Biomedical subjects

E N Marsh

Publications and source records attributed to E N Marsh.

31 records · Page 2Linked to original sources

PubMed1996

Carboxymethylation of MutS-cysteine-15 specifically inactivates adenosylcobalamin-dependent glutamate mutase. Examination of the role of this residue in coenzyme-binding and catalysis.

The sensitivity of adenosylcobalamin (AdoCbl)-dependent glutamate mutase toward thiol-directed reagents has been investigated. Iodoacetate specifically alkylates one cysteine residue, Cys-15, in MutS with concomitant irreversible loss of enzyme activity. Cys-15 lies between the conserved residues Asp-14 and His-16, that are believed to coordinate cobalt to form a Co-His-Asp hydrogen-bonded "triad" when AdoCbl is bound by the enzyme. Although inactive, carboxymethylated MutS still bound AdoCbl with only a 5-fold increase in apparent Kd. To determine whether Cys-15 plays an essential role in catalysis, it was mutated to serine and to alanine. These mutants were active, but both exhibited decreased Vmax and increased apparent Km and Kd for AdoCbl. To mimic the effect of carboxymethylation, Cys15 was mutated to aspartate and, as an isosteric control, to asparagine. Neither of these mutants was active: MutS-C15N bound AdoCbl approximately 10-fold weaker than wild type, whereas MutS-C15N bound AdoCbl over 100 times less strongly than wild type. The results demonstrate both coenzyme-binding and catalysis to be very sensitive to mutations at position 15 that could potentially perturb the Co-His-Asp hydrogen-bonding network.

Adenosine Triphosphatases↗

PubMed1995

Identification of a flavin:NADH oxidoreductase involved in the biosynthesis of actinorhodin. Purification and characterization of the recombinant enzyme.

The biosynthesis of the polyketide antibiotic actinorhodin by Streptomyces coelicolor involves the oxidative dimerization and hydroxylation of a precursor, most likely dihydrokalafungin, as the final steps in its formation. Mutations in the actVB gene block these last steps, and the mutants secrete kalafungin as a shunt product. To investigate the role of the actVB gene in these transformation, we have overexpressed the gene in Escherichia coli and purified and characterized the recombinant protein. ActVB was shown to catalyze the reduction of FMN by NADH to give NAD and FMNH2, which, unusually, is released into solution. The protein contains no chromogenic cofactors and exhibits no requirements for added metal ions. The reaction obeys simple kinetics and proceeds through the formation of a ternary complex; Km values for FMN and NADH are 1.5 and 7.3 microM, respectively, and kcat is about 5 s-1. FAD and riboflavin are also substrates for the enzyme, although they have much higher Km values. The subunit structure of the enzyme was investigated by analytical ultracentrifugation, which showed the protein to exist in rapid equilibrium between monomer and dimer forms. The possible role of this oxidoreductase in the oxidative chemistry of actinorhodin biosynthesis is discussed.

Amino Acid Sequence↗

PubMed1995

Tritium isotope effects in adenosylcobalamin-dependent glutamate mutase: implications for the mechanism.

The transfer of tritium between adenosylcobalamin and substrate in the reaction catalyzed by glutamate mutase was examined to investigate the possibility of a protein-based radical intermediate. There was no evidence that tritium was transferred to the protein during the reaction, as tritium neither became stably bound to the protein nor exchanged with water. The kinetics of tritium transfer from adenosylcobalamin to 3-methylaspartate was investigated. Both the transfer of tritium to product and the exchange of enzyme-bound and free coenzyme contribute to the kinetics of tritium loss from adenosylcobalamin. By varying the experimental conditions, the rates of both coenzyme exchange and tritium transfer could be measured. Exchange of adenosylcobalamin with enzyme is very slow, k off = 0.01 s-1, which may reflect a conformational change in the coenzyme and/or protein involved in forming active holo enzyme. The rate constants for the loss of tritium from adenosylcobalamin and the appearance of tritium in 3-methylaspartate are much faster and very similar, k = 0.67 +/- 0.05 s-1 and k = 0.50 +/- 0.05 s-1, respectively, consistent with the transfer of tritium occurring directly between coenzyme and substrate. The isotope effect, calculated from the rate constants for tritium transfer, and kcat, determined for the overall reaction under the same conditions, are between 13.5 and 18. These values are typical of primary isotope effects seen for enzymes in which hydrogen transfer is substantially rate limiting. A protein radical, therefore, appears unlikely to feature in the mechanism of this enzyme.

