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R G Matthews

Publications and source records attributed to R G Matthews.

At least 55 records · Page 3Linked to original sources
PubMed1996

A protein radical cage slows photolysis of methylcobalamin in methionine synthase from Escherichia coli.

Methionine synthase from Escherichia coli is a B12-dependent enzyme that utilizes a methylcobalamin prosthetic group. In the catalytic cycle, the methyl group of methylcobalamin is transferred to homocysteine, generating methionine and cob(I)-alamin, and cob(I)alamin is then remethylated by a methyl group from methyltetrahydrofolate. Methionine synthase occasionally undergoes side reactions that produce the inactive cob(II)alamin form of the enzyme. One such reaction is photolytic homolysis of the methylcobalamin C-Co bond. Binding to the methionine synthase apoenzyme protects the methylcobalamin cofactor against photolysis, decreasing the rate of this reaction by approximately 50-fold. The X-ray structure of the cobalamin-binding region of methionine synthase suggests how the protein might protect the methylcobalamin cofactor in the resting enzyme. In particular, the upper face (methyl or beta face) of the cobalamin cofactor is in contact with several hydrophobic residues provided by an alpha-helical domain, and these residues could slow photolysis by caging the methyl radical and favoring recombination of the CH3./cob(II)alamin radical pair. We have introduced mutations at three positions in the cap domain; phenylalanine 708, phenylalanine 714, and leucine 715 have each been replaced by alanine. Calculations based on the wild-type structure predict that two of these three mutations (Phe708Ala and Leu715Ala) will increase solvent accessibility to the methylcobalamin cofactor, and in fact these mutations result in dramatic increases in the rate of photolysis. The third mutation, Phe714Ala, is not predicted to increase the accessibility of the cofactor and has only a modest effect on the photolysis rate of the enzyme. These results confirm that the alpha-helical domain covers the cofactor in the resting methylcobalamin enzyme and that residues from this domain can protect the enzyme against photolysis. Further, we show that binding the substrate methyltetrahydrofolate to the wild-type enzyme results in a saturable increase in the rate of photolysis, suggesting that substrate binding induces a conformational change in the protein that increases the accessibility of the methylcobalamin cofactor.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1996

Use of an in vivo titration method to study a global regulator: effect of varying Lrp levels on expression of gltBDF in Escherichia coli.

Most studies of global regulatory proteins are performed in vitro or involve phenotypic comparisons between wild-type and mutant strains. We report the use of strains in which the gene for the leucine-responsive regulatory protein (lrp) is transcribed from isopropyl-beta-D-thiogalactopyranoside (IPTG)-inducible promoters for the purpose of continuously varying the in vivo concentration of Lrp. To obtain a broad range of Lrp concentrations, strains were employed that contained the lrp fusion either in the chromosome (I. C. Blomfield, P. J. Calie, K. J. Eberhardt, M. S. McClain, and B. I. Eisenstein, J. Bacteriol. 175:27-36, 1993) or on a multicopy plasmid. Western blot (immunoblot) analysis with polyclonal antiserum to Lrp confirmed that Lrp levels could be varied more than 70-fold by growing the strains in glucose minimal 3-(N-morpholino)propanesulfonic acid (MOPS) medium containing different amounts of IPTG. Expression of an Lrp-regulated gltB::lacZ operon fusion was measured over this range of Lrp concentrations. beta-Galactosidase activity rose with increasing Lrp levels up to the level of Lrp found in wild-type strains, at which point expression is maximal. The presence of leucine in the medium increased the level of Lrp necessary to achieve half-maximal expression of the gltB::lacZ fusion, as predicted by earlier in vitro studies (B. R. Ernsting, J. W. Denninger, R. M. Blumenthal, and R. G. Matthews, J. Bacteriol. 175:7160-7169, 1993). Interestingly, levels of Lrp greater than those in wild-type cells interfered with activation of gltB::lacZ expression. The growth rate of cultures correlated with the intracellular Lrp concentration: levels of Lrp either lower or higher than wild-type levels resulted in significantly slower growth rates. Thus, the level of Lrp in the cell appears to be optimal for rapid growth in minimal medium, and the gltBDF control region is designed to give maximal expression at this Lrp level.

Bacterial Proteins↗
PubMed1995

Stereospecificity of folate binding to DNA photolyase from Escherichia coli.

