RESUMO
The experimental verification of a proposed biosynthetic pathway for a given natural product is often difficult to obtain with the use of the whole organism (permeability factors) or, in the case of higher plants, a cell-free system. Until the purified enzyme for each step of biosynthesis is available, biosynthetic studies can, however, be carried out, albeit with modest incorporation values, by means of either hydroponic or injection methodology. Where viable seed sources are available it is suggested that improvements of several orders of magnitude in incorporation can be achieved by a short-term incubation (small pool size of precursors; trapping of reactive intermediates) or a long-term feeding (equilibration of precursor with the compartmentalized or induced synthetases). In bacterial, fungal, mammalian, and plant systems, where incorporation efficiencies provide the opportunity to study (13)C enrichment (at least equal to natural abundance of the isotope), we can expect a rapid expansion since the method removes the tedium of carbon-by-carbon degradation. For the next few years, however, the prognosis would seem to favor parallel studies of (13)C and (2)H, and of (14)C/(3)H ratio techniques since the last-mentioned method provides more information concerning the stereoselectivity of labeling processes on the microgram scale.
Assuntos
Alcaloides/biossíntese , Marcação por Isótopo , Plantas/metabolismo , Isótopos de Carbono , Radioisótopos de Carbono , Sistema Livre de Células , Análise de Fourier , Ácidos Levulínicos/metabolismo , Espectroscopia de Ressonância Magnética , Porfobilinogênio/metabolismo , Propionibacterium/metabolismo , Estricnina/biossíntese , Triptofano/metabolismo , Alcaloides de Vinca/biossíntese , Vitamina B 12/biossínteseRESUMO
High-resolution carbon-13 nuclear magnetic resonance (NMR) spectra of enzyme-inhibitor and enzyme-substrate complexes provide detailed structural and stereochemical information on the mechanism of enzyme action. The proteases trypsin and papain are shown to form tetrahedrally coordinated complexes and acyl derivatives with a variety of compounds artificially enriched at the site or sites of interest. These results are compared with the structural information derived from x-ray diffraction. Detailed NMR studies have provided a clearer picture of the ionization state of the residues participating in enzyme-catalyzed processes than other more classical techniques. The dynamics of enzymic catalysis can be observed at sub-zero temperatures by a combination of cryoenzymology and carbon-13 NMR spectroscopy. With these powerful techniques, transient, covalently bound intermediates in enzyme-catalyzed reactions can be detected and their structures rigorously assigned.
Assuntos
Coenzimas/metabolismo , Enzimas/metabolismo , Peptídeo Hidrolases/metabolismo , Sítios de Ligação , Isótopos de Carbono , Carboxipeptidases/metabolismo , Carboxipeptidases A , Catálise , Fenômenos Químicos , Química , Endopeptidases/metabolismo , Congelamento , Frutose-Bifosfato Aldolase/metabolismo , Espectroscopia de Ressonância Magnética , Papaína/metabolismo , Pepsina A/metabolismo , Inibidores de Proteases , Pterinas/metabolismo , Fosfato de Piridoxal/metabolismo , Serina EndopeptidasesRESUMO
Data obtained using a combination of molecular biology and NMR spectroscopy has transformed our thinking about the evolution of the biochemical machinery required for the synthesis of the vital metallopigments: haem, chlorophyll, vitamin B12 and factor F430. One of the most recent advances is the discovery of a unique dipyrromethane cofactor that is bound covalently at the active site of porphobillinogen deaminase, the key enzyme of tetrapyrrole assembly. We will also discuss how the oxidation level and chromophoric arrangement of the uroporphinoid ring, rather than its substitution pattern, provides the necessary molecular recognition for some of the later enzymes, whose function is to decorate the template by C-methylation on the way to the biologically active cofactors.
Assuntos
Pirróis/química , Uroporfirinogênios/química , Sítios de Ligação , Hidroximetilbilano Sintase/química , Ligantes , Metilação , Porfobilinogênio/química , Tetrapirróis , Uroporfirinogênios/metabolismoRESUMO
BACKGROUND: The crystal structure of precorrin-8x methyl mutase (CobH), an enzyme of the aerobic pathway to vitamin B12, provides evidence that the mechanism for methyl migration can plausibly be regarded as an allowed [1,5]-sigmatropic shift of a methyl group from C-11 to C-12 at the C ring of precorrin-8x to afford hydrogenobyrinic acid. RESULTS: The dimeric structure of CobH creates a set of shared active sites that readily discriminate between different tautomers of precorrin-8x and select a discrete tautomer for sigmatropic rearrangement. The active site contains a strictly conserved histidine residue close to the site of methyl migration in ring C of the substrate. CONCLUSION: Analysis of the structure with bound product suggests that the [1,5]-sigmatropic shift proceeds by protonation of the ring C nitrogen, leading to subsequent methyl migration.
