RESUMEN
Engineered microbes can be used for producing value-added chemicals from renewable feedstocks, relieving the dependency on nonrenewable resources such as petroleum. These microbes often are composed of synthetic metabolic pathways; however, one major problem in establishing a synthetic pathway is the challenge of precisely controlling competing metabolic routes, some of which could be crucial for fitness and survival. While traditional gene deletion and/or coarse overexpression approaches do not provide precise regulation, cis-repressors (CRs) are RNA-based regulatory elements that can control the production levels of a particular protein in a tunable manner. Here, we describe a protocol for a generally applicable fluorescence-activated cell sorting technique used to isolate eight subpopulations of CRs from a semidegenerate library in Escherichia coli, followed by deep sequencing that permitted the identification of 15 individual CRs with a broad range of protein production profiles. Using these new CRs, we demonstrated a change in production levels of a fluorescent reporter by over two orders of magnitude and further showed that these CRs are easily ported from E. coli to Pseudomonas putida. We next used four CRs to tune the production of the enzyme PpsA, involved in pyruvate to phosphoenolpyruvate (PEP) conversion, to alter the pool of PEP that feeds into the shikimate pathway. In an engineered P. putida strain, where carbon flux in the shikimate pathway is diverted to the synthesis of the commodity chemical cis,cis-muconate, we found that tuning PpsA translation levels increased the overall titer of muconate. Therefore, CRs provide an approach to precisely tune protein levels in metabolic pathways and will be an important tool for other metabolic engineering efforts.
Asunto(s)
Petróleo , Pseudomonas putida , Escherichia coli/genética , Escherichia coli/metabolismo , Fosfoenolpiruvato/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Ingeniería Metabólica , Ácido Pirúvico/metabolismo , Genómica , ARN/metabolismo , Petróleo/metabolismoRESUMEN
A structure-function characterization of Synechococcus elongatus enolase (SeEN) is presented, representing the first structural report on a cyanobacterial enolase. X-ray crystal structures of SeEN in its apoenzyme form and in complex with phosphoenolpyruvate are reported at 2.05 and 2.30â Å resolution, respectively. SeEN displays the typical fold of enolases, with a conformationally flexible loop that closes the active site upon substrate binding, assisted by two metal ions that stabilize the negatively charged groups. The enzyme exhibits a catalytic efficiency of 1.2 × 105â M-1â s-1 for the dehydration of 2-phospho-D-glycerate, which is comparable to the kinetic parameters of related enzymes. These results expand the understanding of the biophysical features of these enzymes, broadening the toolbox for metabolic engineering applications.
Asunto(s)
Fosfopiruvato Hidratasa , Synechococcus , Cristalografía por Rayos X , Fosfoenolpiruvato/química , Fosfopiruvato Hidratasa/químicaRESUMEN
Three high-resolution X-ray crystal structures of malate dehydrogenase (MDH; EC 1.1.1.37) from the methylotroph Methylobacterium extorquens AM1 are presented. By comparing the structures of apo MDH, a binary complex of MDH and NAD+, and a ternary complex of MDH and oxaloacetate with ADP-ribose occupying the pyridine nucleotide-binding site, conformational changes associated with the formation of the catalytic complex were characterized. While the substrate-binding site is accessible in the enzyme resting state or NAD+-bound forms, the substrate-bound form exhibits a closed conformation. This conformational change involves the transition of an α-helix to a 310-helix, which causes the adjacent loop to close the active site following coenzyme and substrate binding. In the ternary complex, His284 forms a hydrogen bond to the C2 carbonyl of oxaloacetate, placing it in a position to donate a proton in the formation of (2S)-malate.