DNA photolyase from Escherichia coli contains folate ([6S]-5,10-CH(+)-H4Pte(Glu)n = 3-6) and reduced FAD. The folate chromophore acts as an antenna, harvesting light energy which is transferred to the reduced flavin where DNA repair occurs. The folate binding stereospecificity of the enzyme was investigated by reconstituting the apoenzyme with [6R,S]-5,10-CH(+)-H4folate and reduced FAD. The isomer composition of [methyl-3H]-5-CH3-H4folate, released into solution upon reduction of the reconstituted enzyme with [3H]NaBH4, was analyzed by enzymatic and chiral chromatographic methods. Both methods showed that the reconstituted enzyme contained nearly equimolar amounts of [6R]- and [6S]-5,10-CH(+)-H4folate.

Apoenzymes↗
PubMed1995

4-Chlorothreonine is substrate, mechanistic probe, and mechanism-based inactivator of serine hydroxymethyltransferase.

Serine hydroxymethyltransferase catalyzes the cleavage of a variety of beta-hydroxy-L-amino acids to form glycine and aldehyde products. 4-chloro-L-threonine has been synthesized and shown to be both a substrate and a mechanism-based inactivator of serine hydroxymethyltransferase. kcat values for the formation of glycine in the absence of tetrahydrofolate were determined for 4-chloro-L-threonine and other beta-hydroxyamino acid substrates; an inverse relationship between the rate of cleavage of the amino acid and the electrophilicity of the product aldehyde was demonstrated. 4-Chloro-L-threonine inactivates serine hydroxymethyltransferase in a time- and concentration-dependent manner and exhibits saturation of the rate of inactivation at high concentrations. Our evidence suggests that 4-chlorothreonine undergoes aldol cleavage, and generation of chloroacetaldehyde at the active site of the enzyme results in inactivation. Serine or glycine protect the enzyme against inactivation by chlorothreonine, while tetrahydrofolate does not. The enzyme is also protected from inactivation by 2-mercaptoethanol or by alcohol dehydrogenase and NADH. These studies suggest that halothreonine derivatives that generate electrophilic aldehyde products will be effective inhibitors of serine hydroxymethyltransferase and might be potentially useful chemotherapeutic agents.

Alcohol Dehydrogenase↗
PubMed1995

Characterization of nonradioactive assays for cobalamin-dependent and cobalamin-independent methionine synthase enzymes.

Methionine synthase enzymes catalyze methyl group transfer from 5-methyltetrahydrofolate to homocysteine to give methionine and tetrahydrofolate. Assays for this enzyme activity usually monitor transfer of a 14C-methyl group from the N5-position of methyltetrahydrofolate to homocysteine to produce 14C-methionine that must be purified by anion-exchange chromatography. Alternatively, tetrahydrofolate may be derivatized with a formylating agent under acidic conditions to produce methenyltetrahydrofolate. We report optimization of this reaction for assay of cobalamin-dependent methionine synthase to give an economical method for determining enzyme activity that does not require the use of radioactive compounds. By heating for 10 min in 1 N hydrochloric acid containing 12% formic acid, the enzymatic product tetrahydrofolate is converted into methenyltetrahydrofolate, which absorbs light at 350 nm, while residual substrate 5-methyltetrahydrofolate does not contribute to the absorbance at 350 nm. The assay allows the derivatized product to be characterized in situ with a minimal increase in volume upon acidification. The results of the spectrophotometric assay given here have been compared with the radioactive assay to confirm the validity of the derivatization under the assay conditions. We also report the extension of this assay method for use in activity measurements of cobalamin-independent methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

Vitamin-B12-independent methionine synthase from a higher plant (Catharanthus roseus). Molecular characterization, regulation, heterologous expression, and enzyme properties.

Methionine synthases catalyze the formation of methionine by the transfer of a methyl group from 5-methyltetrahydrofolate to homocysteine. This reaction is the last step in L-methionine biosynthesis, and it also serves to regenerate the methyl group of S-adenosylmethionine, a cofactor required for biological methylation reactions. We describe the cloning, expression and characterization of a methionine synthase from the higher plant Catharanthus roseus. cDNAs were identified that encoded a protein of 85 kDa sharing 50% identify with the cobalamin-independent methionine synthase from Escherichia coli (MetE) and 41% identity with a partial sequence of a yeast homolog of MetE. The C. roseus protein was expressed at high levels in E. coli. The enzyme accepts the triglutamate form of methyltetrahydrofolate as a methyl donor but not the monoglutamate form, and it does not require S-adenosylmethionine or cobalamin for activity. The properties indicate that the enzyme is a cobalamin-independent methionine synthase (EC 2.1.1.14). In contrast to the E. coli MetE, the plant protein does not require phosphate or magnesium ions for activity. Immunoblots of plants extracts showed that the protein was localized in the cytosol, and was present in a variety of plant species. A nutritional downshift of the C. roseus cell culture revealed a strong, transient transcriptional activation, but no significant increment in the total level of the protein. The availability of the protein and the cDNA now provide tools to investigate the complexities of methionine biosynthesis in plants.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1995

