RESUMEN
Methylenetetrahydrofolate reductase (MTHFR) catalyzes the conversion of methylenetetrahydrofolate to methyltetrahydrofolate, the major methyl donor for the conversion of homocysteine to methionine. Two common polymorphisms of the human enzyme have been identified: 677C>T, which leads to the substitution of Ala-222 by valine, and 1298A>C, which leads to the replacement of Glu-429 by alanine; the former polymorphism is the most frequent genetic cause of mild hyperhomocysteinemia, a risk factor for cardiovascular disease. By using a baculovirus expression system, recombinant human MTHFR has been expressed at high levels and purified to homogeneity in quantities suitable for biochemical characterization. The Glu429Ala protein has biochemical properties that are indistinguishable from the wild-type enzyme. The Ala222Val MTHFR, however, has an enhanced propensity to dissociate into monomers and to lose its FAD cofactor on dilution; the resulting loss of activity is slowed in the presence of methyltetrahydrofolate or adenosylmethionine. This biochemical phenotype is in good agreement with predictions made on the basis of studies comparing wild-type Escherichia coli MTHFR with a mutant, Ala177Val, homologous to the Ala222Val mutant human enzyme [Guenther, B. D., et al. (1999) Nat. Struct. Biol. 6, 359-365].
Asunto(s)
Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Polimorfismo Genético , Animales , Flavina-Adenina Dinucleótido/metabolismo , Humanos , Cinética , Metilenotetrahidrofolato Reductasa (NADPH2) , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/aislamiento & purificación , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Cobalamin cofactors play critical roles in radical-catalyzed rearrangements and in methyl transfers. This Account focuses on the role of methylcobalamin and its structural homologues, the methylcorrinoids, as intermediaries in methyl transfer reactions, and particularly on the reaction catalyzed by cobalamin-dependent methionine synthase. In these methyl transfer reactions, the cobalt(I) form of the cofactor serves as the methyl acceptor. Biological methyl donors to cobalamin include N5-methyltetrahydrofolate, other methylamines, methanol, aromatic methyl ethers, acetate, and dimethyl sulfide. The challenge for chemists is to determine the enzymatic mechanisms for activation of these unreactive methyl donors and to mimic these amazing biological reactions.
Asunto(s)
Metiltransferasas/química , Vitamina B 12/química , Escherichia coli/enzimologíaRESUMEN
Flavodoxins are electron-transfer proteins that contain the prosthetic group flavin mononucleotide. In Escherichia coli, flavodoxin is reduced by the FAD-containing protein NADPH:ferredoxin (flavodoxin) oxidoreductase; flavodoxins serve as electron donors in the reductive activation of anaerobic ribonucleotide reductase, biotin synthase, pyruvate formate lyase, and cobalamin-dependent methionine synthase. In addition, domains homologous to flavodoxin are components of the multidomain flavoproteins cytochrome P450 reductase, nitric oxide synthase, and methionine synthase reductase. Although three-dimensional structures are known for many of these proteins and domains, very little is known about the structural aspects of their interactions. We address this issue by using NMR chemical shift mapping to identify the surfaces on flavodoxin that bind flavodoxin reductase and methionine synthase. We find that these physiological partners bind to unique overlapping sites on flavodoxin, precluding the formation of ternary complexes. We infer that the flavodoxin-like domains of the cytochrome P450 reductase family form mutually exclusive complexes with their electron-donating and -accepting partners, complexes that require conformational changes for interconversion.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Proteínas Bacterianas/metabolismo , Flavodoxina/metabolismo , NADH NADPH Oxidorreductasas/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Proteínas Bacterianas/química , Sitios de Unión , Escherichia coli/química , Flavodoxina/química , Espectroscopía de Resonancia Magnética , Modelos Moleculares , NADH NADPH Oxidorreductasas/química , Oxidación-Reducción , Unión Proteica , Conformación Proteica , S-Adenosilmetionina/metabolismo , Vitamina B 12/metabolismoRESUMEN
The leucine-responsive regulatory protein (Lrp) binds to three sites centered 252, 216, and 152 bp upstream of the transcription start site of the Escherichia coli glutamate synthase operon (gltBDF) and activates transcription. Activators of sigma(70)-dependent promoters usually bind closer to the -35 hexamer of the core promoter sequence. To study the mechanism by which Lrp-dependent activation occurs over this relatively large distance, the gltBDF upstream region was sequentially replaced with corresponding portions from the well-characterized sigma(70)-dependent promoter lacZYAp. The glt-lac promoter hybrids were placed upstream of lacZ, allowing transcriptional activity to be monitored via beta-galactosidase assays. Even replacing all gltBDF sequences downstream of and including the -35 hexamer did not eliminate Lrp-dependent activation of transcription. When a 91-bp region between the -35 hexamer and the proximal Lrp binding site (-48 to -128) was replaced with heterologous DNA of the same length, transcription was reduced nearly 40-fold. Based on the presence of a consensus binding sequence, this region seemed likely to be a binding site for integration host factor (IHF). Experiments to study the effects of a himD mutant on expression of a gltB::lacZ transcriptional fusion, gel mobility shift analyses, and DNA footprinting assays were used to confirm the direct participation of IHF in gltBDF promoter regulation. Based on these results, we suggest that IHF plays a crucial architectural role, bringing the distant Lrp complex in close proximity to the promoter-bound RNA polymerase.
