SEARCH · PubMed Health

Results for “Biosynthetic Pathways”

Explore indexed PubMed citations for clinical trials, systematic reviews and public health research. Read source abstracts and follow each citation to its original PubMed record.

Quote a phrase for an exact phrase match. Source license links do not imply unrestricted reuse.

At least 937 records · Page 52Linked to original sources

PubMed2005

1-Deoxy-D-xylulose 5-phosphate reductoisomerase (IspC) from Mycobacterium tuberculosis: towards understanding mycobacterial resistance to fosmidomycin.

1-Deoxy-d-xylulose 5-phosphate reductoisomerase (IspC) catalyzes the first committed step in the mevalonate-independent isopentenyl diphosphate biosynthetic pathway and is a potential drug target in some pathogenic bacteria. The antibiotic fosmidomycin has been shown to inhibit IspC in a number of organisms and is active against most gram-negative bacteria but not gram positives, including Mycobacterium tuberculosis, even though the mevalonate-independent pathway is the sole isopentenyl diphosphate biosynthetic pathway in this organism. Therefore, the enzymatic properties of recombinant IspC from M. tuberculosis were characterized. Rv2870c from M. tuberculosis converts 1-deoxy-d-xylulose 5-phosphate to 2-C-methyl-d-erythritol 4-phosphate in the presence of NADPH. The enzymatic activity is dependent on the presence of Mg(2+) ions and exhibits optimal activity between pH 7.5 and 7.9; the K(m) for 1-deoxyxylulose 5-phosphate was calculated to be 47.1 microM, and the K(m) for NADPH was 29.7 microM. The specificity constant of Rv2780c in the forward direction is 1.5 x 10(6) M(-1) min(-1), and the reaction is inhibited by fosmidomycin, with a 50% inhibitory concentration of 310 nM. In addition, Rv2870c complements an inactivated chromosomal copy of IspC in Salmonella enterica, and the complemented strain is sensitive to fosmidomycin. Thus, M. tuberculosis resistance to fosmidomycin is not due to intrinsic properties of Rv2870c, and the enzyme appears to be a valid drug target in this pathogen.

Aldose-Ketose Isomerases↗

PubMed2003

NAD biosynthesis: identification of the tryptophan to quinolinate pathway in bacteria.

Previous studies have demonstrated two different biosynthetic pathways to quinolinate, the universal de novo precursor to the pyridine ring of NAD. In prokaryotes, quinolinate is formed from aspartate and dihydroxyacetone phosphate; in eukaryotes, it is formed from tryptophan. It has been generally believed that the tryptophan to quinolinic acid biosynthetic pathway is unique to eukaryotes; however, this paper describes the use of comparative genome analysis to identify likely candidates for all five genes involved in the tryptophan to quinolinic acid pathway in several bacteria. Representative examples of each of these genes were overexpressed, and the predicted functions are confirmed in each case using unambiguous biochemical assays.

Arylformamidase↗

PubMed1994

Heterospecific cloning of Arabidopsis thaliana cDNAs by direct complementation of pyrimidine auxotrophic mutants of Saccharomyces cerevisiae. I. Cloning and sequence analysis of two cDNAs catalysing the second, fifth and sixth steps of the de novo pyrimidine biosynthesis pathway.

An Arabidopsis thaliana cDNA library was used to complement Saccharomyces cerevisiae pyrimidine auxotrophic mutants. Mutants in all but one (carbamylphosphate synthetase) of the six steps in the de novo pyrimidine biosynthetic pathway could be complemented. We report here the cloning, sequencing and computer analysis of two cDNAs encoding the aspartate transcarbamylase (ATCase; EC 2.1.3.2) and orotate phosphoribosyltransferase-orotidine-5'-phosphate decarboxylase (OPRTase-OMPdecase; EC 2.4.2.10, EC 4.1.1.23) enzymes. These results confirm the presence in A. thaliana of a bifunctional gene whose product catalyses the last two steps of the pyrimidine biosynthetic pathway, as previously suggested by biochemical studies. The ATCase encoding cDNA sequence (PYRB gene) shows an open reading frame (ORF) of 1173 bp coding for 390 amino acids. The cDNA encoding OPRTase-OMPdecase (PYRE-F gene) shows an ORF of 1431 bp coding for 476 amino acids. Computer analysis of the deduced amino acid sequences of both cDNAs shows the expected high similarity with the ATCase, ornithine transcarbamylase (OTCase; EC 2.1.3.3), OPRTase and OMPdecase families. This heterospecific cloning approach increases our understanding of the genetic organization and interspecific functional conservation of the pyrimidine biosynthetic pathway and underlines its usefulness as a model for evolutionary studies.