Adenosine Triphosphatases↗

PubMed1995

A radical approach to enzyme catalysis.

Free radicals are generally perceived as highly reactive species which are harmful to biological systems. There are, however, a number of enzymes that use carbon-based radicals to catalyse a variety of important and unusual reactions. The most prominent example is ribonucleotide reductase, an enzyme which is crucial for the synthesis of DNA. In general, radicals are used to remove hydrogen from unreactive positions in the substrate, and in this way the substrate is activated to undergo chemical transformations that would otherwise be difficult to achieve. Several different mechanisms have evolved which allow enzymes to generate and maintain radicals in increasingly aerobic environments. An unexpected finding is the existence of stable protein-based radicals, residing on a variety of amino-acid side chains, which serve to link the radical-generating and catalytic sites and to store the radical between turnovers.

Catalysis↗

PubMed1994

Adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum. Overexpression in Escherichia coli, purification, and characterization of the recombinant enzyme.

The genes encoding both components, MutE and MutS, of adenosylcobalamin-dependent glutamate mutase from Clostridium tetanomorphum have been over-expressed in Escherichia coli. This has allowed MutE to be obtained in homogeneous form, free of inhibiting cobamides and traces of MutS. MutE binds MutS cooperatively, with a Hill coefficient of 1.3. The recombinant enzyme has an unchanged Km for L-glutamate, but a much higher specific activity than those previously reported for preparations from clostridia. The apparent Km for adenosylcobalamin was dependent upon the concentration of MutS and varied between 18 microM with equimolar concentrations of MutS and MutE and 5.8 microM with a 5-fold molar excess of MutS over MutE present in the assay. The dissociation constant for adenosylcobalamin was measured directly using equilibrium gel filtration. In the presence of equimolar amounts of MutE and MutS, the apparent Kd was 5.4 microM, but this decreased to 1.8 microM when MutS was present at a 5-fold molar excess. No binding of adenosylcobalamin to MutE was observed in the absence of MutS. This suggests that the (minimal) function for MutS, whose role in the reaction has been unclear until now, is to form part of the adenosylcobalamin-binding site. It seems likely that MutS is representative of a cobalamin-binding domain conserved across several cobalamin-dependent enzymes.

Amino Acid Isomerases↗

PubMed1993

Methylmalonyl-CoA mutase from Propionibacterium shermanii: characterization of the cobalamin-inhibited form and subunit-cofactor interactions studied by analytical ultracentrifugation.

A large proportion of adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermannii is isolated in an inactive form which contains a tightly bound cobalamin. Even when the enzyme was denatured in 5.0 M guanidine hydrochloride the cobalamin remained associated with the protein. However, when dithiothreitol was added to the denatured protein, the pink inhibitor was rapidly converted into a yellow-brown compound which could be removed by dialysis. Enzyme activity could be recovered after removal of the denaturant, although surprisingly this did not depend on prior treatment with dithiothreitol. The interaction between the protein and inhibitor was investigated by using analytical ultracentrifugation under denaturing conditions. The sedimentation coefficient s20,w was measured in various concentrations of guanidine hydrochloride. A complicated picture emerged in which at low denaturant concentrations subunit dissociation, partial unfolding and aggregation occur, whereas at high concentration the protein behaves as a monodisperse species. No major differences in sedimentation were observed between the enzyme-cobalamin complex and the cobalamin-free enzyme, suggesting that the inhibitor does not significantly stabilize higher-order structure within the protein.

Dithiothreitol↗

PubMed1993

Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum. Organization of the mut genes.

