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[This retracts the article PMC6042866.].
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Substrate channeling has emerged as a common mechanism for enzymatic intermediate transfer. A conspicuous gap in knowledge concerns the use of covalent lysine imines in the transfer of carbonyl-group-containing intermediates, despite their wideuse in enzymatic catalysis. Here we show how imine chemistry operates in the transfer of covalent intermediates in pyridoxal 5'-phosphate biosynthesis by the Arabidopsis thaliana enzyme Pdx1. An initial ribose 5-phosphate lysine imine is converted to the chromophoric I320 intermediate, simultaneously bound to two lysine residues and partially vacating the active site, which creates space for glyceraldehyde 3-phosphate to bind. Crystal structures show how substrate binding, catalysis and shuttling are coupled to conformational changes around strand ß6 of the Pdx1 (ßα)8-barrel. The dual-specificity active site and imine relay mechanism for migration of carbonyl intermediates provide elegant solutions to the challenge of coordinating a complex sequence of reactions that follow a path of over 20 Å between substrate- and product-binding sites.
Assuntos
Lisina/metabolismo , Vitamina B 6/biossíntese , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/metabolismo , Carbono-Nitrogênio Liases , Lisina/química , Modelos Moleculares , Estrutura Molecular , Transferases de Grupos Nitrogenados/química , Transferases de Grupos Nitrogenados/metabolismo , Vitamina B 6/químicaRESUMO
A gene-level targeted enrichment method for direct detection of epigenetic modifications is described. The approach is demonstrated on the CGG-repeat region of the FMR1 gene, for which large repeat expansions, hitherto refractory to sequencing, are known to cause fragile X syndrome. In addition to achieving a single-locus enrichment of nearly 700,000-fold, the elimination of all amplification steps removes PCR-induced bias in the repeat count and preserves the native epigenetic modifications of the DNA. In conjunction with the single-molecule real-time sequencing approach, this enrichment method enables direct readout of the methylation status and the CGG repeat number of the FMR1 allele(s) for a clonally derived cell line. The current method avoids potential biases introduced through chemical modification and/or amplification methods for indirect detection of CpG methylation events.
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Epigênese Genética , Proteína do X Frágil da Deficiência Intelectual/genética , Análise de Sequência de DNA/métodos , Linhagem Celular , Metilação de DNA , Feminino , Síndrome do Cromossomo X Frágil/genética , Humanos , Sequências de Repetição em TandemRESUMO
MoaA, a radical S-adenosylmethionine enzyme, catalyzes the first step in molybdopterin biosynthesis. This reaction involves a complex rearrangement in which C8 of guanosine triphosphate is inserted between C2' and C3' of the ribose. This study identifies the site of initial hydrogen atom abstraction by the adenosyl radical and advances a mechanistic proposal for this unprecedented reaction.
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Coenzimas/metabolismo , Hidrolases/química , Hidrolases/metabolismo , Metaloproteínas/metabolismo , Pteridinas/metabolismo , Carbono , Catálise , Coenzimas/química , Guanosina Trifosfato/química , Guanosina Trifosfato/metabolismo , Espectroscopia de Ressonância Magnética , Metaloproteínas/química , Modelos Químicos , Cofatores de Molibdênio , Pteridinas/química , Ribose/química , Espectrometria de Massas por Ionização por ElectrosprayRESUMO
This communication describes the development of a thiamin sensor based on the bacterial thiamin binding protein. A triple mutant (C48S, C50S, S62C) of TbpA was labeled on C62 with N-[2-(L-maleimidyl)ethyl]-7-(diethylamino)coumarin-3-carboxamide (MDCC). Thiamin binding to this protein reduced the coumarin fluorescence giving a thiamin sensor with low nanomolar sensitivity.