Asunto(s)
Proteínas Bacterianas/metabolismo , Proteínas de Unión al ADN/metabolismo , Escherichia coli/genética , Glutamato Sintasa/genética , Operón/genética , Factores de Transcripción , Secuencia de Bases , Sitios de Unión , Escherichia coli/enzimología , Proteínas de Escherichia coli , Regulación Bacteriana de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Factores de Integración del Huésped , Proteína Reguladora de Respuesta a la Leucina , Modelos Genéticos , Modelos Moleculares , Datos de Secuencia Molecular , Regiones Promotoras Genéticas , Unión Proteica , Transcripción GenéticaRESUMEN
The flavoprotein methylenetetrahydrofolate reductase (MTHFR) from Escherichia coli catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate) using NADH as the source of reducing equivalents. The enzyme also catalyzes the transfer of reducing equivalents from NADH or CH(3)-H(4)folate to menadione, an artificial electron acceptor. Here, we have determined the midpoint potential of the enzyme-bound flavin to be -237 mV. We have examined the individual reductive and oxidative half-reactions constituting the enzyme's activities. In an anaerobic stopped-flow spectrophotometer, we have measured the rate constants of flavin reduction and oxidation occurring in each half-reaction and have compared these with the observed catalytic turnover numbers measured under steady-state conditions. We have shown that, in all cases, the half-reactions proceed at rates sufficiently fast to account for overall turnover, establishing that the enzyme is kinetically competent to catalyze these oxidoreductions by a ping-pong Bi-Bi mechanism. Reoxidation of the reduced flavin by CH(2)-H(4)folate is substantially rate limiting in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction. In the NADH-menadione oxidoreductase reaction, the reduction of the flavin by NADH is rate limiting as is the reduction of flavin by CH(3)-H(4)folate in the CH(3)-H(4)folate-menadione oxidoreductase reaction. We conclude that studies of individual half-reactions catalyzed by E. coli MTHFR may be used to probe mechanistic questions relevant to the overall oxidoreductase reactions.