Assuntos
Proteínas de Bactérias , Transferases Intramoleculares/química , Transferases Intramoleculares/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Cristalografia por Raios X , Modelos Moleculares , Dados de Sequência Molecular , Conformação Proteica , Homologia de Sequência de Aminoácidos , Especificidade por Substrato , Uroporfirinas/química , Uroporfirinas/metabolismoRESUMO
Tritiated vinblastine was prepared by catalytic exchange and its metabolism was studied in dogs. Plasma levels of drug fell in biphasic mode with initial and secondary phase half-lives of 17 to 38 min and 3 to 5 hr, respectively. Between 28.6 and 79.1% of plasma tritium was precipitable with cold trichloroacetic acid and thus was presumably protein bound. Blood leukocytes had levels of intracellular tritium between 2.4 and 11.8 times those of the coincident plasma samples. Over a 9-day period, urinary excretion accounted for 12.1 to 16.8% and fecal excretion accounted for 30.1 to 36.1% of the administered radioactivity. Ratios of biliary to plasma radioactivity varied between 7.3 and 56.9, with unchanged vinblastine being the mamor component (46.8 to 80.7%) in the bile.
Assuntos
Vimblastina/metabolismo , Animais , Bile/análise , Cães , Fezes/análise , Meia-Vida , Leucócitos/metabolismo , Ligação Proteica , Vimblastina/sangue , Vimblastina/urinaRESUMO
Recent studies on ex vivo synthesis of natural products reveal that even complex multistep pathways can be successfully reconstructed. Genetic engineering of such reconstituted pathways has already been used to generate 'unnatural' natural products related to the original compound. In the future, it may be possible to use these approaches to make natural products that are currently inaccessible to conventional synthesis.
Assuntos
Enzimas/metabolismo , Carboidratos/biossíntese , Catálise , Corrinoides , Porfirinas/biossínteseRESUMO
BACKGROUND: During the biosynthesis of vitamin B12, the aerobic bacterium Pseudomonas denitrificans uses two enzymes, CobG and CobJ, to convert precorrin-3 to the ring-contracted intermediate, precorrin-4. CobG is a monooxygenase that adds a hydroxyl group, derived from molecular oxygen, to C-20, whereas CobJ is bifunctional, inserting a methyl group at C-17 of the macrocycle and catalyzing ring contraction. Molecular oxygen is not available to vitamin B12-producing anaerobic bacteria and members of the ancient Archaea, so the question arises of how these microbes accomplish the key ring-contraction process. RESULTS: Cloning and overexpression of Salmonella typhimurium genes has led to the discovery that a single enzyme, CbiH, is responsible for ring contraction during anaerobic biosynthesis of vitamin B12. The process occurs when CbiH is incubated with precorrin-3, but only in the presence of cobalt. CbiH functions as a C-17 methyltransferase and mediates ring contraction and lactonization to yield the intermediate, cobalt-precorrin-4, isolated as cobalt-factor IV. 13C labeling studies have proved that cobalt-precorrin-4 is incorporated into cobyrinic acid, thereby confirming that cobalt-precorrin-4 is an intermediate in vitamin B12 biosynthesis. CONCLUSIONS: Two distinct mechanisms exist in nature for the ring contraction of porphyrinoids to corrinoids-an ancient anaerobic pathway that requires cobalt complexation prior to nonoxidative rearrangement, and a more recent aerobic route in which molecular oxygen serves as the cofactor. The present results offer a rationale for the main differences between aerobic and anaerobic biosynthesis of vitamin B12. Thus, in anaerobes there is exchange of oxygen at the C-27 acetate site, extrusion of acetaldehyde and early insertion of cobalt, whereas the aerobes show no exchange of oxygen at C-27, extrude acetic acid and insert cobalt very late in the biosynthetic pathway, after ring contraction has occurred. These parallel routes to vitamin B12 have now been clearly distinguished by their differing mechanisms for ring contraction.