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Técnicas Biossensoriais/métodos , Proteínas de Transporte/química , Periplasma/metabolismo , Tiamina/análise , Substituição de Aminoácidos , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Cumarínicos/química , Cinética , Mutação , Estrutura Terciária de ProteínaRESUMO
PURPOSE: We have previously shown that the expression of the thiamine transporter THTR2 is decreased sevenfold in breast cancer, which may leave breast cancer cells vulnerable to acute thiamine starvation. This concept was supported by the observation that MDA231 breast cancer xenografts demonstrated growth inhibition in mice fed a thiamine-free diet. METHODS: We purified recombinant Bacillus thiaminolyticus thiaminase I enzyme, which digests thiamine, to study acute thiamine starvation in breast cancer. RESULTS: Thiaminase I enzyme was cytotoxic in six breast cancer cell lines with IC(50)s ranging from 0.012 to 0.022 U/ml. The growth inhibitory effects of the combination of thiaminase I with either doxorubicin or paclitaxel were also examined. Over a wide range of drug concentrations, thiaminase 1 was consistently synergistic or additive with doxorubicin and paclitaxel in MCF-7, ZR75, HS578T and T47D cell lines, with most combinations having a calculated combination index (CI) of less than 0.8, indicating synergy. Although thiaminase I exposure did not stimulate the energy-sensing signaling kinases AKT, AMPK and GSK-3beta in MCF-7, ZR75, HS578T and T47D cell lines, thiaminase I exposure did stimulate expression of the ER stress response protein GRP78. In summary, thiaminase I is cytotoxic in breast cancer cell lines and triggers the unfolded protein response. CONCLUSION: These findings suggest that THTR2 down-regulation in breast tumors may present a nutritional vulnerability that could be exploited by thiaminase I enzyme therapy.
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Alquil e Aril Transferases/uso terapêutico , Protocolos de Quimioterapia Combinada Antineoplásica/uso terapêutico , Doxorrubicina/uso terapêutico , Paclitaxel/uso terapêutico , Proteínas Quinases Ativadas por AMP/metabolismo , Trifosfato de Adenosina/metabolismo , Alquil e Aril Transferases/administração & dosagem , Animais , Linhagem Celular Tumoral , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/administração & dosagem , Ensaios de Seleção de Medicamentos Antitumorais , Sinergismo Farmacológico , Chaperona BiP do Retículo Endoplasmático , Feminino , Quinase 3 da Glicogênio Sintase/metabolismo , Glicogênio Sintase Quinase 3 beta , Proteínas de Choque Térmico/metabolismo , Humanos , Proteínas de Membrana Transportadoras/metabolismo , Camundongos , Camundongos Nus , Paclitaxel/administração & dosagem , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/uso terapêutico , Deficiência de Tiamina/metabolismo , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Thiaminase induced thiamine deficiency occurs in fish, humans, livestock and wild animals. A non-radioactive thiaminase assay was described in 2007, but a direct comparison with the radioactive 14C-thiamine method which has been in use for more than 30 years has not been reported. The objective was to measure thiaminase activity in forage fish (alewife Alosa pseudoharengus, rainbow smelt Osmerus mordax, and slimy sculpin Cottus cognatus) consumed by predators that manifest thiamine deficiency using both methods. Modifications were made to the colorimetric assay to improve repeatability. Modification included a change in assay pH, enhanced sample clean-up, constant assay temperature (37 °C), increase in the concentration of 4-nitrothiophenol (4NTP) and use of a spectrophotometer fitted with a 0.2 cm cell. A strong relationship between the two assays was found for 51 alewife (R2=0.85), 36 smelt (R2=0.87) and 20 sculpin (R2=0.82). Thiaminase activity in the colorimetric assay was about 1000 times higher than activity measured by the radioactive method. Application of the assay to fish species from which no thiaminase activity has previously been reported resulted in no 4NTP thiaminase activity being found in bloater Coregonus hoyi, lake trout Salvelinus namaycusch, steelhead trout Oncorhynchus mykiss or Chinook salmon Oncorhynchus tshawytscha. In species previously reported to contain thiaminase, 4NTP thiaminase activity was measured in bacteria Paenibacillus thiaminolyticus, gizzard shad Dorosoma cepedianum, bracken fern Pteridium aquilinum, quagga mussel Dreissena bugensis and zebra mussels D. polymorpha.
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S-Adenosylmethionine decarboxylase (AdoMetDC) is a key enzyme in the polyamine biosynthetic pathway. Inhibition of this pathway and subsequent depletion of polyamine levels is a viable strategy for cancer chemotherapy and for the treatment of parasitic diseases. Substrate analogue inhibitors display an absolute requirement for a positive charge at the position equivalent to the sulfonium of S-adenosylmethionine. We investigated the ligand specificity of AdoMetDC through crystallography, quantum chemical calculations, and stopped-flow experiments. We determined crystal structures of the enzyme cocrystallized with 5'-deoxy-5'-dimethylthioadenosine and 5'-deoxy-5'-(N-dimethyl)amino-8-methyladenosine. The crystal structures revealed a favorable cation-pi interaction between the ligand and the aromatic side chains of Phe7 and Phe223. The estimated stabilization from this interaction is 4.5 kcal/mol as determined by quantum chemical calculations. Stopped-flow kinetic experiments showed that the rate of the substrate binding to the enzyme greatly depends on Phe7 and Phe223, thus supporting the importance of the cation-pi interaction.