Amino Acid Sequence↗

PubMed2003

Overexpression of AtCPS and AtKS in Arabidopsis confers increased ent-kaurene production but no increase in bioactive gibberellins.

The plant growth hormone gibberellin (GA) is important for many aspects of plant growth and development. Although most genes encoding enzymes at each step of the GA biosynthetic pathway have been cloned, their regulation is less well understood. To assess how up-regulation of early steps affects the biosynthetic pathway overall, we have examined transgenic Arabidopsis plants that overexpress either AtCPS or AtKS or both. These genes encode the enzymes ent-copalyl diphosphate synthase (CPS) and ent-kaurene synthase, which catalyze the first two committed steps in GA biosynthesis. We find that both CPS and CPS/ent-kaurene synthase overexpressors have greatly increased levels of the early intermediates ent-kaurene and ent-kaurenoic acid, but a lesser increase of later metabolites. These overexpression lines do not exhibit any GA overdose morphology and have wild-type levels of bioactive GAs. Our data show that CPS is limiting for ent-kaurene production and suggest that conversion of ent-kaurenoic acid to GA12 by ent-kaurenoic acid oxidase may be an important rate-limiting step for production of bioactive GA. These results demonstrate the ability of plants to maintain GA homeostasis despite large changes in accumulation of early intermediates in the biosynthetic pathway.

Alkyl and Aryl Transferases↗

PubMed2004

Precursor-directed biosynthesis of epothilone in Escherichia coli.

Engineered biosynthetic pathways provide a powerful method for generating complex molecules. Precursor-directed biosynthesis, which combines chemical synthesis and enzymatic transformations, allows non-native starting materials to be incorporated into biosynthetic pathways. Using this approach, we achieved the production of the anticancer agent epothilone C in Escherichia coli. An E. coli strain was engineered to express the last three modules of the epothilone biosynthetic pathway (epoD-M6, epoE, and epoF) and the substrate required to complement the biosynthetic enzymes was obtained by chemical synthesis. Under high-density cell culture conditions, the E. coli strain processed exogenously fed synthetic substrate into epothilone C at levels comparable to the native host (1 mg/L) and at higher levels than other heterologous hosts. Importantly, this precursor-directed approach will allow chemical modifications to be introduced into the polyketide backbone and may ultimately provide access to epothilone analogues with improved pharmacological properties in quantities sufficient for clinical development.

Antibiotics, Antineoplastic↗

PubMed2000

Seasonal variation of artemisinin and its biosynthetic precursors in plants of Artemisia annua of different geographical origin: proof for the existence of chemotypes.