Assuntos
Oxigênio/metabolismo , Porfirinas/biossíntese , Vitamina B 12/biossíntese , Anaerobiose , Cobalto/metabolismo , Corrinoides , Metilação , Metiltransferases/metabolismo , Conformação Molecular , Porfirinas/metabolismo , Pseudomonas/enzimologia , Pseudomonas/metabolismo , Frações Subcelulares/metabolismo , Uroporfirinas/biossíntese , Uroporfirinas/metabolismo , Vitamina B 12/metabolismoRESUMO
BACKGROUND: Genetically engineered synthesis, in which the gene products, cofactors, and substrates of a complete pathway are combined in vitro in a single flask to give the target, can be a viable alternative to conventional chemical construction of molecules of complex structure and stereochemistry. We chose to attempt to synthesize the metal-free corrinoid hydrogenobyrinic acid, an advanced precursor of vitamin B12. RESULTS: Cloning and overexpression of the genes necessary for the S-adenosyl methionine dependent conversion of 5-aminolevulinic acid (ALA) to precorrin-3 and those required for the synthesis of hydrogenobyrinic acid from precorrin-3 completed the repertoire of the 12 biosynthetic enzymes involved in corrin synthesis. Using these enzymes and the necessary cofactors, the multi-enzyme synthesis of hydrogenobyrinic acid from ALA can be achieved in 20% overall yield in a single reaction vessel, corresponding to an average of at least 90% conversion for each of the 17 steps involved. CONCLUSIONS: By replacing the cell wall with glass, and by mixing the soluble biosynthetic enzymes and necessary cofactors, the major segment of the physiological synthesis of vitamin B12 has been accomplished. Since only those enzymes necessary for the synthesis of hydrogenobyrinic acid from ALA are supplied, none of the intermediates is deflected from the direct pathway. This results in an efficiency which in fact surpasses that of nature.
Assuntos
Uroporfirinas/biossíntese , Vitamina B 12/biossíntese , Escherichia coli/genética , Escherichia coli/metabolismo , Engenharia de Proteínas , Precursores de Proteínas/biossíntese , Precursores de Proteínas/química , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/química , Uroporfirinas/químicaRESUMO
Serum concentrations of immunoreactive neurophysin (IRN) and vasopressin-associated neurophysin (hNpI) were measured before and after the first treatment in a course of electroencephalographically monitored electroconvulsive therapy (ECT) given to 19 depressed patients. The difference (DIFF) between the serum concentrations of IRN and hNpI is equivalent to the concentration of oxytocin-associated neurophysin. Before ECT the six patients who had a good outcome at 2 months after the course of ECT had a mean serum IRN concentration one-half (p less than 0.05) and a mean serum DIFF concentration one-third (p less than 0.05) that of the 13 patients who had a poor outcome. The increase in serum DIFF concentration (but not IRN or hNpI) after the first ECT correlated with the improvement on the Hamilton Rating Scale for Depression (r = -0.73, p less than 0.005) and the Montgomery and Asberg Depression Rating Scale (r = -0.49, p less than 0.05). The peak percentage increase in serum DIFF concentrations after ECT was 4 times greater (p less than 0.001) in the good outcome group than in the poor outcome group. None of the neurophysin responses to ECT correlated with electroencephalogram-measured seizure duration.
Assuntos
Transtorno Depressivo/terapia , Eletroconvulsoterapia , Neurofisinas/sangue , Adulto , Terapia Combinada , Transtorno Depressivo/sangue , Feminino , Seguimentos , Humanos , Masculino , Psicotrópicos/uso terapêutico , RadioimunoensaioRESUMO
The nucleotide sequence of the Candida albicans erg7 gene, which complements erg7 mutants of Saccharomyces cerevisiae and restores oxidosqualene cyclase activity, was determined. The gene encodes a 728-aa protein that displays homology with squalene-hopene cyclase, providing further evidence that erg7 is the gene encoding 2,3-oxidosqualene cyclase.
Assuntos
Candida albicans/genética , Genes Fúngicos , Transferases Intramoleculares , Isomerases/genética , Sequência de Aminoácidos , Sequência de Bases , Candida albicans/enzimologia , DNA Fúngico , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Uroporphyrinogen III synthase from the cyanobacterium Anacystis nidulans was overproduced in Escherichia coli and analyzed by site specific mutagenesis. Of the nine conserved amino acids altered, only a single tyrosine mutant (Y166F) showed any significant decrease in activity suggesting this residue is critical for proper substrate binding and/or catalysis.
