Loss of Met31 and Met32 abolishes all Met4-activated transcription, while only certain target genes, such as sulfate assimilation genes, depend on Cbf1 and Met28 for expression. Unlike Met4 and the other cofactors, Cbf1 remains promoter-bound under inducing and repressing conditions and helps to stabilize Met32, the main platform for Met4, at promoters.
Mass spectrometry technologies for measurement of cellular metabolism are opening new avenues to explore drug activity. Trimethoprim is an antibiotic that inhibits bacterial dihydrofolate reductase (DHFR). Kinetic flux profiling with 15 N-labeled ammonia in Escherichia coli reveals that trimethoprim leads to blockade not only of DHFR but also of another critical enzyme of folate metabolism: folylpoly-γ-glutamate synthetase (FP-γ-GS). Inhibition of FP-γ-GS is not directly due to trimethoprim. Instead, it arises from accumulation of DHFR's substrate dihydrofolate, which we show is a potent FP-γ-GS inhibitor. Thus, owing to the inherent connectivity of the metabolic network, falling DHFR activity leads to falling FP-γ-GS activity in a domino-like cascade. This cascade results in complex folate dynamics, and its incorporation in a computational model of folate metabolism recapitulates the dynamics observed experimentally. These results highlight the potential for quantitative analysis of cellular metabolism to reveal mechanisms of drug action.Enzyme inhibition is among the best established mechanisms of drug action 1 . Nevertheless, even for enzyme inhibitors, the mechanisms of action are often incompletely understood 2 . For example, drugs known to be inhibitors of one enzyme may further bind to unidentified enzymes or indirectly interact with other pathways. In part, this limited understanding is due to the scope and complexity of cellular chemical reactions as well as the associated difficulty of tracking many reactions at once. The recent development of technologies that can measure many cellular metabolites 3-5 and metabolic fluxes 6-8 in parallel may allow for more complete elucidation of actions of enzyme inhibitors. Here we apply LC-MS/MS to explore the effects of the DHFR inhibitor trimethoprim (1) in Escherichia coli by tracking both metabolite concentrations and fluxes throughout the folate pathway.Folates are cofactors that accept or donate one-carbon units for the biosynthesis of essential metabolites including purines, thymine (2), methionine (3) and glycine (4). Folate metabolism is the target of many therapeutics, including antibiotics (trimethoprim and sulfa drugs) 9-11 and anticancer agents (methotrexate, 5, and pemetrexed, 6) 12 . Folates are synthesized from GTP (7), p-aminobenzoic acid (pABA, 8) and glutamates (9) and can exist in three different oxidation/reduction states: PteGlu n (pteroylglutamate, or folate,
Three vegetables, spinach, carrot, and bell peppers were blanched conventionally in water and using pulsed microwave at 95 Ϯ Ϯ Ϯ Ϯ Ϯ 2 C. The effect of various parameters like mass of the product, mobility of the product in the microwave field, and physical geometry on the temperature attained was evaluated. The study also included the kinetics of peroxidase inactivation, temperature, and power distribution during microwave blanching. The study highlights the potential application of microwave blanching in reducing the loss of valuable nutrients. The kinetics of peroxidase inactivation indicated that microwave blanching was comparable to water blanching with higher reaction rate in the case of water blanching. The temperature and absorbed power levels during microwave blanching was influenced by the vegetable itself, its quantity, shape, location in the oven, and the microwave power applied.
Folylpolyglutamate synthetase, which is responsible for the addition of a polyglutamate tail to folate and folate derivatives, is an ATP-dependent enzyme isolated from eukaryotic and bacterial sources, where it plays a key role in the retention of the intracellular folate pool. Here, we report the 2.4-Å resolution crystal structure of the MgATP complex of the enzyme from Lactobacillus casei. The structural analysis reveals that folylpolyglutamate synthetase is a modular protein consisting of two domains, one with a typical mononucleotide-binding fold and the other strikingly similar to the folate-binding enzyme dihydrofolate reductase. We have located the active site of the enzyme in a large interdomain cleft adjacent to an ATP-binding P-loop motif. Opposite this site, in the C domain, a cavity likely to be the folate binding site has been identified, and inspection of this cavity and the surrounding protein structure suggests that the glutamate tail of the substrate may project into the active site. A further feature of the structure is a well defined ⍀ loop, which contributes both to the active site and to interdomain interactions. The determination of the structure of this enzyme represents the first step toward the elucidation of the molecular mechanism of polyglutamylation of folates and antifolates.Both bacteria and eukaryotes require folate coenzymes in a number of important metabolic cycles, where they serve as carriers of one-carbon units, typically in the form of 5,10-methylene-tetrahydrofolate or 10-formyl-tetrahydrofolate. These folate derivatives act as cosubstrates in a host of enzymes involved in one-carbon metabolism (1), including the biosynthesis of methionine and thymidylate and the de novo synthesis of purine. Folate antagonists (or antifolates), on the other hand, are potent inhibitors of purine and thymidylate biosynthesis, blocking DNA synthesis and halting cell growth, and are important in cancer chemotherapy. Methotrexate, for example, is a potent inhibitor of dihydrofolate reductase (DHFR) (2) whereas 5-formyltetrahydrofolate combined with 5-fluorouracil effectively inhibits thymidylate synthase (3). It has been shown (2) that polyglutamylation of these antifolates (addition of L-glutamate groups at the ␥-carboxy of the terminal glutamate) greatly enhances their cytotoxicity and that the enzyme folylpolyglutamate synthetase (FPGS) is responsible for the conversion to the more active form. Consequently, the use of FPGS to produce anticancer drugs with increased cytotoxicity is currently under biochemical and clinical study (2).Endogenous folates in the cell are predominantly in the form of polyglutamate derivatives, which are the normal substrates for enzymes involved in one-carbon metabolism. It has been shown that many folate-dependent enzymes have a higher affinity for polyglutamate substrates (reviewed in refs. 1 and 4); thymidylate synthase, for example, shows a 60% increase in substrate affinity from monoglutamate 5,10-methylene-tetrahydrofolate to the diglutamate derivativ...
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