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PubMed1984

The stereochemistry of the formation of the methyl group in the glutamate mutase-catalysed reaction in Clostridium tetanomorphum.

The adenosylcobalamin-dependent enzyme glutamate mutase from Clostridium tetanomorphum catalyses the reversible rearrangement of (2S)-glutamate to (2S,3S)-3- methylaspartate . In this conversion 6 carbon centers are involved. The stereochemistry of 4 has been elucidated whereas the formation of the methyl group from the methylene group remains to be established. To solve this problem, (2S,3R)- and (2S,3S)-[3,3-2H1,3H]glutamates were prepared via the 2-oxo[3,3-2H2 or 3H] glutarates by incubation with isocitrate dehydrogenase in deuterium oxide or tritiated water. The labelled glutamates were fermented with growing cells of C. tetanomorphum to butyrate and acetate. Butyrate was further degraded to acetate in which methyl group over 90% of the tritium of the starting glutamate was retained. The chirality of the acetates was determined with malate synthase and fumarase. In both samples complete racemisation was found. This result confirms the rule that racemisation occurs in all adenosylcobalamin-dependent rearrangements in which methyl groups are formed. A methylene radical as intermediate could explain these observations. In a control experiment inversion of configuration in the formation of the methine group of (2S,3S)-3-methylaspartate from the methylene group of (2S)-glutamate was confirmed. Glutamates stereospecifically labelled at C-4 were synthesized from chiral acetates via citrate.

Amino Acid Isomerases↗
PubMed1986

Inhibition of fungal and mammalian sterol biosynthesis by 2-aza-2,3-dihydrosqualene.

2-Aza-2,3-dihydrosqualene (I) and a quaternary ammonium derivative (II) inhibited ergosterol biosynthesis in cells and cell-free extracts of the pathogenic yeast Candida albicans as measured by incorporation of radiolabelled precursors. The compounds inhibited squalene epoxidase and 2,3-oxidosqualene cyclase to varying degrees in microsomes from C. albicans and from rat liver. The rat liver epoxidase was 50% inhibited by I at 2.4 microM. In C. albicans cells, but not in cell-free extracts, I also inhibited lanosterol demethylation and led to accumulation of an unidentified polar product.

Animals↗
PubMed1987

A spectrophotometric assay for deacetoxycephalosporin C synthase.

A continuous direct spectrophotometric assay for deacetoxycephalosporin C synthase was developed, based on the absorption at 260 nm characteristic of the dihydrothiazine moiety of cephalosporins. Km values of 0.18 mM for penicillin N and 0.16 mM for alpha-ketoglutarate were determined. A coupled assay using succinate thiokinase, pyruvate kinase and lactate dehydrogenase showed that succinate was a product of both deacetoxycephalosporin C synthase and hydroxylase reactions. The expandase reaction exhibited a 1:1.06 stoichiometry for deacetoxycephalosporin C and succinate.

Acremonium↗
PubMed1991

Coenzyme B12-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Protection by the substrate from inactivation by light.

Partially purified 2-methyleneglutarate mutase from Clostridium barkeri was separated from 3-methylitaconate delta-isomerase by treatment with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS) followed by FPLC on the anion exchange column Mono Q in the presence of the detergent. When purified in the dark, the active mutase contained a corrinoid, most probably coenzyme B12. The enzyme was inactivated by light at the same wavelength (lambda less than 620 nm) and rate as free coenzyme B12. The rate was not influenced by oxygen or by temperature (0-37 degrees C). Reactivation of up to 50% of the original activity was achieved by incubation with coenzyme B12 and dithiothreitol. The substrates 2-methyleneglutarate (up to 40 mM) or (R)-3-methylitaconate specifically protected the enzyme from inactivation by visible light. This effect was enhanced 3-fold by raising the temperature from 0 degrees C to 37 degrees C. The data indicate that during catalysis, the Co-C bond of the coenzyme is cleaved and cannot be affected any more by light.

Carbon-Carbon Double Bond Isomerases↗
PubMed1991

Direct identification and quantification of the cofactor in glutamate semialdehyde aminotransferase from pea leaves.

Glutamate semialdehyde aminotransferase, a key enzyme in the synthetic pathway leading to chlorophyll was purified from pea (Pisum sativum) leaves. Although the preparation contained a single contaminant the enzyme could be unambiguously identified as a dimer of subunit molar mass 45 kDa having an absorption spectrum consistent with the presence of pyridoxamine phosphate as cofactor. The cofactor was released by treatment with strong phosphate at low pH and was identified and quantified fluorimetrically. The specific activity of the enzyme (1.4 mumol.min-1.mg-1; 23 nkatal.mg-1) is very much higher than previously reported.

Chromatography, Gel↗
PubMed1992

Identification of a paramagnetic species as an early intermediate in the coenzyme B12-dependent glutamate mutase reaction. A cob(II)amide?

Highly active and cobamide-free glutamate mutase was obtained from Clostridium cochlearium by a modification of the original purification procedure. After incubation of the enzyme with dithiothreitol, adenosylcobalamin (coenzyme B12) and the substrate (S)-glutamate, a paramagnetic species was observed by EPR-spectroscopy. The signal was maximal within 15 ms after mixing with glutamate. Different signals were detected after incubating the system with the competitive inhibitors (2S,4S)-4-fluoroglutamate or 2-methyleneglutarate instead of the substrate. The former developed with an at least 100-fold lower rate then the signal with glutamate. All three signals are probably due to low-spin cob(II)amide species with an extraordinary low gxy value as compared with cob(II)alamin.