Assuntos
Cianobactérias/enzimologia , Tirosina/metabolismo , Uroporfirinogênio III Sintetase/química , Uroporfirinogênio III Sintetase/metabolismo , Sequência de Aminoácidos , Substituição de Aminoácidos , Catálise , Cromatografia Líquida de Alta Pressão , Sequência Conservada , Eletroforese em Gel de Poliacrilamida , Ativação Enzimática/fisiologia , Dados de Sequência Molecular , Estrutura Molecular , Mutagênese Sítio-Dirigida , Relação Estrutura-Atividade , Tirosina/genética , Uroporfirinogênio III Sintetase/genéticaRESUMO
A cell-free extract of the anaerobic eubacterium, Clostridium thermoaceticum, catalyzes the synthesis of 5-aminolevulinic acid (ALA) from glutamate via the C5 pathway. The enzyme reaction resembles that of higher plants and algae in cofactor requirements and sensitivity to ribonuclease. From the phylogenetic distribution it is proposed that the C5 pathway evolved earlier than the ALA synthase pathway.
Assuntos
Ácido Aminolevulínico/biossíntese , Clostridium/metabolismo , Glutamatos/metabolismo , Ácidos Levulínicos/biossíntese , Carbono/metabolismo , Catálise , Sistema Livre de Células , Espectroscopia de Ressonância MagnéticaRESUMO
Previously, the E. coli cysG gene product had been shown to sequentially methylate uro'gen III to produce precorrin-2, hence it was given the trivial name uro'gen III methylase. We now report that in addition to methylase activity, the CysG protein catalyses both the NAD+ dependent oxidation of precorrin-2 to sirohydrochlorin, but also the insertion of iron into this oxidized intermediate, thereby producing siroheme. Thus CysG is a multifunctional protein solely responsible for siroheme synthesis from uro'gen III in E. coli, and accordingly is renamed siroheme synthase.
Assuntos
Escherichia coli/genética , Genes Bacterianos , Metiltransferases/genética , Complexos Multienzimáticos/genética , Sequência de Aminoácidos , Sítios de Ligação , Escherichia coli/enzimologia , Heme/análogos & derivados , Heme/biossíntese , Espectroscopia de Ressonância Magnética , Metilação , Metiltransferases/química , Metiltransferases/metabolismo , Dados de Sequência Molecular , Complexos Multienzimáticos/química , Complexos Multienzimáticos/metabolismo , NAD/metabolismo , NAD/farmacologia , Homologia de Sequência de Aminoácidos , Uroporfirinogênios/metabolismoRESUMO
Biosynthesis of 5-aminolevulinic acid (ALA) in Chloroflexus aurantiacus, a thermophilic bacterium forming bacteriochlorophyll c, is shown to proceed via the C5 pathway by demonstrating (1) the specific labeling of its chlorin ring with [1 - 13C]glutamate and (2) the enzyme activity to produce ALA from glutamate in a cell-free extract. From the phylogenetic distribution it is suggested that ALA synthetase distributed in some aerobic eubacteria could be monophyletic in origin.
Assuntos
Ácido Aminolevulínico/metabolismo , Bactérias/metabolismo , Proteínas de Bactérias/metabolismo , Bacterioclorofilas/metabolismo , Bacterioclorofilas/química , Bacterioclorofilas/isolamento & purificação , Glutamatos/metabolismo , Espectroscopia de Ressonância Magnética/métodos , Estrutura MolecularRESUMO
The dipyrromethane cofactor of Escherichia coli porphobilinogen deaminase was specifically labelled with 13C by growth of the bacteria in the presence of 5-amino[5-13C]levulinic acid. Using 13C-NMR spectroscopy, the structure of the cofactor was confirmed as a dipyrromethane made up of two linked pyrrole rings each derived from porphobilinogen. The chemical shift data indicate that one of the pyrrole rings of the cofactor is covalently linked to the deaminase enzyme through a cysteine residue. Evidence from protein chemistry studies suggest that cysteine-242 is the covalent binding site for the cofactor.
Assuntos
Amônia-Liases/metabolismo , Cisteína/metabolismo , Escherichia coli/enzimologia , Hidroximetilbilano Sintase/metabolismo , Porfobilinogênio/metabolismo , Ácido Aminolevulínico/metabolismo , Sítios de Ligação , Radioisótopos de Carbono , Fenômenos Químicos , Química , Concentração de Íons de Hidrogênio , Espectroscopia de Ressonância MagnéticaRESUMO
Expression of porphobilinogen deaminase in a hemB- strain of E. coli has permitted the isolation of the apoenzyme, i.e. deaminase lacking the porphobilinogen-derived dipyrromethane cofactor. Incubation of purified apoenzyme with porphobilinogen resulted in reconstitution of the covalently attached dipyrromethane cofactor, indicating no additional cofactors or enzymes are required for biosynthesis of holoenzyme. Electrophoretic and 13C-NMR spectroscopic analyses demonstrate that the apoenzyme exists in a conformationally unstable form which is converted to a highly stable tertiary structure on covalent attachment of the dipyrromethane cofactor.