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Adenosilmetionina Descarboxilase/metabolismo , Compostos de Sulfônio/metabolismo , Adenosilmetionina Descarboxilase/química , Cristalografia por Raios X , Humanos , Cinética , Ligantes , Conformação Proteica , Teoria Quântica , Especificidade por Substrato , TermodinâmicaRESUMO
Pre-steady state, stopped flow analysis of Escherichia coli D-3-phosphoglycerate dehydrogenase was performed by following the fluorescence of protein tryptophan and the fluorescence resonance energy transfer from protein tryptophan to bound NADH. The results indicate that binding of substrates is ordered, with coenzyme, NADH, binding first. Furthermore, the analysis indicated that there are two sets of sites on the tetrameric enzyme that can be differentiated by their kinetic behavior. NADH binding was consistent with an initial binding event followed by a slow conformational change for each site. The slow conformational change is responsible for the apparent tight binding of NADH to the apoenzyme but is too slow to participate in the catalytic cycle when the enzyme is rapidly turning over. Subsequent binding of the substrate, alpha-ketoglutarate, was characterized by a rapid equilibrium binding event followed by a conformational change for each site. Catalysis in the direction of NAD(+) reduction showed a distinct burst of activity followed by a slow rate of turnover, indicating that the rate-limiting step is after hydride transfer. Catalysis in the direction of NADH oxidation did not display burst kinetics, indicating that the rate-limiting step is at or before the hydride transfer step. The burst data indicated that the rate of NAD(+) reduction (3.8 s(-1)) is similar to the k(cat) of the enzyme (2-3 s(-1)) in that direction. However, analysis of the reaction with deuterated NADH failed to show an effect on the velocity of the reaction with a V(H)/V(D)=1.07+/-0.06. None of the other rates determined by stopped flow analysis could account for the k(cat) of the enzyme in either direction (forward k(cat)=0.01 s(-1), reverse k(cat)=2-3 s(-1)), suggesting that the rate-limiting step in both directions is a conformational change in the enzyme that is not detected optically.
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Escherichia coli/enzimologia , Fosfoglicerato Desidrogenase/química , Fosfoglicerato Desidrogenase/fisiologia , Catálise , Escherichia coli/metabolismo , Cinética , Modelos Biológicos , Modelos Químicos , Conformação Molecular , NAD/química , Oxigênio/química , Ligação Proteica , Estrutura Terciária de Proteína , Espectrofotometria/métodos , Especificidade por Substrato , Fatores de TempoRESUMO
The predominant biosynthetic route to vitamin B6 is catalyzed by a single enzyme. The synthase subunit of this enzyme, Pdx1, operates in concert with the glutaminase subunit, Pdx2, to catalyze the complex condensation of ribose 5-phosphate, glutamine and glyceraldehyde 3-phosphate to form pyridoxal 5'-phosphate, the active form of vitamin B6. In previous studies it became clear that many if not all of the reaction intermediates were covalently bound to the synthase subunit, thus making them difficult to isolate and characterize. Here we show that it is possible to follow a single turnover reaction by heteronuclear NMR using (13)C- and (15)N-labeled substrates as well as (15)N-labeled synthase. By denaturing the enzyme at points along the reaction coordinate, we solved the structures of three covalently bound intermediates. This analysis revealed a new 1,5 migration of the lysine amine linking the intermediate to the enzyme during the conversion of ribose 5-phosphate to pyridoxal 5'-phosphate.
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Bacillus subtilis/enzimologia , Glutaminase/metabolismo , Fosfato de Piridoxal/biossíntese , Ribosemonofosfatos/metabolismo , Vitamina B 6/biossíntese , Bacillus subtilis/metabolismo , Isótopos de Carbono , Catálise , Glutaminase/química , Glutaminase/isolamento & purificação , Cinética , Ressonância Magnética Nuclear Biomolecular , Fosfato de Piridoxal/química , Ribosemonofosfatos/química , Ribosemonofosfatos/isolamento & purificação , Especificidade por Substrato , Vitamina B 6/químicaRESUMO
This mini review covers recent advances in the mechanistic enzymology of cofactor biosynthesis.