The gene encoding component E, the large subunit, of adenosylcobalamin (coenzyme B12)-dependent glutamate mutase from Clostridium tetanomorphum has been cloned and sequenced. The mutE gene encodes a protein of 485 amino acid residues, with M(r) 53,708. The mutE gene is situated some 1,400 bp downstream of the mutS gene, which encodes the small subunit of glutamate mutase. Between the two is an open reading frame encoding a protein of 462 amino acids, with M(r) 50,171, and of unknown function. All three genes appear to be transcribed as an operon and lie immediately upstream of the gene encoding beta-methylaspartase, the next enzyme in the pathway of glutamate fermentation. Local homology exists between mutE and a region of beta-methylaspartase which contains an active-site serine residue.

Amino Acid Isomerases↗

PubMed1992

Two isozymes of clavaminate synthase central to clavulanic acid formation: cloning and sequencing of both genes from Streptomyces clavuligerus.

Clavaminate synthase (CS) is an alpha-ketoglutarate-dependent oxygenase central to the biosynthesis of clavulanic acid, a potent inhibitor of beta-lactamases. CS catalyzes the oxidative cyclization/desaturation of proclavaminic acid to clavaminic acid in a two-step process involving the intermediacy of dihydroclavaminic acid [Salowe, S. P., Krol, W. J., Iwata-Reuyl, D., & Townsend, C. A. (1991) Biochemistry 30, 2281-2292]. During the purification of CS to homogeneity from Streptomyces clavuligerus [Salowe, S. P., Marsh, E. N., & Townsend, C. A. (1990) Biochemistry 29, 6499-6508], two forms of the enzyme capable of carrying out the complete reaction having very similar molecular weights and kinetic properties were isolated by Mono-Q chromatography. The gene for each has been cloned, sequenced, and found to be significantly homologous (87% identity). The two genes so isolated, cs1 and cs2, have open reading frames of 975 and 978 nucleotides, respectively, encoding proteins of M(r) 35,347 and 35,774. These genes are located in different loci of the genome separated by > 20 kbp. This separation is large for a natural product biosynthetic pathway in bacteria where gene duplication and limited divergence are typically observed to occur within narrower confines of a gene cluster. Sequence comparisons made between cs1/cs2 and other genes encoding iron-dependent proteins involved in penicillin and cephalosporin biosynthesis in the same organism show minimal homology. Further sequence alignments made to other non-heme iron oxygenases reveal unexpected dissimilarity within the alpha-ketoglutarate-dependent class itself. The limited data available suggests evolutionary convergence among these proteins.

Amino Acid Sequence↗

PubMed1992

Cloning and sequencing of glutamate mutase component S from Clostridium tetanomorphum. Homologies with other cobalamin-dependent enzymes.

The gene encoding component S, the small subunit, of glutamate mutase, an adenosylcobalamin (coenzyme B12)-dependent enzyme from Clostridium tetanomorphum has been cloned and its nucleotide sequence determined. The mutS gene encodes a protein of 137 amino acid residues, with M(r) 14,748. The deduced amino acid sequence showed homology with the C-terminal portion of adenosylcobalamin-dependent methylmalonyl-CoA mutase [1989, Biochem. J. 260, 345-352] and a region of cobalamin-dependent methionine synthase which has been shown to bind cobalamin [1989, J. Biol. Chem 264, 13888-13895].

5-Methyltetrahydrofolate-Homocysteine S-Methyltran↗

PubMed1990

Purification and characterization of clavaminate synthase from Streptomyces clavuligerus: an unusual oxidative enzyme in natural product biosynthesis.