Asunto(s)
Adenosina Difosfato Ribosa/química , Proteínas Bacterianas/química , Malato Deshidrogenasa/química , Malatos/química , Methylobacterium extorquens/química , NAD/química , Ácido Oxaloacético/química , Adenosina Difosfato Ribosa/metabolismo , Secuencia de Aminoácidos , Apoenzimas/química , Apoenzimas/genética , Apoenzimas/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Clonación Molecular , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Vectores Genéticos/química , Vectores Genéticos/metabolismo , Enlace de Hidrógeno , Cinética , Malato Deshidrogenasa/genética , Malato Deshidrogenasa/metabolismo , Malatos/metabolismo , Methylobacterium extorquens/enzimología , Modelos Moleculares , NAD/metabolismo , Ácido Oxaloacético/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Dominios y Motivos de Interacción de Proteínas , Multimerización de Proteína , Protones , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Especificidad por SustratoRESUMEN
Mycolactone is the exotoxin produced by Mycobacterium ulcerans and is the virulence factor behind the neglected tropical disease Buruli ulcer. The toxin has a broad spectrum of biological effects within the host organism, stemming from its interaction with at least two molecular targets and the inhibition of protein uptake into the endoplasmic reticulum. Although it has been shown that the toxin can passively permeate into host cells, it is clearly lipophilic. Association with lipid carriers would have substantial implications for the toxin's distribution within a host organism, delivery to cellular targets, diagnostic susceptibility, and mechanisms of pathogenicity. Yet the toxin's interactions with, and distribution in, lipids are unknown. Herein we have used coarse-grained molecular dynamics simulations, guided by all-atom simulations, to study the interaction of mycolactone with pure and mixed lipid membranes. Using established techniques, we calculated the toxin's preferential localization, membrane translocation, and impact on membrane physical and dynamical properties. The computed water-octanol partition coefficient indicates that mycolactone prefers to be in an organic phase rather than in an aqueous environment. Our results show that in a solvated membrane environment the exotoxin mainly localizes in the water-membrane interface, with a preference for the glycerol moiety of lipids, consistent with the reported studies that found it in lipid extracts of the cell. The calculated association constant to the model membrane is similar to the reported association constant for Wiskott-Aldrich syndrome protein. Mycolactone is shown to modify the physical properties of membranes, lowering the transition temperature, compressibility modulus, and critical line tension at which pores can be stabilized. It also shows a tendency to behave as a linactant, a molecule that localizes at the boundary between different fluid lipid domains in membranes and promotes inter-mixing of domains. This property has implications for the toxin's cellular access, T-cell immunosuppression, and therapeutic potential.
Asunto(s)
Toxinas Bacterianas/química , Úlcera de Buruli/microbiología , Macrólidos/química , Mycobacterium ulcerans/química , Animales , Transporte Biológico , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Exotoxinas/química , Glicerol/química , Humanos , Membrana Dobles de Lípidos , Lípidos/química , Espectroscopía de Resonancia Magnética , Simulación de Dinámica Molecular , Octanoles/química , Transporte de Proteínas , Programas Informáticos , Estrés Mecánico , Temperatura , Virulencia , Factores de Virulencia/metabolismo , Agua/químicaRESUMEN
A 1.1 Å resolution, room-temperature X-ray structure and a 2.1 Å resolution neutron structure of a chitin-degrading lytic polysaccharide monooxygenase domain from the bacterium Jonesia denitrificans (JdLPMO10A) show a putative dioxygen species equatorially bound to the active site copper. Both structures show an elongated density for the dioxygen, most consistent with a Cu(II)-bound peroxide. The coordination environment is consistent with Cu(II). In the neutron and X-ray structures, difference maps reveal the N-terminal amino group, involved in copper coordination, is present as a mixed ND2 and ND-, suggesting a role for the copper ion in shifting the pKa of the amino terminus.
Asunto(s)
Cobre/química , Oxigenasas de Función Mixta/química , Oxígeno/química , Polisacáridos/química , Dominio Catalítico , Cristalografía por Rayos X , Conformación Proteica , ProtonesRESUMEN
The Protein Crystallography Station (PCS), located at the Los Alamos Neutron Scattering Center (LANSCE), was the first macromolecular crystallography beamline to be built at a spallation neutron source. Following testing and commissioning, the PCS user program was funded by the Biology and Environmental Research program of the Department of Energy Office of Science (DOE-OBER) for 13 years (2002-2014). The PCS remained the only dedicated macromolecular neutron crystallography station in North America until the construction and commissioning of the MaNDi and IMAGINE instruments at Oak Ridge National Laboratory, which started in 2012. The instrument produced a number of research and technical outcomes that have contributed to the field, clearly demonstrating the power of neutron crystallo-graphy in helping scientists to understand enzyme reaction mechanisms, hydrogen bonding and visualization of H-atom positions, which are critical to nearly all chemical reactions. During this period, neutron crystallography became a technique that increasingly gained traction, and became more integrated into macromolecular crystallography through software developments led by investigators at the PCS. This review highlights the contributions of the PCS to macromolecular neutron crystallography, and gives an overview of the history of neutron crystallography and the development of macromolecular neutron crystallography from the 1960s to the 1990s and onwards through the 2000s.