A candidate genetic risk factor for vascular disease: a common mutation in methylenetetrahydrofolate reductase.

Hyperhomocysteinaemia has been identified as a risk factor for cerebrovascular, peripheral vascular and coronary heart disease. Elevated levels of plasma homocysteine can result from genetic or nutrient-related disturbances in the trans-sulphuration or re-methylation pathways for homocysteine metabolism. 5, 10-Methylenetetrahydrofolate reductase (MTHFR) catalyzes the reduction of 5, 10-methylenetetrahydrofolate to 5-methyltetrahydrofolate, the predominant circulatory form of folate and carbon donor for the re-methylation of homocysteine to methionine. Reduced MTHFR activity with a thermolabile enzyme has been reported in patients with coronary and peripheral artery disease. We have identified a common mutation in MTHFR which alters a highly-conserved amino acid; the substitution occurs at a frequency of approximately 38% of unselected chromosomes. The mutation in the heterozygous or homozygous state correlates with reduced enzyme activity and increased thermolability in lymphocyte extracts; in vitro expression of a mutagenized cDNA containing the mutation confirms its effect on thermolability of MTHFR. Finally, individuals homozygous for the mutation have significantly elevated plasma homocysteine levels. This mutation in MTHFR may represent an important genetic risk factor in vascular disease.

Adult↗
PubMed1995

The leucine-responsive regulatory protein of Escherichia coli negatively regulates transcription of ompC and micF and positively regulates translation of ompF.

The two major porins of Escherichia coli K-12 strains, OmpC and OmpF, are inversely regulated with respect to one another. The expression of OmpC and OmpF has been shown to be influenced by the leucine-responsive regulatory protein (Lrp): two-dimensional gel electrophoresis of proteins from strains with and strains without a functional Lrp protein revealed that OmpC expression is increased in an lrp strain, while OmpF expression is decreased. In agreement with these findings, we now present evidence that transcriptional (operon) fusions of lacZ+ to ompC and micF are negatively regulated by Lrp. Lrp binds specifically to the intergenic region between micF and ompC, as indicated by mobility shift assays and by DNase I footprinting. The expression of an ompF'-lacZ+ gene (translational) fusion is increased 3.7-fold in an lrp+ background compared with an lrp background, but expression of an ompF-lacZ+ operon fusion is not. Studies of in vivo expression of the outer membrane porins during growth on glucose minimal medium showed that the OmpF/OmpC ratio is higher in lrp+ strains than it is in isogenic lrp strains. The effect of Lrp was not seen in a strain containing a deletion of micF. Our studies suggest that the positive effect of Lrp on OmpF expression stems from a negative effect of Lrp on the expression of micF, an antisense RNA that inhibits ompF translation.

Bacterial Outer Membrane Proteins↗
PubMed1994

How a protein binds B12: A 3.0 A X-ray structure of B12-binding domains of methionine synthase.

The crystal structure of a 27-kilodalton methylcobalamin-containing fragment of methionine synthase from Escherichia coli was determined at 3.0 A resolution. This structure depicts cobalamin-protein interactions and reveals that the corrin macrocycle lies between a helical amino-terminal domain and an alpha/beta carboxyl-terminal domain that is a variant of the Rossmann fold. Methylcobalamin undergoes a conformational change on binding the protein; the dimethylbenzimidazole group, which is coordinated to the cobalt in the free cofactor, moves away from the corrin and is replaced by a histidine contributed by the protein. The sequence Asp-X-His-X-X-Gly, which contains this histidine ligand, is conserved in the adenosylcobalamin-dependent enzymes methylmalonyl-coenzyme A mutase and glutamate mutase, suggesting that displacement of the dimethylbenzimidazole will be a feature common to many cobalamin-binding proteins. Thus the cobalt ligand, His759, and the neighboring residues Asp757 and Ser810, may form a catalytic quartet, Co-His-Asp-Ser, that modulates the reactivity of the B12 prosthetic group in methionine synthase.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Nitrous oxide degradation by cobalamin-dependent methionine synthase: characterization of the reactants and products in the inactivation reaction.