The time course of the levels of artemisinin, its biosynthetic precursors and the biosynthetically related sesquiterpenes was monitored during a vegetation period of Artemisia annua plants of different geographical origin. Considerable differences in contents of artemisinin and its direct precursors artemisinic acid and dihydroartemisinic acid were found between these A. annua's. For the first time the A. annua plants of different geographical origin were found to belong to different chemotypes. A chemotype with a high artemisinin level was found to have also a high dihydroartemisinic acid level but a relatively low artemisinic acid level. Reversibly, a chemotype with low levels of artemisinin and dihydroartemisinic acid contained a high artemisinic acid level. Artemisinic acid is considered to be the direct precursor of dihydroartemisinic acid in the biosynthetic pathway of artemisinin. The observed accumulation of artemisinic acid in one of the A. annua chemotypes may indicate the presence of a rate-limiting step in the biosynthetic pathway of artemisinin. The enzymatic reduction of artemisinic acid into dihydroartemisinic acid is probably a "bottle neck" in the biosynthetic pathway of artemisinin in varieties with high artemisinic acid and consequentially low artemisinin levels. After a night-frost period, the level of artemisinin was increased, in the Vietnamese A. annua plants, while the dihydroartemisinic acid level was decreased. This phenomenon is in accordance with our hypothesis that stress triggers the conversion of dihydroartemisinic acid to artemisinin. It is suggested that the presence of high levels of dihydroartemisinic acid may be an adaptation to stress conditions (e.g., night-frost), during which relatively high levels of 1O2 are formed. Dihydroartemisinic acid gives the plant protection by reacting with these reactive oxygen species yielding artemisinin as stable end-product.

Artemisia↗

PubMed1998

Down-regulation of cholesterol biosynthesis in sitosterolemia: diminished activities of acetoacetyl-CoA thiolase, 3-hydroxy-3-methylglutaryl-CoA synthase, reductase, squalene synthase, and 7-dehydrocholesterol delta7-reductase in liver and mononuclear leukocytes.

Sitosterolemia is a recessively inherited disorder characterized by abnormally increased plasma and tissue plant sterol concentrations. Patients have markedly reduced whole body cholesterol biosynthesis associated with suppressed hepatic, ileal, and mononuclear leukocyte 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, the rate-controlling enzyme in cholesterol biosynthetic pathway, coupled with significantly increased low density lipoprotein (LDL) receptor expression. To investigate the mechanism of down-regulated cholesterol biosynthesis, we assayed several other key enzymes in the cholesterol biosynthetic pathway including acetoacetyl-CoA thiolase, HMG-CoA synthase, squalene synthase, and 7-dehydrocholesterol delta7-reductase activities in liver and freshly isolated mononuclear leukocytes from four sitosterolemic patients and 19 controls. Hepatic acetoacetyl-CoA thiolase, HMG-CoA synthase, reductase, and squalene synthase activities were significantly decreased (P < 0.05) -39%, -54%, -76%, and -57%, respectively, and 7-dehydrocholesterol delta7-reductase activity tended to be lower (-35%) in the sitosterolemic compared with control subjects. The reduced HMG-CoA synthase, reductase, and squalene synthase activities were also found in mononuclear leukocytes from a sitosterolemic patient. Thus, reduced cholesterol synthesis is caused not only by decreased HMG-CoA reductase but also by the coordinate down-regulation of entire pathway of cholesterol biosynthesis. These results suggest that inadequate cholesterol production in sitosterolemia is due to abnormal down-regulation of early, intermediate, and late enzymes in the cholesterol biosynthetic pathway rather than a single inherited defect in the HMG-CoA reductase gene.

Acetyl-CoA C-Acetyltransferase↗

PubMed2003

[Advances in molecular regulation of artemisinin biosynthesis].

