Metabolic engineering in yeast demonstrates that S-adenosylmethionine controls flux through the methylenetetrahydrofolate reductase reaction in vivo.

One-carbon flux into methionine and S-adenosylmethionine (AdoMet) is thought to be controlled at the methylenetetrahydrofolate reductase (MTHFR) step. Mammalian MTHFRs are inhibited by AdoMet in vitro, and it has been proposed that methyl group biogenesis is regulated in vivo by this feedback loop. In this work, we used metabolic engineering in the yeast Saccharomyces cerevisiae to test this hypothesis. Like mammalian MTHFRs, the yeast MTHFR encoded by the MET13 gene is NADPH-dependent and is inhibited by AdoMet in vitro. This contrasts with plant MTHFRs, which are NADH-dependent and AdoMet-insensitive. To manipulate flux through the MTHFR reaction in yeast, the chromosomal copy of MET13 was replaced by an Arabidopsis MTHFR cDNA (AtMTHFR-1) or by a chimeric sequence (Chimera-1) comprising the yeast N-terminal domain and the AtMTHFR-1 C-terminal domain. Chimera-1 used both NADH and NADPH and was insensitive to AdoMet, supporting the view that the C-terminal domain is responsible for AdoMet inhibition. Engineered yeast expressing Chimera-1 accumulated 140-fold more AdoMet and 7-fold more methionine than did the wild-type and grew normally. Yeast expressing AtMTHFR-1 accumulated 8-fold more AdoMet. This is the first in vivo evidence that the AdoMet sensitivity and pyridine nucleotide preference of MTHFR control methylneogenesis. (13)C labeling data indicated that glycine cleavage becomes a more prominent source of one-carbon units when Chimera-1 is expressed. Possibly related to this shift in one-carbon fluxes, total folate levels are doubled in yeast cells expressing Chimera-1.     

Molecular phylogenetic analysis of methylenetetrahydrofolate reductase family of proteins

Methylenetetrahydrofolate reductase (MTHFR) family of proteins catalyze the conversion of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. They contain a flavin adenine dinucleotide (FAD) as the cofactor and the enzyme in eukaryotes, except in yeast, is known to be allosterically regulated by S-adenosylmethionine. Some cardiovascular diseases, neural tube defects, neuropsychiatric diseases and certain type of cancers in humans are associated with certain polymorphisms of MTHFR. Here, we analyzed 57 of MTHFR polypeptide sequences by multiple sequence alignment and determined previously unrecognized conserved residues that may have a functional or structural importance. A previously unrecognized ATP synthase motif was found in all of the examined plant MTHFRs, suggesting a different functional capability to the plant MTHFRs in addition to the known function. On a phylogenetic tree built, eukaryotic MTHFR proteins formed a clear cluster separated from prokaryotic and archeal relatives. The sequence identities among the eukaryotic MTHFRs were less divergent than the bacterial MTHFRs.     

Biochemical and genetic analysis of methylenetetrahydrofolate reductase in Leishmania metabolism and virulence

Methylenetetrahydrofolate reductase (MTHFR; EC 1.5.1.20) is the sole enzyme responsible for generation of 5-methyltetrahydrofolate, which is required for methionine synthesis and provision of methyl groups via S-adenosylmethionine. Genome analysis showed that Leishmania species, unlike Trypanosoma brucei and Trypanosoma cruzi, contain genes encoding MTHFR and two distinct methionine synthases. Leishmania MTHFR differed from those in other eukaryotes by the absence of a C-terminal regulatory domain. L. major MTHFR was expressed in yeast and recombinant enzyme was produced in Escherichia coli. MTHFR was not inhibited by S-adenosylmethionine and, uniquely among folate-metabolizing enzymes, showed dual-cofactor specificity with NADH and NADPH under physiological conditions. MTHFR null mutants (mthfr(-)) lacked 5-methyltetrahydrofolate, the most abundant intracellular folate, and could not utilize exogenous homocysteine for growth. Under conditions of methionine limitation mthfr(-) mutant cells grew poorly, whereas their growth was normal in standard culture media. Neither in vitro MTHFR activity nor the growth of mthfr(-) mutants or MTHFR overexpressors were differentially affected by antifolates known to inhibit parasite growth via targets beyond dihydrofolate reductase and pteridine reductase 1. In a mouse model of infection mthfr(-) mutants showed good infectivity and virulence, indicating that sufficient methionine is available within the parasitophorous vacuole to meet the needs of the parasite.     