Asunto(s)
Escherichia coli/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Catálisis , Flavina-Adenina Dinucleótido/química , Cinética , Metilenotetrahidrofolato Reductasa (NADPH2) , Modelos Químicos , NAD/química , NAD(P)H Deshidrogenasa (Quinona)/química , Oxidación-Reducción , Espectrofotometría/métodos , Tetrahidrofolatos/química , Vitamina K/químicaRESUMEN
The flavoprotein Escherichia coli methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5,10-methylenetetrahydrofolate (CH(2)-H(4)folate) to 5-methyltetrahydrofolate (CH(3)-H(4)folate). The X-ray crystal structure of the enzyme has revealed the amino acids at the flavin active site that are likely to be relevant to catalysis. Here, we have focused on two conserved residues, Asp 120 and Glu 28. The presence of an acidic residue (Asp 120) near the N1-C2=O position of the flavin distinguishes MTHFR from all other known flavin oxidoreductases and suggests an important function for this residue in modulating the flavin reactivity. Modeling of the CH(3)-H(4)folate product into the enzyme active site also suggests roles for Asp 120 in binding of folate and in electrostatic stabilization of the putative 5-iminium cation intermediate during catalysis. In the NADH-menadione oxidoreductase assay and in the isolated reductive half-reaction, the Asp120Asn mutant enzyme is reduced by NADH 30% more rapidly than the wild-type enzyme, which is consistent with a measured increase in the flavin midpoint potential. Compared to the wild-type enzyme, the mutant showed 150-fold decreased activity in the physiological NADH-CH(2)-H(4)folate oxidoreductase reaction and in the oxidative half-reaction involving CH(2)-H(4)folate, but the apparent K(d) for CH(2)-H(4)folate was relatively unchanged. Our results support a role for Asp 120 in catalysis of folate reduction and perhaps in stabilization of the 5-iminium cation. By analogy to thymidylate synthase, which also uses CH(2)-H(4)folate as a substrate, Glu 28 may serve directly or via water as a general acid catalyst to aid in 5-iminium cation formation. Consistent with this role, the Glu28Gln mutant was unable to catalyze the reduction of CH(2)-H(4)folate and was inactive in the physiological oxidoreductase reaction. The mutant enzyme was able to bind CH(3)-H(4)folate, but reduction of the FAD cofactor was not observed. In the NADH-menadione oxidoreductase assay, the mutant demonstrated a 240-fold decrease in activity.
Asunto(s)
Ácido Aspártico/química , Escherichia coli/enzimología , Ácido Fólico/química , Ácido Glutámico/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Sustitución de Aminoácidos/genética , Asparagina/química , Asparagina/genética , Ácido Aspártico/genética , Catálisis , Activación Enzimática/genética , Escherichia coli/genética , Ácido Glutámico/genética , Glutamina/genética , Cinética , Metilenotetrahidrofolato Deshidrogenasa (NAD+) , Metilenotetrahidrofolato Reductasa (NADPH2) , Mutagénesis Sitio-Dirigida , NAD/química , NAD(P)H Deshidrogenasa (Quinona)/química , Oxidación-Reducción , Oxidorreductasas/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/antagonistas & inhibidores , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Espectrofotometría/métodos , Vitamina K/químicaRESUMEN
Cobalamin-dependent methionine synthase (MetH) catalyzes the methylation of homocysteine using methyltetrahydrofolate as the methyl donor. The cobalamin cofactor serves as an intermediate carrier of the methyl group from methyltetrahydrofolate to homocysteine. In the two half-reactions that comprise turnover for MetH, the cobalamin is alternatively methylated by methyltetrahydrofolate and demethylated by homocysteine to form methionine. Upon binding to the protein, the usual dimethylbenzimidazole ligand is replaced by the imidazole side chain of His759 [Drennan, C. L., Huang, S., Drummond, J. T., Matthews, R. G., and Ludwig, M. L. (1994) Science 266, 1669-1674]. Despite the ligand replacement that accompanies binding of cobalamin to the holo-MetH protein, a MetH(2-649) fragment of methionine synthase that contains the regions that bind homocysteine and methyltetrahydrofolate utilizes exogenously supplied cobalamin in methyl transfer reactions akin to those of the catalytic cycle. However, the interactions of MetH(2-649) with endogenous cobalamin are first order in cobalamin, while the half-reactions catalyzed by the holoenzyme are zero order in cobalamin, so rate constants for reactions of bound and exogenous cobalamins cannot be compared. In this paper, we investigate the catalytic rate enhancements generated by binding cobalamin to MetH after dividing the protein in half and reacting MetH(2-649) with a second fragment, MetH(649-1227), that harbors the cobalamin cofactor. The second-order rate constant for demethylation of methylcobalamin by Hcy is elevated 60-fold and that for methylation of cob(I)alamin is elevated 120-fold. Thus, binding of cobalamin to MetH is essential for efficient catalysis.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Vitamina B 12/análogos & derivados , Vitamina B 12/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Sitios de Unión/genética , Catálisis , Activación Enzimática/genética , Escherichia coli/enzimología , Escherichia coli/genética , Vectores Genéticos , Homocisteína/química , Cinética , Metilación , Mutagénesis Insercional , Fragmentos de Péptidos/biosíntesis , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/aislamiento & purificación , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Especificidad por Sustrato/genéticaRESUMEN