RESUMEN
Malyl-CoA lyase (MCL) is an Mg2+-dependent enzyme that catalyzes the reversible cleavage of (2S)-4-malyl-CoA to yield acetyl-CoA and glyoxylate. MCL enzymes, which are found in a variety of bacteria, are members of the citrate lyase-like family and are involved in the assimilation of one- and two-carbon compounds. Here, the 1.56â Å resolution X-ray crystal structure of MCL from Methylobacterium extorquens AM1 with bound Mg2+ is presented. Structural alignment with the closely related Rhodobacter sphaeroides malyl-CoA lyase complexed with Mg2+, oxalate and CoA allows a detailed analysis of the domain motion of the enzyme caused by substrate binding. Alignment of the structures shows that a simple hinge motion centered on the conserved residues Phe268 and Thr269 moves the C-terminal domain by about 30° relative to the rest of the molecule. This domain motion positions a conserved aspartate residue located in the C-terminal domain in the active site of the adjacent monomer, which may serve as a general acid/base in the catalytic mechanism.
Asunto(s)
Acilcoenzima A/química , Proteínas Bacterianas/química , Magnesio/química , Methylobacterium extorquens/química , Oxo-Ácido-Liasas/química , Acilcoenzima A/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Dominio Catalítico , Cationes Bivalentes , Clonación Molecular , Coenzima A/química , Coenzima A/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Expresión Génica , Magnesio/metabolismo , Methylobacterium extorquens/enzimología , Modelos Moleculares , Ácido Oxálico/química , Ácido Oxálico/metabolismo , Oxo-Ácido-Liasas/genética , Oxo-Ácido-Liasas/metabolismo , Plásmidos/química , Plásmidos/metabolismo , Unión Proteica , Conformación Proteica en Hélice alfa , Conformación Proteica en Lámina beta , Dominios y Motivos de Interacción de Proteínas , Estructura Cuaternaria de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Rhodobacter sphaeroides/química , Rhodobacter sphaeroides/enzimología , Especificidad por SustratoRESUMEN
Bacteria and fungi express lytic polysaccharide monooxgyenase (LPMO) enzymes that act in conjunction with canonical hydrolytic sugar-processing enzymes to rapidly convert polysaccharides such as chitin, cellulose and starch to single monosaccharide products. In order to gain a better understanding of the structure and oxidative mechanism of these enzymes, large crystals (1-3 mm(3)) of a chitin-processing LPMO from the Gram-positive soil bacterium Jonesia denitrificans were grown and screened for their ability to diffract neutrons. In addition to the collection of neutron diffraction data, which were processed to 2.1 Å resolution, a high-resolution room-temperature X-ray diffraction data set was collected and processed to 1.1 Å resolution in space group P212121. To our knowledge, this work marks the first successful neutron crystallographic experiment on an LPMO. Joint X-ray/neutron refinement of the resulting data will reveal new details of the structure and mechanism of this recently discovered class of enzymes.
Asunto(s)
Oxigenasas de Función Mixta/química , Difracción de Neutrones/métodos , Polisacáridos Bacterianos/química , Cristalización , Cristalografía por Rayos X , Bacterias Grampositivas/enzimología , Oxigenasas de Función Mixta/aislamiento & purificación , Polisacáridos Bacterianos/aislamiento & purificación , TemperaturaRESUMEN
The most abundant carbohydrate product of cellulosic biomass pyrolysis is the anhydrosugar levoglucosan (1,6-anhydro-ß-d-glucopyranose), which can be converted to glucose 6-phosphate by levoglucosan kinase (LGK). In addition to the canonical kinase phosphotransfer reaction, the conversion requires cleavage of the 1,6-anhydro ring to allow ATP-dependent phosphorylation of the sugar O6 atom. Using x-ray crystallography, we show that LGK binds two magnesium ions in the active site that are additionally coordinated with the nucleotide and water molecules to result in ideal octahedral coordination. To further verify the metal binding sites, we co-crystallized LGK in the presence of manganese instead of magnesium and solved the structure de novo using the anomalous signal from four manganese atoms in the dimeric structure. The first metal is required for catalysis, whereas our work suggests that the second is either required or significantly promotes the catalytic rate. Although the enzyme binds its sugar substrate in a similar orientation to the structurally related 1,6-anhydro-N-acetylmuramic acid kinase (AnmK), it forms markedly fewer bonding interactions with the substrate. In this orientation, the sugar is in an optimal position to couple phosphorylation with ring cleavage. We also observed a second alternate binding orientation for levoglucosan, and in these structures, ADP was found to bind with lower affinity. These combined observations provide an explanation for the high Km of LGK for levoglucosan. Greater knowledge of the factors that contribute to the catalytic efficiency of LGK can be used to improve applications of this enzyme for levoglucosan-derived biofuel production.