Amino Acid Isomerases↗
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↗
PubMed1993

Cloning and sequencing of glutamate mutase component E from Clostridium tetanomorphum.

The nucleotide sequence of the large subunit E of glutamate mutase of Clostridium tetanomorphum was determined. The protein consists of 483 amino acids and is not made in a precursor form, thus excluding the possibility of subunit E being a pyruvoyl enzyme. It shows no homology to any other protein in the database, and while binding coenzyme B12, a conspicuous B12 binding motif, shared amongst other proteins, is not detectable at the sequence level.

Amino Acid Isomerases↗
PubMed1993

Differential inhibition of fungal oxidosqualene cyclase by 6E and 6Z isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene.

Inhibitory properties of 6E (compound 1) and 6Z (compound 2) isomers of 2,3-epoxy-10-aza-10,11-dihydrosqualene against oxidosqualene-lanosterol cyclase were assayed on microsomes and whole cells of Saccharomyces cerevisiae and Candida albicans. Only the 6E isomer (compound 1), bearing a correct substrate-like configuration, strongly inhibited the enzyme both in microsomes and cell cultures. The difference between compounds 1 and 2 (which had an unfavorable geometry) was especially evident when measuring [14C]acetate incorporation into non-saponifiable lipids extracted from treated cells. While isomer Z was totally ineffective at up to 30 microM, in cells treated with 5 microM isomer E, labelled oxidosqualene, the level of which was negligible in the control, rose to over 60% of the non-saponifiable lipids.

Candida albicans↗
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↗
PubMed1995

Coordination of a histidine residue of the protein-component S to the cobalt atom in coenzyme B12-dependent glutamate mutase from Clostridium cochlearium.

Electron paramagnetic resonance (EPR) spectroscopy of glutamate mutase from Clostridium cochlearium was performed in order to test the idea, that a histidine residue of component S replaces the dimethylbenzimidazole ligand of the Co-atom during binding of coenzyme B12 to the enzyme. The shapes and the superhyperfine splitting of the gz-lines of the Co(II) EPR spectra were used as indicators of the interaction of the axial base nitrogen with the Co-atom. A mixture of completely 15N-labelled component S, unlabelled component E, coenzyme B12 and glutamate gave slightly sharper gz-lines than that with unlabelled component S. A more dramatic change was observed in the Co(II) spectrum of the inactivated enzyme containing tightly bound cob(II)alamin, in which unlabelled component S caused a threefold superhyperfine-splitting of the gz-line, whereas the 15N-labelled protein only caused a twofold splitting, as expected for a direct interaction of a nitrogen of the enzyme with the Co-atom. By using a sample of 15N-labelled component S, in which only the histidines were 14N-labelled, the EPR spectra showed no difference to those with unlabelled component S. The experiments indeed demonstrate a replacement of the dimethylbenzimidazole ligand in coenzyme B12 by a histidine when bound to glutamate mutase. The most likely candidate is H16, which is conserved among the carbon skeleton rearranging mutases and methionine synthase.

Amino Acid Isomerases↗
PubMed1995

beta-Glucosylarginine: a new glucose-protein bond in a self-glucosylating protein from sweet corn.

In the search for a protein primer for starch synthesis, an autocatalytic self-glucosylating protein has been isolated from sweet corn. Several tryptic peptides were obtained from the [14C]glucosylated protein and were sequenced, corresponding to over 40% of the estimated total sequence (molecular mass 42 kDa). There is no homology with the amino acid sequence of the autocatalytic glycogen primer, glycogenin, nor in respect of the nature of the union between the autocatalytically added glucose and the protein, which, in the case of the corn protein, now named amylogenin, is a novel glucose-protein bond, a single beta-glucose residue joined to an arginine residue.

Amino Acid Sequence↗
PubMed1996

An isochorismate hydroxymutase isogene in Escherichia coli.

The pivotal step in enterobactin and menaquinone biosynthesis is the conversion of chorismate to isochorismate. Circumstantial evidence pointed to Escherichia coli isochorismate hydroxymutase isogenes being responsible for this conversion. While the gene involved in enterobactin synthesis (entC) was known, the corresponding gene for menaquinone biosynthesis (menF) was not but has now been identified and sequenced. The amino acid sequence of MenF is 23.5% identical and 57.8% similar to that of EntC.

Amino Acid Sequence↗
PubMed1991

Purification of the coenzyme B12-containing 2-methyleneglutarate mutase from Clostridium barkeri by high-performance liquid chromatography.

Two methods are described by which the enzymes 2-methyleneglutarate mutase and 3-methylitaconate delta-isomerase from Clostridium barkeri have been separated by high-performance liquid chromatography on a much larger scale than reported previously. First, the mutase eluted before the delta-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulphonate (CHAPS) followed by high-performance anion-exchange chromatography on Mono Q in the presence of the same detergent. Second, an even better separation, although with a lower yield of mutase, was obtained by hydrophobic interaction chromatography on phenyl-Sepharose HiLoad, whereby the enzymes were eluted in the reverse order. Final high-performance anion-exchange chromatography of the latter preparation on Mono Q at pH 8 gave highly purified 2-methyleneglutarate mutase (greater than 95% purity) which had a pink-orange colour (lambda max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B12 (adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).

Amino Acid Sequence↗