Assuntos
Amônia-Liases/metabolismo , Hidroximetilbilano Sintase/metabolismo , Porfobilinogênio/metabolismo , Apoenzimas/metabolismo , Apoproteínas/metabolismo , Escherichia coli/enzimologia , Espectroscopia de Ressonância Magnética , Peso MolecularRESUMO
Uroporphyrinogen III methylase was purified from a recombinant hemB-strain of E. coli harbouring a plasmid containing the cysG gene. N-terminal analysis of this purified protein gave an amino acid sequence corresponding to that predicted from the genetic code. From the u.v./visible spectrum of the reaction catalysed by this SAM dependent methylase it was possible to observe the sequential appearance of the chromophores of a dipyrrocorphin and subsequently of a pyrrocorphin. Confirmation of this transformation was obtained from 13C-NMR studies when it was demonstrated, for the first time directly, that uroporphyrinogen is initially converted into dihydrosirohydrochlorin (precorrin-2) and then, by further methylation, into a novel trimethylpyrrocorphin.
Assuntos
Escherichia coli/enzimologia , Metiltransferases/metabolismo , Porfirinas/biossíntese , Uroporfirinas/biossíntese , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Espectroscopia de Ressonância Magnética , Metilação , Dados de Sequência Molecular , Estrutura Molecular , Espectrofotometria , Vitamina B 12/biossínteseRESUMO
Nine of the cbi genes from the 17.5 kb cob operon of Salmonella typhimurium previously shown by genetic studies to be involved in the biosynthesis of cobinamide from precorrin-2, have been subcloned and expressed in Escherichia coli. Seven of the gene products were found in the soluble fraction of cell lysates and have been purified. The gene products corresponding to cbi E, F, H and L were shown by SAM binding and by homology with other SAM-binding proteins to be candidates for the methyltransferases of vitamin B12 biosynthesis. The enzymatic functions of the gene products of cbiL and cbiF are associated with C-methylation at C-20 of precorrin-2 and C-11 of precorrin-3.
Assuntos
Proteínas de Bactérias/genética , Genes Bacterianos/genética , Salmonella typhimurium/enzimologia , Salmonella typhimurium/genética , Vitamina B 12/biossíntese , Sequência de Aminoácidos , Proteínas de Bactérias/isolamento & purificação , Clonagem Molecular , Escherichia coli/genética , Espectroscopia de Ressonância Magnética , Metiltransferases/biossíntese , Metiltransferases/genética , Dados de Sequência Molecular , S-Adenosilmetionina/metabolismo , Uroporfirinas/metabolismoRESUMO
In the vitamin B12 biosynthetic pathway the enzymes responsible for the conversion of precorrin-3 to precorrin-4 have been identified as the gene products of cobG and cobJ from Pseudomonas denitrificans. CobG catalyzes the oxidation of precorrin-3 to precorrin-3x (a hydroxy lactone) whereas CobJ is a SAM-dependent C-17 methyl transferase and is necessary for ring contraction. A mechanism for ring contraction is proposed.
Assuntos
Proteínas de Bactérias , Metiltransferases/metabolismo , Oxigenases/metabolismo , Vitamina B 12/biossíntese , Sequência de Bases , DNA de Cadeia Simples , Metilação , Metiltransferases/genética , Dados de Sequência Molecular , Oxirredução , Oxigenases/genética , Pseudomonas/enzimologia , S-Adenosilmetionina/metabolismo , Uroporfirinas/biossínteseRESUMO
Metabolism in the erythrocytes of normal mice and mice infected with Babesia microti has been monitored non-invasively by high resolution 13C NMR spectroscopy. The conversion of [U-13C]glucose to lactate in both normal and infected cells together with the effect of the trypanocidal drug, 4,4'-diamidinodiazoaminobenzene diaceturate, on glycolytic rates were monitored. These studies show that erythrocytes utilize [U-13C]glucose at a rate of 3 X 10(-12) mumol cell-1 min-1 at 35 degrees C while parasitized cells consume 2.9 X 10(-11) mumol cell-1 min-1 and produce lactate as the sole end-product. This rate decreases to 9 X 10(-12) mumol cell-1 min-1 on the addition of 0.75 mM drug.