Asunto(s)
Celulosa/química , Proteínas Fúngicas/química , Glucosa-6-Fosfato/química , Glucosa/análogos & derivados , Lipomyces/química , Fosfotransferasas/química , Biocatálisis , Biomasa , Celulosa/metabolismo , Cristalografía por Rayos X , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Expresión Génica , Glucosa/química , Glucosa/metabolismo , Glucosa-6-Fosfato/metabolismo , Cinética , Lipomyces/enzimología , Magnesio/química , Magnesio/metabolismo , Manganeso/química , Manganeso/metabolismo , Modelos Moleculares , Fosforilación , Fosfotransferasas/genética , Fosfotransferasas/metabolismo , Unión Proteica , Conformación Proteica , Multimerización de Proteína , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMEN
Until recently, engineering strategies for altering gene expression have focused on transcription control using strong inducible promoters or one of several methods to knock down wasteful genes. Recently, synthetic riboregulators have been developed for translational regulation of gene expression. Here, we report a new modular synthetic riboregulator class that has the potential to finely tune protein expression and independently control the concentration of each enzyme in an engineered metabolic pathway. This development is important because the most straightforward approach to altering the flux through a particular metabolic step is to increase or decrease the concentration of the enzyme. Our design includes a cis-repressor at the 5' end of the mRNA that forms a stem-loop helix, occluding the ribosomal binding sequence and blocking translation. A trans-expressed activating-RNA frees the ribosomal-binding sequence, which turns on translation. The overall architecture of the riboregulators is designed using Watson-Crick base-pairing stability. We describe here a cis-repressor that can completely shut off translation of antibiotic-resistance reporters and a trans-activator that restores translation. We have established that it is possible to use these riboregulators to achieve translational control of gene expression over a wide dynamic range. We have also found that a targeting sequence can be modified to develop riboregulators that can, in principle, independently regulate translation of many genes. In a selection experiment, we demonstrated that by subtly altering the sequence of the trans-activator it is possible to alter the ratio of the repressed and activated states and to achieve intermediate translational control.
Asunto(s)
Escherichia coli , Regulación Bacteriana de la Expresión Génica/genética , Conformación de Ácido Nucleico , Biosíntesis de Proteínas/genética , ARN Bacteriano , Riboswitch/genética , Escherichia coli/genética , Escherichia coli/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
Human carbonic anhydrase II (HCA II) uses a Zn-bound OH(-)/H2O mechanism to catalyze the reversible hydration of CO2. This catalysis also involves a separate proton transfer step, mediated by an ordered solvent network coordinated by hydrophilic residues. One of these residues, Tyr7, was previously shown to be deprotonated in the neutron crystal structure at pH 10. This observation indicated that Tyr7 has a perturbed pKa compared with free tyrosine. To further probe the pKa of this residue, NMR spectroscopic measurements of [(13)C]Tyr-labeled holo HCA II (with active-site Zn present) were preformed to titrate all Tyr residues between pH 5.4-11.0. In addition, neutron studies of apo HCA II (with Zn removed from the active site) at pH 7.5 and holo HCA II at pH 6 were conducted. This detailed interrogation of tyrosines in HCA II by NMR and neutron crystallography revealed a significantly lowered pKa of Tyr7 and how pH and Tyr proximity to Zn affect hydrogen-bonding interactions.