A pivotal step in the biosynthetic pathway to the beta-lactamase inhibitor clavulanic acid is the conversion of proclavaminic acid to clavaminic acid in a reaction requiring Fe2+, alpha-ketoglutarate, and oxygen [Elson, S. W., Baggaley, K. H., Gillett, J., Holland, S., Nicholson, N. H., Sime, J. T., & Woroniecki, S. R. (1987) J. Chem. Soc., Chem. Commun., 1736-1738]. Clavaminate synthase, the enzyme that catalyzes this oxidative cyclization/desaturation, has been purified to homogeneity from clavulanic acid producing cells of Streptomyces clavuligerus (ATCC 27064). The enzyme behaved as a monomer during gel filtration and migrated with Mr 47,000 during denaturing gel electrophoresis. After ion-exchange FPLC two active forms of the protein were resolved that differed slightly in kinetic constants and apparent molecular weight. Kinetic comparisons with the four possible diastereomers of proclavaminate confirmed the absolute configuration of the substrate to be 2S,3R. The stoichiometry of the overall transformation was determined to be proclavaminate + 2(alpha-ketoglutarate) + 2O2----clavaminate + 2(succinate) + 2CO2 + 2H2O. In the absence of proclavaminate a slow decarboxylation of alpha-ketoglutarate to succinate and CO2 was observed in an uncoupled reaction which resulted in enzyme inactivation. Steady-state kinetic studies were undertaken for an initial description of the enzyme's catalytic cycle. The double-reciprocal plot with alpha-ketoglutarate as the variable substrate was linear; this supports the proposal that two stepwise oxidations of proclavaminate occur, each with the consumption of alpha-ketoglutarate and oxygen and the release of succinate, CO2, and H2O. The intersecting initial velocity plots obtained from pairwise variation of substrate concentrations were consistent with a sequential kinetic mechanism for the first oxidation. Similarities observed between clavaminate synthase and alpha-ketoglutarate-dependent dioxygenases argue for a common mechanism of oxygen activation. However, the nature of the interactions of the substrates in the active site of clavaminate synthase apparently redirects the conventional hydroxylase activity of dioxygenases to the construction of a strained bicyclic skeleton driven by the overall reduction of dioxygen.

Aza Compounds↗

PubMed1989

Methylmalonyl-CoA mutase from Propionibacterium shermanii. Evidence for the presence of two masked cysteine residues.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii contains no intramolecular disulphide bridges, but two of the six thiol groups in the heterodimer are only revealed after reduction of the denatured enzyme with dithiothreitol. The available evidence suggests that they are present in disulphide linkages to unknown thiols of low Mr. The two specifically masked cysteine residues are Cys-535 in the alpha-subunit and Cys-517 in the beta-subunit, which occupy exactly homologous positions in each chain.

Amino Acid Sequence↗

PubMed1989

Cloning and structural characterization of the genes coding for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii.

The structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been cloned, with the use of synthetic oligonucleotides as primary hybridization probes. The genes are closely linked and are transcribed in the same direction. Nucleotide sequence analysis of 4.5 kb of DNA encompassing both genes allowed us to infer the complete amino acid sequence of the two subunits: the beta-subunit is the product of the upstream gene, and consists of 638 amino acid residues (Mr 69465) and the alpha-subunit consists of 728 amino acid residues (Mr 80,147). There is a very close structural homology between the two subunits, reflecting the probable duplication of a common ancestral gene. A sequence present only in the alpha-subunit is significantly homologous to a portion of the sequence of the methylmalonyl-CoA-binding subunit of transcarboxylase from P. shermanii [Samols, Thornton, Murtif, Kumar, Haase & Wood (1988) J. Biol. Chem. 263, 6461-6464], and this homologous region may form part of the CoA ester-binding site in both enzymes.

Amino Acid Sequence↗

PubMed1989

Subunit interactions in Propionibacterium shermanii methylmalonyl-CoA mutase studied by analytical ultracentrifugation.

The effect of increasing ionic strength on adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii was studied by using analytical ultracentrifugation. Both sedimentation-velocity and low-speed sedimentation-equilibration measurements show that the enzyme dissociates progressively into its two dissimilar subunits with increasing ionic strength. Equilibrium between the alpha beta-dimer and the separated subunits is rapidly established under these conditions. Dissociation is accompanied by loss of enzymic activity, but the position of the equilibrium is unaffected by the presence of either substrate or adenosylcobalamin cofactor.

Acyl Coenzyme A↗