X-ray absorption spectroscopy has been used to investigate binding of selenohomocysteine to cobalamin-independent (MetE) and cobalamin-dependent (MetH) methionine synthase enzymes of Escherichia coli. We have shown previously [Peariso et al. (1998) J. Am. Chem. Soc. 120, 8410-8416] that the Zn sites in both enzymes show an increase in the number of sulfur ligands when homocysteine binds. The present data provide direct evidence that this change is due to coordination of the substrate to the Zn. Addition of L-selenohomocysteine to either MetE or the N-terminal fragment of MetH, MetH(2-649), causes changes in the zinc X-ray absorption near-edge structure that are remarkably similar to those observed following the addition of L-homocysteine. Zinc EXAFS spectra show that the addition of L-selenohomocysteine changes the coordination environment of the zinc in MetE from 2S + 2(N/O) to 2S + 1(N/O) + 1Se and in MetH(2-649) from 3S + 1(N/O) to 3S + 1Se. The Zn-S, Zn-Se, and Se-S bond distances determined from the zinc and selenium EXAFS data indicate that the zinc sites in substrate-bound MetE and MetH(2-649) both have an approximately tetrahedral geometry. The selenium edge energy for selenohomocysteine shifts to higher energy when binding to either methionine synthase enzyme, suggesting that there is a slight decrease in the effective charge of the selenium. Increases in the Zn-Cys bond distances upon selenohomocysteine binding together with identical magnitudes of the shifts to higher energy in the Se XANES spectra of MetE and MetH(2-649) suggest that the Lewis acidity of the Zn sites in these enzymes appears the same to the substrate and is electronically buffered by the Zn-Cys interaction.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Cisteína/análogos & derivados , Proteínas de Escherichia coli , Selenio/química , Vitamina B 12/química , Zinc/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Proteínas Bacterianas/metabolismo , Sitios de Unión , Cisteína/metabolismo , Escherichia coli/enzimología , Metiltransferasas , Compuestos de Organoselenio/metabolismo , Selenocisteína/análogos & derivados , Análisis Espectral/métodos , Especificidad por Sustrato , Rayos X , Zinc/metabolismoRESUMEN
A single-step convenient synthesis of L-selenohomocysteine (SeHcy) from L-selenomethionine (SeMet) using sodium in liquid ammonia is described. Methionine synthases convert SeHcy to SeMet at rates comparable to their rates of conversion of L-homocysteine (Hcy) to L-methionine (Met). This study suggests that SeHcy generated from SeMet metabolism can be efficiently recycled to SeMet in mammals.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Cisteína/análogos & derivados , Cisteína/síntesis química , Cisteína/metabolismo , Compuestos de Organoselenio/síntesis química , Compuestos de Organoselenio/metabolismo , Selenio/metabolismo , Selenometionina/metabolismo , Animales , Anticarcinógenos/síntesis química , Anticarcinógenos/metabolismo , Cromatografía Líquida de Alta Presión , Femenino , Humanos , Cinética , Masculino , Estructura Molecular , Oxidación-Reducción , Selenocisteína/análogos & derivados , Selenometionina/químicaRESUMEN
N5-Methyltetrahydrofolate (CH(3)-H(4)folate) donates a methyl group to the cob(I)alamin cofactor in the reaction catalyzed by cobalamin-dependent methionine synthase (MetH, EC 2.1.1.3). Nucleophilic displacement of a methyl group attached to a tertiary amine is a reaction without an obvious precedent in bioorganic chemistry. Activation of CH(3)-H(4)folate by protonation prior to transfer of the methyl group has been the favored mechanism. Protonation at N5 would lead to formation of an aminium cation, and quaternary amines such as 5,5-dimethyltetrahydropterin have been shown to transfer methyl groups to cob(I)alamin. Because CH(3)-H(4)folate is an enamine, protonation could occur either at N5 to form an aminium cation or on a conjugated carbon with formation of an iminium cation. We used (13)C distortionless enhancement by polarization transfer (DEPT) NMR spectroscopy to infer that CH(3)-H(4)folate in aqueous solution protonates at N5, not on carbon. CH(3)-H(4)folate must eventually protonate at N5 to form the product