Two Aspergillus nidulans genes encoding methylenetetrahydrofolate reductases are up-regulated by homocysteine

Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, a co-substrate in the synthesis of methionine from homocysteine. We have cloned and characterized two Aspergillus nidulans genes encoding MTHFRs: metA and metF. Mutations in either gene result in methionine requirement; the metA-encoded enzyme is responsible for only 10-15% of total MTHFR activity. These two enzymes belong to different classes of MTHFRs. Mutations in metA but not in the metF gene are suppressed by mutations resulting in enhancement of homocysteine synthesis. The expression of both genes is up-regulated by homocysteine.     

Expression analysis of Arabidopsis thaliana NAD-dependent isocitrate dehydrogenase genes shows the presence of a functional subunit that is mainly expressed in the pollen and absent from vegetative organs

NAD-dependent isocitrate dehydrogenase (IDH) is a Krebs cycle enzyme situated in mitochondria. In Arabidopsis thaliana, five genes encode functional IDH subunits that can be classed into two groups based on gene structure and subunit amino acid sequence. Arabidopsis contains two 'catalytic' and three 'regulatory' subunits according to their homology with yeast IDH. To date, an active IDH is believed to be heteromeric, containing at least one of each subunit type. This was verified in Arabidopsis by the complementation of yeast IDH mutants with the different Arabidopsis IDH-encoding cDNAs. Indeed, a single 'catalytic' and 'regulatory' subunit was sufficient to restore acetate growth of the yeast IDH double mutant. To gain information on possible IDH subunit interactions in planta, Arabidopsis IDH gene expression was analysed by Northern blot, PCR on cDNA libraries, in silico and in 'promoter'-reporter gene transgenic plants. Four of the IDH genes were expressed in all plant organs tested, while one gene (At4g35650) was not expressed in vegetative organs but was mainly expressed in the pollen. In leaves, the IDH genes were highly expressed in the veins, and to a lesser extent in mesophyll cells. The data are discussed with respect to IDH in other plant species.     

Isolation and characterization of cDNAs encoding wheat 3-hydroxy-3-methylglutaryl coenzyme A reductase.

The enzyme 3-hydroxy-3-methylglutaryl coenzyme A reductase (HMGR, EC 1.1.1.34) is a key enzyme in the isoprenoid biosynthetic pathway. We have isolated partial cDNAs from wheat (Triticum aestivum) using the polymerase chain reaction. Comparison of deduced amino acid sequences of these cDNAs shows that they represent a small family of genes that share a high degree of sequence homology among themselves as well as among genes from other organisms including tomato, Arabidopsis, hamster, human, Drosophila, and yeast. Southern blot analysis reveals the presence of at least four genes. Our results concerning the tissue-specific expression as well as developmental regulation of these HMGR cDNAs highlight the important role of this enzyme in the growth and development of wheat.     

Functional characterization of human methylenetetrahydrofolate reductase in Saccharomyces cerevisiae.

Human methylenetetrahydrofolate reductase (MTHFR, EC 1.5.1.20) catalyzes the reduction of 5,10-methylenetetrahydrofolate to 5-methyltetrahydrofolate. 5-Methyltetrahydrofolate is a major methyl donor in the remethylation of homocysteine to methionine. Impaired MTHFR can cause high levels of homocysteine in plasma, which is an independent risk factor for vascular disease and neural tube defects. We have functionally characterized wild-type and several mutant alleles of human MTHFR in yeast, Saccharomyces cerevisiae. We have shown that yeast MET11 is a functional homologue of human MTHFR. Expression of the human MTHFR cDNA in a yeast strain deleted for MET11 can restore the strain's MTHFR activity in vitro and complement its methionine auxotrophic phenotype in vivo. To understand the domain structure of human MTHFR, we have truncated the C terminus (50%) of the protein and demonstrated that expressing an N-terminal human MTHFR in met11(-) yeast cells rescues the growth phenotype, indicating that this region contains the catalytic domain of the enzyme. However, the truncation leads to the reduced protein levels, suggesting that the C terminus may be important for protein stabilization. We have also functionally characterized four missense mutations identified from patients with severe MTHFR deficiency and two common missense polymorphisms found at high frequency in the general population. Three of the four missense mutations are unable to complement the auxotrophic phenotype of met11(-) yeast cells and show less than 7% enzyme activity of the wild type in vitro. Both of the two common polymorphisms are able to complement the growth phenotype, although one exhibited thermolabile enzyme activity in vitro. These results shall be useful for the functional characterization of MTHFR mutations and analysis structure/function relationship of the enzyme.     

