis of solution-phase structures of wild-type and variant MtuIPMS indicates that substitutions of these residues have little effect on overall protein conformation, a result also observed for addition of the feedback inhibitor leucine to the wild-type enzyme. All variants had increased catalytic efficiency relative to wild-type MtuIPMS, and those with an Asp444 substitution displayed increased affinity for the substrate AcCoA. All variants also showed reduced sensitivity to leucine and altered biphasic reaction kinetics when compared with those of the wild-type enzyme. It is proposed that substituting residues at the asymmetric domain interface increases flexibility in the protein, particularly affecting the AcCoA binding site and the response to leucine, without penalty on catalysis.

QTL analysis using SNP markers developed by next-generation sequencing for identification of candidate genes controlling 4-methylthio-3-butenyl glucosinolate contents in roots of radish, Raphanus sativus L.

SNP markers for QTL analysis of 4-MTB-GSL contents in radish roots were developed by determining nucleotide sequences of bulked PCR products using a next-generation sequencer. DNA fragments were amplified from two radish lines by multiplex PCR with six primer pairs, and those amplified by 2,880 primer pairs were mixed and sequenced. By assembling sequence data, 1,953 SNPs in 750 DNA fragments, 437 of which have been previously mapped in a linkage map, were identified. A linkage map of nine linkage groups was constructed with 188 markers, and five QTLs were detected in two F(2) populations, three of them accounting for more than 50% of the total phenotypic variance being repeatedly detected. In the identified QTL regions, nine SNP markers were newly produced. By synteny analysis of the QTLs regions with Arabidopsis thaliana and Brassica rapa genome sequences, three candidate genes were selected, i.e., RsMAM3 for production of aliphatic glucosinolates linked to GSL-QTL-4, RsIPMDH1 for leucine biosynthesis showing strong co-expression with glucosinolate biosynthesis genes linked to GSL-QTL-2, and RsBCAT4 for branched-chain amino acid aminotransferase linked to GSL-QTL-1. Nucleotide sequences and expression of these genes suggested their possible function in 4MTB-GSL biosynthesis in radish roots.

The slow-onset nature of allosteric inhibition in α-isopropylmalate synthase from Mycobacterium tuberculosis is mediated by a flexible loop.

The identification of structure-function relationships in allosteric enzymes is essential to describing a molecular mechanism for allosteric processes. The enzyme α-isopropylmalate synthase from Mycobacterium tuberculosis (MtIPMS) is subject to slow-onset, allosteric inhibition by l-leucine. Here we report that alternate amino acids act as rapid equilibrium noncompetitive inhibitors of MtIPMS failing to display biphasic inhibition kinetics. Amino acid substitutions on a flexible loop covering the regulatory binding pocket generate enzyme variants that have significant affinity for l-leucine but lack biphasic inhibition kinetics. Taken together, these results are consistent with the flexible loop mediating the slow-onset step of allosteric inhibition.

Removal of the C-terminal regulatory domain of α-isopropylmalate synthase disrupts functional substrate binding.

α-Isopropylmalate synthase (α-IPMS) catalyzes the metal-dependent aldol reaction between α-ketoisovalerate (α-KIV) and acetyl-coenzyme A (AcCoA) to give α-isopropylmalate (α-IPM). This reaction is the first committed step in the biosynthesis of leucine in bacteria. α-IPMS is homodimeric, with monomers consisting of (ß/α)(8) barrel catalytic domains fused to a C-terminal regulatory domain, responsible for binding leucine and providing feedback regulation for leucine biosynthesis. In these studies, we demonstrate that removal of the regulatory domain from the α-IPMS enzymes of both Neisseria meningitidis (NmeIPMS) and Mycobacterium tuberculosis (MtuIPMS) results in enzymes that are unable to catalyze the formation of α-IPM, although truncated NmeIPMS was still able to slowly hydrolyze AcCoA. The lack of catalytic activity of these truncation variants was confirmed by complementation studies with Escherichia coli cells lacking the α-IPMS gene, where transformation with the plasmids encoding the truncated α-IPMS enzymes was not able to rescue α-IPMS activity. X-ray crystal structures of both truncation variants reveal that both proteins are dimeric and that the catalytic sites of the proteins are intact, although the divalent metal ion that is thought to be responsible for activating substrate α-KIV is displaced slightly relative to its position in the substrate-bound, wild-type structure. Isothermal titration calorimetry and WaterLOGSY nuclear magnetic resonance experiments demonstrate that although these truncation variants are not able to catalyze the reaction between α-KIV and AcCoA, they are still able to bind the substrate α-KIV. It is proposed that the regulatory domain is crucial for ensuring protein dynamics necessary for competent catalysis.

Expression pattern of the glucosinolate side chain biosynthetic genes MAM1 and MAM3 of Arabidopsis thaliana in different organs and developmental stages.

Aliphatic glucosinolates, secondary metabolites known to be involved in plant defence, make up the majority of the glucosinolate content of Arabidopsis thaliana, and their structural diversity arises in part from chain elongations of methionine before the formation of the glucosinolate core structure. The key enzymatic step in determining the length of the chain is the condensation of acetyl-coenzyme A with a series of ω-methylthio-2-oxoalkanoic acids, catalyzed by methylthioalkylmalate (MAM) synthases. The existence of two MAM synthases has been previously reported in A. thaliana, ecotype Columbia-0. MAM1 catalyses the condensation step of the first three elongation cycles while MAM3 catalyzes the condensation step of all six elongation cycles. We studied the expression patterns of MAM1 and MAM3 genes in different organs and developmental stages using promoter-GUS fusion lines and qRT-PCR. The promoter-GUS lines revealed MAM1 and MAM3 expression in varying degrees in all organs, but this was generally restricted to the phloem, except in wounded tissue where expression was general. No difference was found between the two genes. The qRT-PCR measurements showed that expression was generally highest in seedlings and vegetative parts at the reproductive phase, but low in flowers and fruits. Since high amounts of glucosinolates accumulate in flowers and fruits, these data indicate possible transport from vegetative to reproductive organs. The expression of MAM1 was different than that of MAM3 with MAM3 having relative more expression in seedlings and roots than MAM1.