Artemisinin, a new and a very potent antimalarial drug, is produced by the Chinese medicinal herb Artemisia annua L. It is a sesquiterpene lactone with an endoperoxide bridge and is active against chloroquine resistant forms of Plasmodium falciparum. The relatively low yield (0.01% - 0.6%) of artemisinin in A. annua is a serious limitation to the commercialization of the drug. Therefore, a through understanding of the biosynthetic pathway and the characterization of the involved enzymes are important for the biology production of artemisinin. This review is focused on the recent progress in the molecular regulation of artemisinin biosynthesis from the following aspects: the biosynthetic pathway of artemisinin, the key enzymes involved in artemisinin biosynthesis, and the molecular regulation of artemisinin biosynthesis. The biosynthetic pathway of artemisinin belongs to the isoprenoid metabolite pathway, the key enzymes involved in the biosynthesis of artemisinin include: 3-hydroxy-3-methylglutaryl-CoA reductase (HMGR), farnesyl diphosphate synthase (FDPS), and amorpha-4, 11-diene synthase, of which amorpha-4, 11-diene synthase catalyzes the cyclisation of the ubiquitous precursor farnesyl diphosphate to the highly specific olefinic sesquiter-pene skeletons and has been postulated as the regulatory step in the biosynthesis of artemisinin. Recently the gene encoding of the amorpha-4, 11-diene synthase has been cloned and the functional expressions have been studied by several research teams, therefore, the breakthroughs in production of artemisinin could hopefully be achieved by metabolic engineering of the plant, in particular, by over-expressing enzyme(s) catalyzing the rate limiting step(s) of artemisinin biosynthesis or by inhibiting the enzyme(s) of other pathway competing for its precursors. Besides, the effects of the heterogenesis isoprenoid pathway related genes on artemisinin biosynthesis of the transformed plants were also discussed.

Alkyl and Aryl Transferases↗

PubMed1975

Pyrimidine biosynthetic enzymes of Salmonella typhimurium, repressed specifically by growth in the presence of cytidine.

The repressive effects of exogenous cytidine on growing cells was examined in a specially constructed strain in which the pool sizes of endogenous uridine 5'-diphosphate and uridine 5'-triphosphate cannot be varied by the addition of uracil and/or uridine to the medium. Five enzymes of the pyrimidine biosynthetic pathway and one enzyme of the arginine biosynthetic pathway were assayed from cells grown under a variety of conditions. Cytidine repressed the synthesis of dihydroorotase (encoded by pyrC), dihydroorotate dehydrogenase (encoded by pyrD), and ornithine transcarbamylase (encoded by argI). Moreover, aspartate transcarbamylase (encoded by pyrB) became further derepressed upon cytidine addition, whereas no change occurred in the levels of the last two enzymes (encoded by pyrE and pyrF) of the pyrimidine pathway. Quantitative nucleotide pool determinations have provided evidence that any individual ribo- or deoxyribonucleoside mono-, di-, or triphosphate of cytosine or uracil is not a repressing metabolite for the pyrimidine biosynthetic enzymes. Other nucleotide derivatives or ratios must be considered.

Adenine Nucleotides↗

PubMed2003

Lignin: genetic engineering and impact on pulping.

Lignin is a major component of wood, the most widely used raw material for the production of pulp and paper. Although the biochemistry and molecular biology underpinning lignin production are better understood than they are for the other wood components, recent work has prompted a number of re-evaluations of the lignin biosynthetic pathway. Some of the work on which these revisions have been based involved the investigation of transgenic plants with modified lignin biosynthesis. In addition to their value in elucidating the lignin biosynthetic pathway, such transgenic plants are also being produced with the aim of improving plant raw materials for pulp and paper production. This review describes how genetic engineering has yielded new insights into how the lignin biosynthetic pathway operates and demonstrates that lignin can be improved to facilitate pulping. The current technologies used to produce paper are presented in this review, followed by a discussion of the impact of lignin modification on pulp production. Fine-tuned modification of lignin content, composition, or both is now achievable and could have important economic and environmental benefits.

Gene Expression Regulation, Plant↗

PubMed2001

Physiological characterisation of Penicillium chrysogenum strains expressing the expandase gene from Streptomyces clavuligerus during batch cultivations. Growth and adipoyl-7-aminodeacetoxycephalosporanic acid production.

The production of adipoyl-7-aminodeacetoxy-cephalosporanic acid (ad-7-ADCA) was studied, using two recombinant strains of Penicillium chrysogenum carrying the expandase gene from Streptomyces clavuligerus. The adipoyl-side chain of this compound may easily be removed using an amidase; and this process therefore represents a new route for the production of 7-ADCA, which serves as a precursor for the production of many semi-synthetic cephalosporins. In this study, one low- and one high-yielding strains were characterised and the specific productivities of ad-7-ADCA and byproducts of the biosynthetic pathway were compared. The fluxes through the biosynthetic pathway were quantified and it was found that there was a 30% higher flux through the expandase in the high-yielding strain. In both strains, there was a significant degradation of adipate. Furthermore, the initial adipate concentration in batch cultures was shown to have a positive effect on the formation of ad-7-ADCA.