H(4)folate; however, this protonation could occur either upon formation of the binary enzyme-CH(3)-H(4)folate complex or later in the reaction mechanism. Protonation at N5 is accompanied by substantial changes in the visible absorbance spectrum of CH(3)-H(4)folate. We have measured the spectral changes associated with binding of CH(3)-H(4)folate to a catalytically competent fragment of MetH over the pH range from 5.5 to 8.5. These studies indicate that CH(3)-H(4)folate is bound in the unprotonated form throughout this pH range and that protonated CH(3)-H(4)folate does not bind to the enzyme. Our observations are rationalized by sequence homologies between the folate-binding region of MetH and dihydropteroate synthase, which suggest that the pterin ring is bound in the hydrophobic core of an alpha(8)beta(8) barrel in both enzymes. The results from these studies are difficult to reconcile with an S(N)2 mechanism for methyl transfer and suggest that the presence of the cobalamin cofactor is important for CH(3)-H(4)folate activation. We propose that protonation of N5 occurs after carbon-nitrogen bond cleavage, and we invoke a mechanism involving oxidative addition of Co(1+) to the N5-methyl bond to rationalize our results.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Protones , Tetrahidrofolatos/química , Vitamina B 12/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Secuencias de Aminoácidos , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Sitios de Unión/genética , Secuencia Conservada , Dihidropteroato Sintasa/química , Humanos , Datos de Secuencia Molecular , Mutagénesis Sitio-Dirigida , Resonancia Magnética Nuclear Biomolecular/métodos , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Estructura Secundaria de Proteína , Homología de Secuencia de Aminoácido , Soluciones , Espectrofotometría Ultravioleta , Especificidad por Sustrato/genéticaAsunto(s)
Proteínas de Unión al ADN/metabolismo , Escherichia coli/metabolismo , Histidina/química , Factores de Transcripción , Secuencia de Aminoácidos , Secuencia de Bases , Cartilla de ADN , Proteínas de Unión al ADN/química , Proteínas de Escherichia coli , Proteína Reguladora de Respuesta a la LeucinaRESUMEN
Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, forming tetrahydrofolate and methionine. The Escherichia coli enzyme, like its mammalian homologue, is occasionally inactivated by oxidation of the cofactor to cob(II)alamin. To return to the catalytic cycle, the cob(II)alamin forms of both the bacterial and mammalian enzymes must be reductively remethylated. Reduced flavodoxin donates an electron for this reaction in E. coli, and S-adenosylmethionine serves as the methyl donor. In humans, the electron is thought to be provided by methionine synthase reductase, a protein containing a domain with a significant degree of homology to flavodoxin. Because of this homology, studies of the interactions between E. coli flavodoxin and methionine synthase provide a model for the mammalian system. To characterize the binding interface between E. coli flavodoxin and methionine synthase, we have employed site-directed mutagenesis and chemical cross-linking using carbodiimide and N-hydroxysuccinimide. Glutamate 61 of flavodoxin is identified as a cross-linked residue, and lysine 959 of the C-terminal activation domain of methionine synthase is assigned as its partner. The mutation of lysine 959 to threonine results in a diminished level of cross-linking, but has only a small effect on the affinity of methionine synthase for flavodoxin. Identification of these cross-linked residues provides evidence in support of a docking model that will be useful in predicting the effects of mutations observed in mammalian homologues of E. coli flavodoxin and methionine synthase.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/metabolismo , Flavodoxina/metabolismo , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Secuencia de Aminoácidos , Sitios de Unión/genética , Reactivos de Enlaces Cruzados/metabolismo , Activación Enzimática/genética , Escherichia coli/enzimología , Escherichia coli/genética , Etildimetilaminopropil Carbodiimida/metabolismo , Flavodoxina/química , Lisina/química , Lisina/genética , Lisina/metabolismo , Sustancias Macromoleculares , Datos de Secuencia Molecular , Peso Molecular , Mutagénesis Sitio-Dirigida , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Succinimidas/metabolismo , Vitamina B 12/química , Vitamina B 12/metabolismoRESUMEN
Genome sequences are the blueprints of diverse life forms but they reveal little information about how cells make coherent responses to environmental changes. The combined use of gene fusions, gene chips, 2-D polyacrylamide gel electrophoresis, mass spectrometry and 'old-fashioned' microbial physiology will provide the means to reveal a cell's regulatory networks and how those networks are integrated.