Cobalamin-dependent methionine synthase catalyzes the remethylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. The enzyme is susceptible to inactivation by the anaesthetic gas, nitrous oxide, through either short-term exposure to high levels or chronic exposure to low levels of this agent. We have studied the chemical reaction wherein the bound cobalamin prosthetic group of the enzyme from Escherichia coli catalyzes the degradation of nitrous oxide. By poising the enzyme at low ambient potentials in an electrochemical cell, the concentration of enzyme in the highly reactive cob(I)alamin state can be controlled, and the observed rate of inactivation is directly dependent upon the amount of enzyme in this redox state. The inactivation consumes both nitrous oxide and electrons, while nitrogen gas is evolved. The inactivation process is also directly dependent upon the proton concentration, but the effect of pH was found to be on the enzyme, revealing a redox-linked base presumed to be on or near the cobalamin. The chemical reaction between enzyme-bound cob(I)alamin and nitrous oxide that leads to inactivation is therefore independent of pH. In a single turnover experiment, where enzyme-bound cob(I)alamin is generated from methylcobalamin by methyl transfer to the substrate homocysteine, the cob(I)alamin decays to cob(II)alamin, suggesting that the degradation of nitrous oxide involves one-electron reduction of nitrous oxide. We propose that the inactivation chemistry is likely to be a one-electron reduction of nitrous oxide, which leads to the formation of a highly reactive oxidant, such as hydroxyl radical, and subsequent enzyme damage.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Nitrous oxide inactivation of cobalamin-dependent methionine synthase from Escherichia coli: characterization of the damage to the enzyme and prosthetic group.

Nitrous oxide, or laughing gas, is an anaesthetic agent that inactivates cobalamin-dependent methionine synthase. This enzyme uses the highly reactive, enzyme-bound cob(I)alamin oxidation state of the prosthetic group to effect methyl group transfer from 5-methyltetrahydrofolate to homocysteine to form tetrahydrofolate and methionine. The cob(I)alamin is capable of reductively degrading nitrous oxide, and here we characterize the modifications that occur to the Escherichia coli enzyme following electrochemical inactivation. Methionine synthase was inactivated on a milligram scale by equilibrating enzyme containing bound cob(II)alamin with a reduced electrochemical mediator to give the reactive cob(I)alamin state under an anaerobic atmosphere of nitrous oxide. The primary damage occurs to a 37.2-kDa domain that binds S-adenosylmethionine (AdoMet), and inactive enzyme can no longer be reductively methylated using AdoMet. The damage is oxidative, and it includes the covalent addition of the mediator, triquat, to the enzyme selectively at valine 1177, as well as the formation of a covalent cross-link between peptides containing the only two cysteines within this domain. Spectrally, the prosthetic group bound to inactive enzyme resembles cob(II)alamin, although some loss in absorbance is apparent. When the enzyme was reconstituted with [57Co]cobalamin and the inactivation repeated, the cobalamin was recovered unmodified in approximately 75% yield, but two products derived from the cobalamin were also observed. We interpret the finding of oxidatively modified products as strong evidence that reductive degradation of nitrous oxide releases a potent oxidant, presumably hydroxyl radical or its equivalent, that is capable of modifying sites proximal to the cobalamin.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Cobalamin-dependent methionine synthase: the structure of a methylcobalamin-binding fragment and implications for other B12-dependent enzymes.

Cobalamin-dependent methionine synthase is a large enzyme composed of structurally and functionally distinct regions. Recent studies have begun to define the roles of several regions of the protein. In particular, the structure of a 27 kDa cobalamin-binding fragment of the enzyme from Escherichia coli has been determined by X-ray crystallography, and has revealed the motifs and interactions responsible for recognition of the cofactor. The amino acid sequences of several adenosylcobalamin-dependent enzymes, the methylmalonyl coenzyme A mutases and glutamate mutases, show homology with the cobalamin-binding region of methionine synthase and retain conserved residues that are determinants for the binding of the prosthetic group, suggesting that these mutases and methionine synthase share common three-dimensional structures.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1994

Human methylenetetrahydrofolate reductase: isolation of cDNA, mapping and mutation identification.