Asunto(s)
Anhidrasas Carbónicas/química , Cristalografía por Rayos X/métodos , Espectroscopía de Resonancia Magnética/métodos , Neutrones , Tirosina/química , Catálisis , Dominio Catalítico , Cationes , Enzimas/química , Humanos , Enlace de Hidrógeno , Concentración de Iones de Hidrógeno , Conformación Proteica , Protones , Electricidad Estática , Agua/químicaRESUMEN
BACKGROUND: The clustering of genes in a pathway and the co-location of functionally related genes is widely recognized in prokaryotes. We used these characteristics to predict the metabolic involvement for a Transcriptional Regulator (TR) of unknown function, identified and confirmed its biological activity. RESULTS: A software tool that identifies the genes encoded within a defined genomic neighborhood for the subject TR and its homologs was developed. The output lists of genes in the genetic neighborhoods, their annotated functions, the reactants/products, and identifies the metabolic pathway in which the encoded-proteins function. When a set of TRs of known function was analyzed, we observed that their homologs frequently had conserved genomic neighborhoods that co-located the metabolically related genes regulated by the subject TR. We postulate that TR effectors are metabolites in the identified pathways; indeed the known effectors were present. We analyzed Bxe_B3018 from Burkholderia xenovorans, a TR of unknown function and predicted that this TR was related to the glycine, threonine and serine degradation. We tested the binding of metabolites in these pathways and for those that bound, their ability to modulate TR binding to its specific DNA operator sequence. Using rtPCR, we confirmed that methylglyoxal was an effector of Bxe_3018. CONCLUSION: These studies provide the proof of concept and validation of a systematic approach to the discovery of the biological activity for proteins of unknown function, in this case a TR. Bxe_B3018 is a methylglyoxal responsive TR that controls the expression of an operon composed of a putative efflux system.
Asunto(s)
Regulación de la Expresión Génica , Genoma , Genómica , Células Procariotas/metabolismo , Transcripción Genética , Biología Computacional/métodos , Orden Génico , Sitios Genéticos , Genómica/métodos , Metabolómica , Unión Proteica , Reproducibilidad de los Resultados , Programas Informáticos , Factores de Transcripción , Interfaz Usuario-ComputadorRESUMEN
The 1,3-dithiane is a protected formaldehyde anion equivalent that could serve as a useful labeled synthon. We report a facile synthesis of 1,3-[2-(13)C]- and 1,3-[2-(13)C, 2-(2)H2]dithiane in two steps from [(13)C]- or [(13) C, (2)H3 ]methyl phenyl sulfoxide. We have previously reported the high yield synthesis of [(13)C]methyl phenyl sulfide from [(13)C]MEOH and the oxidation of [(13)C]methyl phenyl sulfide to [(13)C]methyl phenyl sulfoxide. Here, we describe the facile exchange of deuterium from (2) H2 O into [(13)C]methyl phenyl sulfoxide to yield [(13)C, (2)H3]methyl phenyl sulfoxide. Thus, from [(13)C]MEOH and (2)H2O, all possible C2 stable isotopomers of 1,3-dithiane are available. Our synthetic route is also amenable to preparation of radiolabeled 1,3-dithianes.
Asunto(s)
Isótopos de Carbono/síntesis química , Isótopos de Carbono/aislamiento & purificación , Quinolizinas/síntesis química , Quinolizinas/aislamiento & purificación , Compuestos de Azufre/síntesis química , Compuestos de Azufre/aislamiento & purificación , Marcaje Isotópico/métodos , Radiofármacos/síntesis química , Radiofármacos/aislamiento & purificaciónRESUMEN
We have developed large-scale efficient procedures for the conversion of commercially available [(13) C]- or [(2) H3 ,(13) C]methanol and (13) CO2 or (13) C-labeled bromoacetic acid to 2-(phenylthio)[1,2-(13) C2 ]-, [1-(13) C]-, and [2-(13) C]acetic acid. The resulting derivatives are versatile, chemically stable, and nonvolatile two-carbon labeling precursors. We have used the (13) C-isotopomers of 2-(phenylthio)acetic acid in the synthesis of (13) C-labeled acrylic acid, methacrylic acid, and trans-crotonic acid.
Asunto(s)
Glicolatos/síntesis química , Sulfonas/síntesis química , Sulfóxidos/síntesis química , Isótopos de Carbono/síntesis química , Marcaje Isotópico/métodosRESUMEN
The enzyme 2-keto-3-deoxy-9-O-phosphonononic acid phosphatase (KDN9P phosphatase) functions in the pathway for the production of 2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, a sialic acid that is important for the survival of commensal bacteria in the human intestine. The enzyme is a member of the haloalkanoate dehalogenase superfamily and represents a good model for the active-site protonation state of family members. Crystals of approximate dimensions 1.5 × 1.0 × 1.0â mm were obtained in space group P2(1)2(1)2, with unit-cell parameters a = 83.1, b = 108.9, c = 75.7â Å. A complete neutron data set was collected from a medium-sized H/D-exchanged crystal at BIODIFF at the Heinz Maier-Leibnitz Zentrum (MLZ), Garching, Germany in 18â d. Initial refinement to 2.3â Å resolution using only neutron data showed significant density for catalytically important residues.