Adipates↗

PubMed2005

Unexpected behavior of coniferin in lignin biosynthesis of Ginkgo biloba L.

To gain insight into the behavior of monolignol glucoside in Ginkgo biloba L., we examined glucosides potentially involved in lignin biosynthetic pathway. Coniferin (coniferyl alcohol 4O-beta-D-glucoside) is a strong candidate for the storage form of monolignol. Coniferaldehyde glucoside may also have a role in lignin biosynthesis; this was examined with tracer experiments using labeled glucosides fed to stem segments. A series of tracer experiments showed that coniferin and coniferaldehyde glucoside were modified into coniferyl alcohol and then efficiently incorporated into lignin under the experimental conditions used. Interestingly, more than half of the administered coniferin underwent an oxidation to the aldehyde form before its aglycone; coniferyl alcohol was polymerized into lignin. This suggests that there is an alternative pathway for coniferin to enter the monolignol biosynthetic pathway, in addition to the direct pathway beginning with the deglucosylation of coniferin catalyzed by beta-glucosidase. Enzymatic assays revealed that coniferaldehyde glucoside was produced enzymatically from coniferin, and that coniferaldehyde glucoside can be deglucosylated to yield coniferaldehyde, which could be fated to become coniferyl alcohol . Albeit the findings cannot be taken as proof for the in-planta functioning, these results present a possibility for the existence of alternative pathway in which some of the stored coniferin is oxidized to coniferaldehyde glucoside, which is deglucosylated to generate coniferaldehyde that joins the monolignol biosynthesis pathway.

Cinnamates↗

PubMed1984

Biosynthesis of astromicin and related antibiotics. I. Biosynthetic studies by bioconversion experiments.

Biosynthesis of astromicin, a unique pseudodisaccharide aminoglycoside antibiotic containing 1,4-diaminocyclitol component, was investigated by isolating a variety of possible precursor compounds from mutants of Micromonospora olivasterospora in which biosynthetic pathways for astromicin were blocked. Washed mycelia of M. olivasterospora mutants converted these compounds to astromicin, which was detected by thin-layer chromatography. Since astromicin possesses one glycyl and three methyl groups, [14C]glycine and [14C]methionine should be incorporated into precursors to form astromicin. To confirm the biosynthetic pathway, formation of labeled astromicin from the precursors was examined using [1-14C]-glycine or [methyl-14C]methionine. From above results, we propose the biosynthetic pathway for astromicin as shown in Fig. 2.

Aminoglycosides↗

PubMed1993

Characterization of the cobalamin (vitamin B12) biosynthetic genes of Salmonella typhimurium.

Salmonella typhimurium synthesizes cobalamin (vitamin B12) de novo under anaerobic conditions. Of the 30 cobalamin synthetic genes, 25 are clustered in one operon, cob, and are arranged in three groups, each group encoding enzymes for a biochemically distinct portion of the biosynthetic pathway. We have determined the DNA sequence for the promoter region and the proximal 17.1 kb of the cob operon. This sequence includes 20 translationally coupled genes that encode the enzymes involved in parts I and III of the cobalamin biosynthetic pathway. A comparison of these genes with the cobalamin synthetic genes from Pseudomonas denitrificans allows assignment of likely functions to 12 of the 20 sequenced Salmonella genes. Three additional Salmonella genes encode proteins likely to be involved in the transport of cobalt, a component of vitamin B12. However, not all Salmonella and Pseudomonas cobalamin synthetic genes have apparent homologs in the other species. These differences suggest that the cobalamin biosynthetic pathways differ between the two organisms. The evolution of these genes and their chromosomal positions is discussed.