The S-methylmethionine cycle in angiosperms: ubiquity, antiquity and activity

Angiosperms synthesize S-methylmethionine (SMM) from methionine (Met) and S-adenosylmethionine (AdoMet) in a unique reaction catalyzed by Met S-methyltransferase (MMT). SMM serves as methyl donor for Met synthesis from homocysteine, catalyzed by homocysteine S-methyltransferase (HMT). MMT and HMT together have been proposed to constitute a futile SMM cycle that stops the free Met pool from being depleted by an overshoot in AdoMet synthesis. Arabidopsis and maize have one MMT gene, and at least three HMT genes that belong to two anciently diverged classes and encode enzymes with distinct properties and expression patterns. SMM, and presumably its cycle, must therefore have originated before dicot and monocot lineages separated. Arabidopsis leaves, roots and developing seeds all express MMT and HMTs, and can metabolize [35S]Met to [35S]SMM and vice versa. The SMM cycle therefore operates throughout the plant. This appears to be a general feature of angiosperms, as digital gene expression profiles show that MMT and HMT are co-expressed in leaves, roots and reproductive tissues of maize and other species. An in silico model of the SMM cycle in mature Arabidopsis leaves was developed from radiotracer kinetic measurements and pool size data. This model indicates that the SMM cycle consumes half the AdoMet produced, and suggests that the cycle serves to stop accumulation of AdoMet, rather than to prevent depletion of free Met. Because plants lack the negative feedback loops that regulate AdoMet pool size in other eukaryotes, the SMM cycle may be the main mechanism whereby plants achieve short-term control of AdoMet level.     

Two anthranilate synthase genes in Arabidopsis: defense-related regulation of the tryptophan pathway.

Arabidopsis thaliana has two genes, ASA1 and ASA2, encoding the alpha subunit of anthranilate synthase, the enzyme catalyzing the first reaction in the tryptophan biosynthetic pathway. As a branchpoint enzyme in aromatic amino acid biosynthesis, anthranilate synthase has an important regulatory role. The sequences of the plant genes are homologous to their microbial counterparts. Both predicted proteins have putative chloroplast transit peptides at their amino termini and conserved amino acids involved in feedback inhibition by tryptophan. ASA1 and ASA2 cDNAs complement anthranilate synthase alpha subunit mutations in the yeast Saccharomyces cerevisiae and in Escherichia coli, confirming that both genes encode functional anthranilate synthase proteins. The distributions of ASA1 and ASA2 mRNAs in various parts of Arabidopsis plants are overlapping but nonidentical, and ASA1 mRNA is approximately 10 times more abundant in whole plants. Whereas ASA2 is expressed at a constitutive basal level, ASA1 is induced by wounding and bacterial pathogen infiltration, suggesting a novel role for ASA1 in the production of tryptophan pathway metabolites as part of an Arabidopsis defense response. Regulation of key steps in aromatic amino acid biosynthesis in Arabidopsis appears to involve differential expression of duplicated genes.     

Identification of AtPIS, a phosphatidylinositol synthase from Arabidopsis.

Phosphatidylinositol synthase is the enzyme responsible for the synthesis of phosphatidylinositol, a key phospholipid component of all eukaryotic membranes and the precursor of messenger molecules involved in signal transduction pathways for calcium-dependent responses in the cell. Using the amino acid sequence of the yeast enzyme as a probe, we identified an Arabidopsis expressed sequence tag potentially encoding the plant enzyme. Sequencing the entire cDNA confirmed the homology between the two proteins. Functional assays, performed by overexpression of the plant cDNA in Escherichia coli, a bacteria which lacks phosphatidylinositol and phosphatidylinositol synthase activity, showed that the plant protein induced the accumulation of phosphatidylinositol in the bacterial cells. Analysis of the enzymatic activity in vitro showed that synthesis of phosphatidylinositol occurs when CDP-diacylglycerol and myo-inositol only are provided as substrates, that it requires manganese or magnesium ions for activity, and that it is at least in part located to the bacterial membrane fraction. These data allowed us to conclude that the Arabidopsis cDNA codes for a phosphatidylinositol synthase. A single AtPIS genetic locus was found, which we mapped to Arabidopsis chromosome 1.     