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Bactérias/enzimologia , Coenzimas/biossíntese , Leveduras/enzimologia , Bactérias/metabolismo , Leveduras/metabolismoRESUMO
Two routes for the de novo biosynthesis of pyridoxal-5'-phosphate (PLP) have been discovered and reconstituted in vitro. The most common pathway that organisms use is dependent upon the activity of just two enzymes, known as Pdx1 (YaaD) and Pdx2 (YaaE) in bacteria. Pdx2 has been shown to have glutaminase activity and most likely channels ammonia to the active site of the PLP synthase subunit, Pdx1, where ribose-5-phosphate (R5P), glyceraldehyde-3-phosphate (G3P), and ammonia are condensed in a complex series of reactions. In this report we investigated the early steps in the mechanism of PLP formation. Under pre-steady-state conditions, a chromophoric intermediate (I320) is observed that accumulates upon addition of only two of the substrates, R5P and glutamine. The intermediate is covalently bound to the protein. We synthesized C5 monodeuterio (pro-R, pro-S) and dideuterio R5P and showed that there is a primary kinetic isotope effect on the formation of this intermediate using the pro-R but not the pro-S labeled isomer. Furthermore, it was shown that the phosphate unit of R5P is eliminated rather than hydrolyzed in route to intermediate formation and also that there is an observed C5-deuterium kinetic isotope effect on this elimination step. Interestingly, it was observed that the formation of the intermediate could be triggered in the absence of Pdx2 by the addition of high concentrations of NH4Cl to a preincubated solution of Pdx1 and R5P. The formation of I320 was also monitored using high-resolution electrospray ionization Fourier transform mass spectrometry and revealed a species of mass 34,304 Da (Pdx1 + 95 Da). These results allow us to narrow the mechanistic possibilities for the complex series of reactions involved in PLP formation.
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Glutaminase/metabolismo , Fosfato de Piridoxal/biossíntese , Escherichia coli/enzimologia , Glutaminase/química , Glutaminase/isolamento & purificação , Glutamina/metabolismo , Fosfato de Piridoxal/química , Ribosemonofosfatos/metabolismoRESUMO
ATP-binding cassette (ABC) transporters are responsible for the transport of a wide variety of water-soluble molecules and ions into prokaryotic cells. In Gram-negative bacteria, periplasmic-binding proteins deliver ions or molecules such as thiamin to the membrane-bound ABC transporter. The gene for the thiamin-binding protein tbpA has been identified in both Escherichia coli and Salmonella typhimurium. Here we report the crystal structure of TbpA from E. coli with bound thiamin monophosphate. The structure was determined at 2.25 A resolution using single-wavelength anomalous diffraction experiments, despite the presence of nonmerohedral twinning. The crystal structure shows that TbpA belongs to the group II periplasmic-binding protein family. Equilibrium binding measurements showed similar dissociation constants for thiamin, thiamin monophosphate, and thiamin pyrophosphate. Analysis of the binding site by molecular modeling demonstrated how TbpA binds all three forms of thiamin. A comparison of TbpA and thiaminase-I, a thiamin-degrading enzyme, revealed structural similarity between the two proteins, especially in domain 1, suggesting that the two proteins evolved from a common ancestor.
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Alquil e Aril Transferases/química , Alquil e Aril Transferases/genética , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/genética , Sítios de Ligação , Cristalografia por Raios X , Dimerização , Evolução Molecular , Cinética , Modelos Moleculares , Tiamina/química , Tiamina Pirofosfato/químicaRESUMO
The toxicity of nucleoside analogs used for the treatment of human immunodeficiency virus infection is due primarily to the inhibition of replication of the mitochondrial genome by the human mitochondrial DNA polymerase (Pol gamma). The severity of clinically observed toxicity correlates with the kinetics of incorporation versus excision of each analog as quantified by a toxicity index, spanning over six orders of magnitude. Here we show that the rate of excision of dideoxycytidine (zalcitabine; ddC) was reduced fourfold (giving a half-life of approximately 2.4 h) by the addition of a physiological concentration of deoxynucleoside triphosphates (dNTPs) due to the formation of a tight ternary enzyme-DNA-dNTP complex at the polymerase site. In addition, we provide a more accurate measurement of the rate of excision and show that the low rate of removal of ddCMP results from both the unfavorable transfer of the primer strand from the polymerase to the exonuclease site and the inefficient binding and/or hydrolysis at the exonuclease site. The analogs ddC, stavudine, and ddATP (a metabolite of didanosine) each bind more tightly at the polymerase site during incorporation than normal nucleotides, and this tight binding contributes to slower excision by the proofreading exonuclease, leading to increased toxicity toward mitochondrial DNA.