Asunto(s)
Bacterias/genética , Bacterias/metabolismo , Biología Computacional/métodos , Regulación Bacteriana de la Expresión Génica , Bases de Datos Factuales , Electroforesis en Gel Bidimensional , Genoma Bacteriano , Espectrometría de Masas/métodos , Análisis de Secuencia por Matrices de Oligonucleótidos , Biosíntesis de Proteínas , Transcripción GenéticaRESUMEN
Elevated plasma homocysteine levels are associated with increased risk for cardiovascular disease and neural tube defects in humans. Folate treatment decreases homocysteine levels and dramatically reduces the incidence of neural tube defects. The flavoprotein methylenetetrahydrofolate reductase (MTHFR) is a likely target for these actions of folate. The most common genetic cause of mildly elevated plasma homocysteine in humans is the MTHFR polymorphism A222V (base change C677-->T). The X-ray analysis of E. coli MTHFR, reported here, provides a model for the catalytic domain that is shared by all MTHFRs. This domain is a beta8alpha8 barrel that binds FAD in a novel fashion. Ala 177, corresponding to Ala 222 in human MTHFR, is near the bottom of the barrel and distant from the FAD. The mutation A177V does not affect Km or k(cat) but instead increases the propensity for bacterial MTHFR to lose its essential flavin cofactor. Folate derivatives protect wild-type and mutant E. coli enzymes against flavin loss, and protect human MTHFR and the A222V mutant against thermal inactivation, suggesting a mechanism by which folate treatment reduces homocysteine levels.
Asunto(s)
Escherichia coli/enzimología , Ácido Fólico/metabolismo , Hiperhomocisteinemia/enzimología , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Sitios de Unión , Flavina-Adenina Dinucleótido/metabolismo , Ácido Fólico/farmacología , Humanos , Metilenotetrahidrofolato Reductasa (NADPH2) , Modelos Moleculares , Datos de Secuencia Molecular , Mutación , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Polimorfismo Genético , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Relación Estructura-Actividad , Difracción de Rayos XRESUMEN
A K-12 strain of Escherichia coli that overproduces methylenetetrahydrofolate reductase (MetF) has been constructed, and the enzyme has been purified to apparent homogeneity. A plasmid specifying MetF with six histidine residues added to the C terminus has been used to purify histidine-tagged MetF to homogeneity in a single step by affinity chromatography on nickel-agarose, yielding a preparation with specific activity comparable to that of the unmodified enzyme. The native protein comprises four identical 33-kDa subunits, each of which contains a molecule of noncovalently bound flavin adenine dinucleotide (FAD). No additional cofactors or metals have been detected. The purified enzyme catalyzes the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, using NADH as the reductant. Kinetic parameters have been determined at 15 degreesC and pH 7.2 in a stopped-flow spectrophotometer; the Km for NADH is 13 microM, the Km for CH2-H4folate is 0.8 microM, and the turnover number under Vmax conditions estimated for the reaction is 1,800 mol of NADH oxidized min-1 (mol of enzyme-bound FAD)-1. NADPH also serves as a reductant, but exhibits a much higher Km. MetF also catalyzes the oxidation of methyltetrahydrofolate to methylenetetrahydrofolate in the presence of menadione, which serves as an electron acceptor. The properties of MetF from E. coli differ from those of the ferredoxin-dependent methylenetetrahydrofolate reductase isolated from the homoacetogen Clostridium formicoaceticum and more closely resemble those of the NADH-dependent enzyme from Peptostreptococcus productus and the NADPH-dependent enzymes from eukaryotes.