Amino Acid Sequence↗

PubMed1998

Biosynthesis and regulation of coronatine, a non-host-specific phytotoxin produced by Pseudomonas syringae.

Many P. syringae pathovars are known to produce low-molecular-weight, diffusible toxins in infected host plants. These phytotoxins reproduce some of the symptoms of the relevant bacterial disease and are effective at very low concentrations. Phytotoxins generally enhance the virulence of the P. syringae pathovar which produces them, but are not required for pathogenesis. Genes encoding phytotoxin production have been identified and cloned from several P. syringae pathovars. With the exception of coronatine, toxin biosynthetic gene clusters are generally chromosomally encoded. In several pathovars, the toxin biosynthetic gene cluster also contains a resistance gene which functions to protect the producing strain from the biocidal effects of the toxin. In the case of phaseolotoxin, a resistance gene (argK) has been utilized to engineer phaseolotoxin-resistant tobacco plants. Although P. syringae phytotoxins can induce very similar effects in plants (chlorosis and necrosis), their biosynthesis and mode of action can be quite different. Knowledge of the biosynthetic pathways to these toxins and the cloning of the structural genes for their biosynthesis has relevance to the development of new bioactive compounds with altered specificity. For example, polyketides constitute a huge family of structurally diverse natural products including antibiotics, chemotherapeutic compounds, and antiparasitics. Most of the research on polyketide synthesis in bacteria has focused on compounds synthesized by Streptomyces or other actinomycetes. It is also important to note that it is now possible to utilize a genetic rather than synthetic approach to biosynthesize novel polyketides with altered biological properties (Hutchinson and Fujii, 1995; Kao et al., 1994; Donadio et al., 1993; Katz and Donadio, 1993). Most of the reprogramming or engineering of novel polyketides has been done using actinomycete PKSs, but much of this technology could also be applied to polyketides synthesized by Pseudomonas when sufficient sequence information is available. It is important to note that Pseudomonas produces a variety of antimicrobial compounds from the polyketide pathway, including mupirocin (pseudomonic acid) (Feline et al., 1977), pyoluteorin (Cuppels et al., 1986), and 2-4 diacetylphloroglucinol (Phl) (Bangera and Thomashow, 1996). Pseudomonic acid is valued for its pharmaceutical properties as an antibiotic (Aldridge, 1992), whereas pyoluteorin and Phl have antifungal properties (Howell and Stipanovic, 1980; Keel et al., 1992). A thorough understanding of the biosynthetic pathway to polyketide phytotoxins such as coronatine may ultimately lead to the development of novel compounds with altered biological properties. Thus, specific genes in the biosynthetic pathways of P. syringae phytotoxins could be deployed in other systems to develop new compounds with a wide range of activities.

Amino Acids↗

PubMed1984

Divergence in cholesterol biosynthetic rates and 3-hydroxy-3-methylglutaryl-CoA reductase activity as a consequence of granulocyte versus monocyte-macrophage differentiation in HL-60 cells.

Addition of dimethyl sulfoxide or phorbol myristate acetate (PMA) to HL-60 cell cultures induces granulocytic or monocyte-macrophage differentiation, respectively, in HL-60 cells. Dimethyl sulfoxide-induced granulocyte differentiation in HL-60 cells is associated with a decrease in cellular 3-hydroxy-3-methylglutaryl (HMG)-CoA reductase activity and with a decrease in the incorporation of [14C]acetate and mevalonate into products of the cholesterol biosynthetic pathway. PMA-induced monocyte-macrophage differentiation in HL-60 cells is associated with a rapid and profound fall in cell proliferation but nonetheless is accompanied by a dose-dependent increase in cellular HMG-CoA reductase activity and [14C]acetate incorporation into the cholesterol biosynthetic pathway. In addition, PMA induces an increase in [14C]mevalonate incorporation into cholesterol and its precursors, suggesting that post-HMG-CoA reductase events in cholesterol biosynthesis are also enhanced. Mature peripheral blood human monocytes possess an active cholesterol biosynthetic pathway, whereas mature human granulocytes are almost entirely lacking in the ability to synthesize post-squalene products. Our results with HL-60 cells indicate that this divergence in sterol-synthesizing ability between two cell lineages, which normally also derive from a common stem cell, can be observed as an early event in the differentiation process.