Asunto(s)
Proteínas Bacterianas/química , Magnesio/química , Monoéster Fosfórico Hidrolasas/química , Protones , Ácidos Siálicos/química , Proteínas Bacterianas/genética , Sitios de Unión , Dominio Catalítico , Cationes Bivalentes , Cristalografía , Medición de Intercambio de Deuterio , Escherichia coli/genética , Expresión Génica , Ligandos , Modelos Moleculares , Difracción de Neutrones , Monoéster Fosfórico Hidrolasas/genética , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Dispersión del Ángulo Pequeño , Especificidad por SustratoRESUMEN
We have developed a high-throughput approach using frontal affinity chromatography coupled to mass spectrometry (FAC-MS) for the identification and characterization of the small molecules that modulate transcriptional regulator (TR) binding to TR targets. We tested this approach using the methionine biosynthesis regulator (MetJ). We used effector mixtures containing S-adenosyl-L-methionine (SAM) and S-adenosyl derivatives as potential ligands for MetJ binding. The differences in the elution time of different compounds allowed us to rank the binding affinity of each compound. Consistent with previous results, FAC-MS showed that SAM binds to MetJ with the highest affinity. In addition, adenine and 5'-deoxy-5'-(methylthio)adenosine bind to the effector binding site on MetJ. Our experiments with MetJ demonstrate that FAC-MS is capable of screening complex mixtures of molecules and identifying high-affinity binders to TRs. In addition, FAC-MS experiments can be used to discriminate between specific and nonspecific binding of the effectors as well as to estimate the dissociation constant (K(d)) for effector-TR binding.
Asunto(s)
Proteínas Bacterianas/metabolismo , Ensayos Analíticos de Alto Rendimiento/métodos , Metionina/biosíntesis , Proteínas Represoras/metabolismo , Adenina/metabolismo , Proteínas Bacterianas/genética , Sitios de Unión , Cromatografía de Afinidad/métodos , Clonación Molecular , ADN Bacteriano/genética , Desoxiadenosinas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos , Vectores Genéticos/genética , Ligandos , Metionina/genética , Unión Proteica , Proteínas Represoras/genética , S-Adenosilmetionina/metabolismo , Espectrometría de Masa por Ionización de Electrospray/métodos , Tionucleósidos/metabolismo , Factores de Tiempo , Transcripción GenéticaRESUMEN
This perspective reviews the potential for stable isotope labelling to examine the metabolic transformations of drugs. The increased sensitivity and widespread availability of modern nuclear magnetic resonance (NMR) and high-resolution mass spectrometers will increase the application of stable isotopes to study drug metabolism. Creating mass doublets by mixing a natural isotopic abundance compound with a labelled isotopomer and applying stable isotope filtering to high resolution mass spectrometry allows one to rapidly identify drug metabolites in very complex samples, such as blood or urine. Applying this approach to drug metabolism will require a significant synthesis effort. The relatively small number of (13) C, (15) N, or (17,18) O-labelled precursors exacerbates this problem, making the synthesis of the labelled drug often more difficult than that of the parent compound. We have developed new strategies for stable isotope labelling of complex molecules based on the rich chemistry of [(13) C]methyl phenyl sulfide, where the phenylthio group acts as a stable, non-volatile carrier for the valuable (13) C-label. For example we have used [(13) C]methyl phenyl sulfide to prepare the three possible (13) C-isotopomers ([1-(13) C]-, [2-(13) C]-, [1,2-(13) C(2) ]) of the two carbon precursors, ethyl 2-(phenylthio) acetate and ethyl N,N-dimethyl oxamate. In each case, these two-carbon labelling precursors are asymmetric and the differential reactivity of the carbons allows for either/or (13) C-labelling in the products. We demonstrate the utility of these two carbon precursors in the synthesis of aromatic ring-labelled N-(4-hydroxyphenyl)acetamide (acetaminophen or paracetamol).