Asunto(s)
5,10-Metilenotetrahidrofolato Reductasa (FADH2) , Proteínas de Escherichia coli , Escherichia coli/enzimología , Oxidorreductasas/aislamiento & purificación , Oxidorreductasas/metabolismo , Cromatografía de Afinidad , Cromatografía Líquida de Alta Presión , Cromatografía por Intercambio Iónico , Clonación Molecular , Flavina-Adenina Dinucleótido/análisis , Histidina , Cinética , Sustancias Macromoleculares , Metilenotetrahidrofolato Deshidrogenasa (NAD+) , Peso Molecular , Oxidorreductasas/genética , Reacción en Cadena de la Polimerasa , Proteínas Recombinantes/química , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismoRESUMEN
Cobalamin-independent methionine synthase (MetE) from Escherichia coli catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. It contains 1 equiv of zinc that is essential for its catalytic activity. Extended X-ray absorption fine structure analysis of the zinc-binding site has suggested tetrahedral coordination with two sulfur (cysteine) and one nitrogen or oxygen ligands provided by the enzyme and an exchangeable oxygen or nitrogen ligand that is replaced by the homocysteine thiol group in the enzyme-substrate complex [González, J. C., Peariso, K., Penner-Hahn, J. E., and Matthews, R. G. (1996) Biochemistry 35, 12228-34]. Sequence alignment of MetE homologues shows that His641, Cys643, and Cys726 are the only conserved residues. We report here the construction, expression, and purification of the His641Gln, Cys643Ser, and Cys726Ser mutants of MetE. Each mutant displays significantly impaired activity and contains less than 1 equiv of zinc upon purification. Furthermore, each mutant binds zinc with lower binding affinity (K(a) approximately 10(14) M(-)(1)) compared to the wild-type enzyme (K(a) > 10(16) M(-)(1)). All the MetE mutants are able to bind homocysteine. X-ray absorption spectroscopy analysis of the zinc-binding sites in the mutants indicates that the four-coordinate zinc site is preserved but that the ligand sets are changed. Our results demonstrate that Cys643 and Cys726 are two of the zinc ligands in MetE from E. coli and suggest that His641 is a third endogenous ligand. The effects of the mutations on the specific activities of the mutant proteins suggest that zinc and homocysteine binding alone are not sufficient for activity; the chemical nature of the ligands is also a determining factor for catalytic activity in agreement with model studies of the alkylation of zinc-thiolate complexes.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , Escherichia coli/enzimología , Zinc/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/biosíntesis , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Secuencia de Aminoácidos , Sitios de Unión , Quelantes , Homocisteína/química , Ligandos , Datos de Secuencia Molecular , Mutación , Alineación de Secuencia , Vitamina B 12/química , Zinc/análisisRESUMEN
Chemotherapeutic drugs targeted at folate-dependent reactions have typically been directed at a limited number of target enzymes: dihydrofolate reductase, thymidylate synthase, and GAR and AICAR transformylase. This review discusses two other potential targets for chemotherapeutic inhibition: cobalamin-dependent methionine synthase and serine hydroxymethyltransferase. Brief reviews of the catalytic properties of these two enzymes are presented, and possible strategies for chemotherapeutic intervention are discussed.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/antagonistas & inhibidores , Quimioterapia , Inhibidores Enzimáticos/farmacología , Glicina Hidroximetiltransferasa/antagonistas & inhibidores , Adenosina/análogos & derivados , Adenosina/farmacología , Diseño de Fármacos , Humanos , Cinética , Estructura Molecular , Treonina/análogos & derivados , Treonina/metabolismo , Transcobalaminas/metabolismoRESUMEN
Cobalamin-dependent methionine synthase catalyzes the transfer of a methyl group from methyltetrahydrofolate to homocysteine, generating tetrahydrofolate and methionine. During this primary turnover cycle, the enzyme alternates between the active methylcobalamin and cob(I)alamin forms of the enzyme. Formation of the cob(II)alamin prosthetic group by oxidation of cob(I)alamin or photolysis of methylcobalamin renders the enzyme inactive. Methionine synthase from E. coli catalyzes its own reactivation by a reductive methylation that involves electron transfer from reduced flavodoxin and methyl transfer from AdoMet. This process has been proposed to involve formation of a transient