Methylenetetrahydrofolate reductase (MTHFR) catalyses the reduction of methylenetetrahydrofolate to methyltetrahydrofolate, a cofactor for homocysteine methylation to methionine. MTHFR deficiency, an autosomal recessive disorder, results in homocysteinemia. Using degenerate oligonucleotides based on porcine peptide sequence data, we isolated a 90-bp cDNA by PCR from pig liver RNA. This cDNA was used to isolate a human cDNA, the predicted amino acid sequence of which shows strong homology to porcine MTHFR and to bacterial metF genes. The human gene has been localized to chromosome 1p36.3. Two mutations were identified in MTHFR-deficient patients: a missense mutation (Arg to Gln), in a residue conserved in bacterial enzymes, and a nonsense mutation (Arg to Ter).

Amino Acid Sequence↗
PubMed1994

The leucine-responsive regulatory protein, a global regulator of metabolism in Escherichia coli.

The leucine-responsive regulatory protein (Lrp) regulates the expression of more than 40 genes and proteins in Escherichia coli. Among the operons that are positively regulated by Lrp are operons involved in amino acid biosynthesis (ilvIH, serA)), in the biosynthesis of pili (pap, fan, fim), and in the assimilation of ammonia (glnA, gltBD). Negatively regulated operons include operons involved in amino acid catabolism (sdaA, tdh) and peptide transport (opp) and the operon coding for Lrp itself (lrp). Detailed studies of a few members of the regulon have shown that Lrp can act directly to activate or repress transcription of target operons. A substantial fraction of operons regulated by Lrp are also regulated by leucine, and the effect of leucine on expression of these operons requires a functional Lrp protein. The patterns of regulation are surprising and interesting: in some cases activation or repression mediated by Lrp is antagonized by leucine, in other cases Lrp-mediated activation or repression is potentiated by leucine, and in still other cases leucine has no effect on Lrp-mediated regulation. Current research is just beginning to elucidate the detailed mechanisms by which Lrp can mediate such a broad spectrum of regulatory effects. Our view of the role of Lrp in metabolism may change as more members of the regulon are identified and their regulation characterized, but at this point Lrp seems to be important in regulating nitrogen metabolism and one-carbon metabolism, permitting adaptations to feast and to famine.

Amino Acid Sequence↗
PubMed1993

Flavodoxin is required for the activation of the anaerobic ribonucleotide reductase.

The inactive anaerobic ribonucleotide reductase from Escherichia coli is transformed by a multienzyme system and S-adenosylmethionine + NADPH into a radical protein that is enzymatically active. One of the activating enzyme components was earlier shown to be ferredoxin (flavodoxin):NADP+ reductase. Here we present evidence that flavodoxin, but not ferredoxin, also is a component of the system. Light reduced deazaflavin can substitute for the flavodoxin system. An additional unidentified low-molecular weight component further stimulates the reaction.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗
PubMed1993

Electrospray mass spectrometric analysis of the domains of a large enzyme: observation of the occupied cobalamin-binding domain and redefinition of the carboxyl terminus of methionine synthase.

Cobalamin-dependent methionine synthase from Escherichia coli catalyzes the methylation of homocysteine to form methionine, using methyltetrahydrofolate as the primary methyl donor. We have used electrospray mass spectrometry as a powerful tool for characterizing separable fragments obtained by proteolysis of this monomeric 136.1-kDa enzyme. A central 28.0-kDa domain, reported to bind the cobalamin, has been purified to homogeneity in 30% yield. We were able to detect the domain with bound cobalamin by electrospray mass spectrometry at neutral pH. Mass analysis of a 37.2-kDa carboxyl-terminal domain was grossly inconsistent with either of the two amino acid sequences from previously published DNA sequences. We then used electrospray mass spectrometry to analyze peptides generated by a lysyl endoproteolytic digest of a C-terminal fragment, and we have constructed a peptide map that accounts for > 95% of the peptide mass derived from this domain. The correct translational end of this protein (27 residues downstream from the previously predicted ultimate residue) has been established, and sequence conflicts within the two published DNA sequences have been resolved (GenBank Accession Number J04975). Resequencing the DNA near the carboxyl terminus ruled out a frameshifted reading of the DNA and suggested that a cytosine had twice been incorrectly inserted late in the reading frame. The strategies reported here for sequence confirmation, localization of coenzyme-binding regions, and identification of chemically modified peptides within a large protein are potentially applicable to the characterization of many other proteins.

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