Asunto(s)
Marcaje Isotópico/métodos , Espectroscopía de Resonancia Magnética/métodos , Espectrometría de Masas/métodos , Preparaciones Farmacéuticas/metabolismo , Humanos , Preparaciones Farmacéuticas/análisisRESUMEN
MOTIVATION: Our knowledge of the metabolites in cells and their reactions is far from complete as revealed by metabolomic measurements that detect many more small molecules than are documented in metabolic databases. Here, we develop an approach for predicting the reactivity of small-molecule metabolites in enzyme-catalyzed reactions that combines expert knowledge, computational chemistry and machine learning. RESULTS: We classified 4843 reactions documented in the KEGG database, from all six Enzyme Commission classes (EC 1-6), into 80 reaction classes, each of which is marked by a characteristic functional group transformation. Reaction centers and surrounding local structures in substrates and products of these reactions were represented using SMARTS. We found that each of the SMARTS-defined chemical substructures is widely distributed among metabolites, but only a fraction of the functional groups in these substructures are reactive. Using atomic properties of atoms in a putative reaction center and molecular properties as features, we trained support vector machine (SVM) classifiers to discriminate between functional groups that are reactive and non-reactive. Classifier accuracy was assessed by cross-validation analysis. A typical sensitivity [TP/(TP+FN)] or specificity [TN/(TN+FP)] is ≈0.8. Our results suggest that metabolic reactivity of small-molecule compounds can be predicted with reasonable accuracy based on the presence of a potentially reactive functional group and the chemical features of its local environment. AVAILABILITY: The classifiers presented here can be used to predict reactions via a web site (http://cellsignaling.lanl.gov/Reactivity/). The web site is freely available.
Asunto(s)
Inteligencia Artificial , Metaboloma , Metabolómica/métodos , Biocatálisis , Clasificación/métodos , Biología Computacional/métodos , Bases de Datos Factuales , Enzimas/clasificación , Redes y Vías Metabólicas , Estructura MolecularRESUMEN
The biosynthesis of the 3,4-dihydroxybenzoate moieties of the siderophore petrobactin, produced by B. anthracis str. Sterne, was probed by isotopic feeding experiments in iron-deficient media with a mixture of unlabeled and D-[(13)C6]glucose at a ratio of 5:1 (w/w). After isolation of the labeled siderophore, analysis of the isotopomers was conducted via one-dimensional (1)H and (13)C NMR spectroscopy, as well as (13)C-(13)C DQFCOSY spectroscopy. Isotopic enrichment and (13)C-(13)C coupling constants in the aromatic ring of the isolated siderophore suggested the predominant route for the construction of the carbon backbone of 3,4-DHB (1) involved phosphoenol pyruvate and erythrose-4-phosphate as ultimate precursors. This observation is consistent with that expected if the shikimate pathway is involved in the biosynthesis of these moieties. Enrichment attributable to phosphoenol pyruvate precursors was observed at C1 and C6 of the aromatic ring, as well as into the carboxylate group, while scrambling of the label into C2 was not. This pattern suggests 1 was biosynthesized from early intermediates of the shikimate pathway and not through later shikimate intermediates or aromatic amino acid precursors.
Asunto(s)
Bacillus anthracis/química , Bacillus anthracis/metabolismo , Benzamidas/química , Benzamidas/metabolismo , Hidroxibenzoatos/química , Hidroxibenzoatos/metabolismo , Espectroscopía de Resonancia Magnética , Estructura Molecular , Ácido Shikímico/químicaRESUMEN
MOTIVATION: Stable isotope labeling of small-molecule metabolites (e.g. (13)C-labeling of glucose) is a powerful tool for characterizing pathways and reaction fluxes in a metabolic network. Analysis of isotope labeling patterns requires knowledge of the fates of individual atoms and moieties in reactions, which can be difficult to collect in a useful form when considering a large number of enzymatic reactions. RESULTS: We report carbon-fate maps for 4605 enzyme-catalyzed reactions documented in the KEGG database. Every fate map has been manually checked for consistency with known reaction mechanisms. A map includes a standardized structure-based identifier for each reactant (namely, an InChI string); indices for carbon atoms that are uniquely derived from the metabolite identifiers; structural data, including an identification of homotopic and prochiral carbon atoms; and a bijective map relating the corresponding carbon atoms in substrates and products. Fate maps are defined using the BioNetGen language (BNGL), a formal model-specification language, which allows a set of maps to be automatically translated into isotopomer mass-balance equations. AVAILABILITY: The carbon-fate maps and software for visualizing the maps are freely available (http://cellsignaling.lanl.gov/FateMaps/).