cob(I)alamin intermediate that is then trapped by methyl transfer from AdoMet. During aerobic growth of E. coli, electrons for this process are ultimately derived from NADPH, and electron transfer does not generate a detectable level of cob(I)alamin due to the large potential difference between the NADPH/NADP+ couple and the cob(I)alamin/cob(II)alamin couple. In this paper, we show that even in the presence of the strong reductant flavodoxin hydroquinone, cob(I)alamin is not observed as a significant intermediate. We demonstrate, however, that this is due to a rate-limiting reorganization of the cobalt ligand environment from five-coordinate to four-coordinate cob(II)alamin. Mutation of aspartate 757 to glutamate results in a cob(II)alamin enzyme that is approximately 70% four-coordinate, and reductive methylation of this enzyme using flavodoxin hydroquinone as the electron donor proceeds through a kinetically competent cob(I)alamin intermediate. Furthermore, wild-type cob(I)alamin enzyme produced by chemical reduction reacts with AdoMet in a kinetically competent reaction. We provide evidence that methyl transfer from AdoMet to cob(I)alamin enzyme results initially in formation of a five-coordinate methylcobalamin enzyme that slowly decays to the active six-coordinate methylcobalamin enzyme. We propose a kinetic scheme for reductive methylation of wild-type cob(II)alamin enzyme by adenosylmethionine and flavodoxin hydroquinone in which slow conformational changes mask the relatively fast electron and methyl transfer steps.
Asunto(s)
5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/química , S-Adenosilmetionina/química , Transcobalaminas/química , 5-Metiltetrahidrofolato-Homocisteína S-Metiltransferasa/genética , Sustitución de Aminoácidos/genética , Ácido Aspártico/genética , Cobalto/química , Transporte de Electrón , Activación Enzimática , Escherichia coli/enzimología , Escherichia coli/genética , Ácido Glutámico/genética , Histidina/química , Cinética , Metilación , Modelos Químicos , Mutagénesis Sitio-Dirigida , Oxidación-Reducción , Vitamina B 12/análogos & derivados , Vitamina B 12/químicaRESUMEN
Methionine synthase (MetH) from Escherichia coli catalyzes the synthesis of methionine from homocysteine and methyltetrahydrofolate via two methyl transfer reactions that are mediated by the endogenous cobalamin cofactor. After binding both substrates in a ternary complex, the enzyme transfers a methyl group from the methylcobalamin cofactor to homocysteine, generating cob(I)alamin enzyme and methionine. The enzyme then catalyzes methyl transfer from methyltetrahydrofolate to the cob(I)alamin cofactor, forming methylcobalamin cofactor and tetrahydrofolate prior to the release of both products. The cob(I)alamin form of the enzyme occasionally undergoes oxidation to an inactive cob(II)alamin species; the enzyme also catalyzes its own reactivation. Electron transfer from reduced flavodoxin to the cob(II)alamin cofactor is thought to generate cob(I)alamin enzyme, which is then trapped by methyl transfer from adenosylmethionine to the cobalt, restoring the enzyme to the active methylcobalamin form. Thus the enzyme is potentially able to catalyze two methyl transfers to the cob(I)alamin cofactor: methyl transfer from methyltetrahydrofolate during primary turnover and methyl transfer from adenosylmethionine during activation. It has recently been shown that methionine synthase is constructed from at least four separable regions that are responsible for binding each of the three substrates and the cobalamin cofactor, and it has been proposed that changes in positioning of the substrate binding regions vis-à-vis the cobalamin binding region could allow the enzyme to control which substrate has access to the cofactor. In this paper, we offer evidence that methionine synthase exists in two different conformations that interconvert in the cob(II)alamin oxidation state. In the primary turnover conformation, the enzyme reacts with homocysteine and methyltetrahydrofolate but is unreactive toward adenosylmethionine and flavodoxin. In the reactivation conformation, the enzyme is active toward adenosylmethionine and flavodoxin but unreactive toward methyltetrahydrofolate. The two conformations differ in the susceptibility of the substrate-binding regions to tryptic proteolysis. We propose a model in which conformational changes control access to the cobalamin cofactor and are the primary means of controlling cobalamin reactivity in methionine synthase.