Cell Differentiation↗

PubMed2006

GlycoVis: visualizing glycan distribution in the protein N-glycosylation pathway in mammalian cells.

Glycosylation has profound effects on the quality of recombinant proteins produced in mammalian cells. The biosynthetic pathways of N-linked glycans on glycoproteins involves a relatively small number of enzymes and nucleotide sugars. Many of these glycoconjugate enzymes can utilize multiple N-glycans as substrates, thus generating a large number of glycan intermediates, and making the biosynthetic pathway resemble a network with diverging and converging paths. The N-glycans on secreted glycoprotein molecules include not only terminal glycans, but also pathway intermediates. To better assess the glycan distribution and the potential route of their synthesis, we created GlycoVis, a visualization program that displays the distribution and the potential reaction paths leading to each N-glycan on the reaction network. The substrate specificities of the enzymes involved were organized into a relationship matrix. With the input of glycan distribution data, the program outputs a reaction pathway map which labels the relative abundance levels of different glycans with different colors. The program also traces all possible reaction paths leading to each glycan and identifies each pathway on the map. Glycoform distribution of Chinese Hamster Ovary cell-derived tissue plasminogen activator (TPA), and human and mouse IgG were used as illustrations for the application of GlycoVis. In addition, the intracellular and secreted IgG from an NS0 producer cell line were isolated, and their glycoform profiles were displayed using GlycoVis for comparison. This visualization tool facilitates the analysis of potential reaction paths utilized under different physiological or culture conditions, and may provide insight on the potential targets for metabolic engineering.

Algorithms↗

PubMed2004

3-Isopropylmalate is the major endogenous substrate of the Saccharomyces cerevisiae trans-aconitate methyltransferase.

The Saccharomyces cerevisiae Tmt1 gene product is the yeast homologue of the Escherichia coli enzyme that catalyzes the methyl esterification of trans-aconitate, a thermodynamically favored isomer of cis-aconitate and an inhibitor of the citric acid cycle. It has been proposed that methylation may attenuate trans-aconitate inhibition of aconitase and other enzymes of the cycle. Although trans-aconitate is a minor endogenous substrate of the Tmt1 enzyme in extracts of S. cerevisiae, the major endogenous substrate has yet to be identified. We show here that a trimethylsilylated derivative of the major methylated endogenous product of Tmt1 in yeast extracts has an identical gas chromatography retention time and an identical electron impact mass spectrum as one of the two possible monomethyl ester derivatives of (2R,3S)-3-isopropylmalate. (2R,3S)-3-Isopropylmalate is an intermediate of the leucine biosynthetic pathway that shares similar intermediates and reaction chemistry with the portion of the citric acid cycle from oxaloacetate to alpha-ketoglutarate via cis-aconitate. The Tmt1 methyltransferase recognizes (2R,3S)-3-isopropylmalate with similar kinetics as it does trans-aconitate, with respective K(m) values of 127 and 53 microM and V(max) values of 59 and 70 nmol min(-1) mg(-1) of protein in a Tmt1-overexpressed yeast extract. However, we found that isopropylfumarate, the direct homologue of trans-aconitate in the leucine biosynthetic pathway, was at best a very poor substrate for the Tmt1 yeast enzyme. Similarly, the direct homologue of 3-isopropylmalate in the citric acid cycle, isocitrate, is also a very poor substrate. This apparent change in specificity between the intermediates of these two pathways can be understood in terms of the binding of these substrates to the active site. These results suggest that the Tmt1 methyltransferase may work in two different pathways in two different ways: for detoxification in the citric acid cycle and for a possibly novel biosynthetic branch reaction of the leucine biosynthetic pathway.

Aconitic Acid↗