Assuntos
Fármacos Anti-HIV/metabolismo , DNA Polimerase Dirigida por DNA/metabolismo , Exonucleases/metabolismo , Mitocôndrias/enzimologia , Inibidores da Transcriptase Reversa/metabolismo , Zalcitabina/metabolismo , Desoxicitidina Monofosfato/metabolismo , Didesoxinucleotídeos/metabolismo , HumanosRESUMO
Native nucleotides show a hyperbolic concentration dependence of the pre-steady-state rate of incorporation while maintaining concentration-independent amplitude due to fast, largely irreversible pyrophosphate release. The kinetics of 3'-azido-2',3'-dideoxythymidine (AZT) incorporation exhibit an increase in amplitude and a decrease in rate as a function of nucleotide concentration, implying that pyrophosphate release must be slow so that nucleotide binding and incorporation are thermodynamically linked. Here we develop assays to measure pyrophosphate release and show that it is fast following incorporation of thymidine 5'-triphosphate (TTP). However, pyrophosphate release is slow (0.0009 s(-1)) after incorporation of AZT. Modeling of the complex kinetics resolves nucleotide binding (230 microM) and chemistry forward and reverse reactions, 0.38 and 0.22 s(-1), respectively. This unique mechanism increases selectivity against AZT incorporation by allowing reversal of the reaction and release of substrate, thereby reducing kcat/K(m) (7 x 10(-6) microM(-1) s(-1)). Other azido-nucleotides (AZG, AZC and AZA) and 8-oxo-7,8-dihydroguanosine-5'-triphosphate (8-oxo-dGTP) show this same phenomena.
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DNA Polimerase Dirigida por DNA/metabolismo , Mitocôndrias/enzimologia , Inibidores da Transcriptase Reversa/metabolismo , Zidovudina/metabolismo , DNA Polimerase gama , DNA Polimerase Dirigida por DNA/química , Difosfatos/metabolismo , Humanos , Cinética , Nucleotídeos de Timina/metabolismoRESUMO
An alternative method for measuring thiaminase I activity in complex samples is described. This assay is based on the selective consumption of the highly chromophoric 4-nitrothiophenolate by thiaminase I, resulting in a large decrease in absorbance at 411nm. This new assay is simple and sensitive, and it requires only readily available chemicals and a visible region spectrophotometer. In addition, the assay is optimized for high-throughput analysis in a 96-well format with complex biological samples.
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Alquil e Aril Transferases/análise , Alquil e Aril Transferases/metabolismo , Misturas Complexas/química , Hidrolases/análise , Hidrolases/metabolismo , Cinética , Sensibilidade e Especificidade , Espectrofotometria , Especificidade por Substrato , Compostos de Sulfidrila/metabolismoRESUMO
We have examined the kinetics of incorporation of acyclovir triphosphate by the herpes simplex virus-1 DNA polymerase holoenzyme (Pol-UL42) and the human mitochondrial DNA polymerase using transient kinetic methods. For each enzyme, we compared the kinetic parameters for acyclovir to those governing incorporation of dGTP. The favorable ground state dissociation constant (6 microM) and rate of polymerization (10 s(-1)) afford efficient incorporation of acyclovir triphosphate by the Pol-UL42 enzyme. A discrimination factor of approximately 50 favors dGTP over acyclovir triphosphate, mostly due to a faster maximum rate of dGTP incorporation. Once incorporated, acyclovir is removed with a half-life of approximately 1 h in the presence of a normal concentration of deoxynucleoside triphosphates, leading to a high toxicity index (16,000) toward viral replication. To assess the potential for toxicity toward the host we examined the incorporation and removal of acyclovir triphosphate by the human mitochondrial DNA polymerase. These results suggest moderate inhibition of mitochondrial DNA replication defining a toxicity index of 380. This value is much higher than the value of 1.5 determined for tenofovir, another acyclic nucleoside analog. The enzymatic therapeutic index is only 42 in favoring inhibition of the viral polymerase over polymerase gamma, whereas that for tenofovir is greater than 1,200. Mitochondrial toxicity is relatively low because acyclovir is activated only in infected cells by the promiscuous viral thymidine kinase and otherwise, mitochondrial toxicity would accumulate during long term treatment.