Structures of NADH and CH3-H4folate complexes of Escherichia coli methylenetetrahydrofolate reductase reveal a spartan strategy for a ping-pong reaction

Methylenetetrahydrofolate reductases (MTHFRs; EC 1.7.99.5) catalyze the NAD(P)H-dependent reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using flavin adenine dinucleotide (FAD) as a cofactor. The initial X-ray structure of Escherichia coli MTHFR revealed that this 33-kDa polypeptide is a (betaalpha)(8) barrel that aggregates to form an unusual tetramer with only 2-fold symmetry. Structures of reduced enzyme complexed with NADH and of oxidized Glu28Gln enzyme complexed with CH(3)-H(4)folate have now been determined at resolutions of 1.95 and 1.85 A, respectively. The NADH complex reveals a rare mode of dinucleotide binding; NADH adopts a hairpin conformation and is sandwiched between a conserved phenylalanine, Phe223, and the isoalloxazine ring of FAD. The nicotinamide of the bound pyridine nucleotide is stacked against the si face of the flavin ring with C4 adjoining the N5 of FAD, implying that this structure models a complex that is competent for hydride transfer. In the complex with CH(3)-H(4)folate, the pterin ring is also stacked against FAD in an orientation that is favorable for hydride transfer. Thus, the binding sites for the two substrates overlap, as expected for many enzymes that catalyze ping-pong reactions, and several invariant residues interact with both folate and pyridine nucleotide substrates. Comparisons of liganded and substrate-free structures reveal multiple conformations for the loops beta2-alpha2 (L2), beta3-alpha3 (L3), and beta4-alpha4 (L4) and suggest that motions of these loops facilitate the ping-pong reaction. In particular, the L4 loop adopts a "closed" conformation that allows Asp120 to hydrogen bond to the pterin ring in the folate complex but must move to an "open" conformation to allow NADH to bind.     

Diversity of Arabidopsis Genes Encoding Precursors for Phytosulfokine, a Peptide Growth Factor

Phytosulfokine-alpha (PSK-alpha), a unique plant peptide growth factor, was originally isolated from conditioned medium of asparagus (Asparagus officinalis) mesophyll cell cultures. PSK-alpha has several biological activities including promoting plant cell proliferation. Four genes that encode precursors of PSK-alpha have been identified from Arabidopsis. Analysis of cDNAs for two of these, AtPSK2 and AtPSK3, shows that both of these genes consist of two exons and one intron. The predicted precursors have N-terminal signal peptides and only a single PSK-alpha sequence located close to their carboxyl termini. Both precursors contain dibasic processing sites flanking PSK, analogous to animal and yeast prohormones. Although the PSK domain including the sequence of PSK-alpha and three amino acids preceding it are perfectly conserved, the precursors bear very limited similarity among Arabidopsis and rice (Oryza sativa), suggesting a new level of diversity among polypeptides that are processed into the same signaling molecule in plants, a scenario not found in animals and yeast. Unnatural [serine-4]PSK-beta was found to be secreted by transgenic Arabidopsis cells expressing a mutant of either AtPSK2 or AtPSK3 cDNAs, suggesting that both AtPSK2 and AtPSK3 encode PSK-alpha precursors. AtPSK2 and AtPSK3 were expressed demonstrably not only in cultured cells but also in intact plants, suggesting that PSK-alpha may be essential for plant cell proliferation in vivo as well as in vitro. Overexpression of either precursor gene allowed the transgenic calli to grow twice as large as the controls. However, the transgenic cells expressing either antisense cDNA did not dramatically decrease mitogenic activity, suggesting that these two genes may act redundantly.     

Arabidopsis contains nine long-chain acyl-coenzyme a synthetase genes that participate in fatty acid and glycerolipid metabolism

Long-chain acyl-coenzyme A (CoA) synthetases (LACSs) activate free fatty acids to acyl-CoA thioesters and as such play critical roles in fatty acid metabolism. This important class of enzymes factors prominently in several fatty acid-derived metabolic pathways, including phospholipid, triacylglycerol, and jasmonate biosynthesis and fatty acid beta-oxidation. In an effort to better understand the factors that control fatty acid metabolism in oilseeds, we have sought to identify and characterize genes that encode LACSs in Arabidopsis. Nine cDNAs were identified, cloned, and tested for their ability to complement a LACS-deficient strain of yeast (Saccharomyces cerevisiae). Seven of the nine successfully restored growth, whereas two cDNAs encoding putative peroxisomal isoforms did not. Lysates from yeast cells overexpressing each of the nine cDNAs were active in LACS enzyme assays using oleic acid as a substrate. The substrate specificities of the enzymes were determined after overexpression in LACS-deficient Escherichia coli. Most of the LACS enzymes displayed highest levels of activity with the fatty acids that make up the common structural and storage lipids in Arabidopsis tissues. Analysis of the tissue-specific expression profiles for these genes revealed one flower-specific isoform, whereas all others were expressed in various tissues throughout the plant. These nine cDNAs are thought to constitute the entire LACS family in Arabidopsis, and as such, will serve as powerful tools in the study of acyl-CoA metabolism in oilseeds.