2-methyleneglutarate Mutase Research Articles

Glutamate mutase and 2-methyleneglutarate mutase.

Glutamate mutase converts ( S )-glutamate to ( S , S )-3-methylaspartate, whereas 2-methyleneglutarate mutase isomerizes 2-methyleneglutarate to ( R )-3-methylitaconate. Both enzymes occur in amino acid fermenting clostridia, require coenzyme B 12 (adenosylcobalamin) and catalyze a reversible, radical carbon skeleton rearrangement. The enzymes are assayed using the consecutive enzymes in their fermentation pathways: methylaspartase and methylitaconate isomerase, which produce the higher absorbing mesaconate and 2,3-dimethylmaleate, respectively. Although the quaternary structures of glutamate mutase and 2-methyleneglutarate mutase are different, the binding mode of coenzyme B 12 and their catalytic mechanism appear to be very similar. The crystal structure of glutamate mutase and EPR spectroscopy of both enzymes during catalysis revealed a distance of 6.6 Å between the C-H bond of the substrate to be cleaved and the Co-atom of cob(II)alamin. Methods are described for facile production of both enzymes in Escherichia coli and purification.

Molecular and functional analysis of nicotinate catabolism in Eubacterium barkeri

The anaerobic soil bacterium Eubacterium barkeri catabolizes nicotinate to pyruvate and propionate via a unique fermentation. A full molecular characterization of nicotinate fermentation in this organism was accomplished by the following results: (i) A 23.2-kb DNA segment with a gene cluster encoding all nine enzymes was cloned and sequenced, (ii) two chiral intermediates were discovered, and (iii) three enzymes were found, completing the hitherto unknown part of the pathway. Nicotinate dehydrogenase, a (nonselenocysteine) selenium-containing four-subunit enzyme, is encoded by ndhF (FAD subunit), ndhS (2 x [2Fe-2S] subunit), and by the ndhL/ndhM genes. In contrast to all enzymes of the xanthine dehydrogenase family, the latter two encode a two-subunit molybdopterin protein. The 6-hydroxynicotinate reductase, catalyzing reduction of 6-hydroxynicotinate to 1,4,5,6-tetrahydro-6-oxonicotinate, was purified and shown to contain a covalently bound flavin cofactor, one [2Fe-2S](2+/1+) and two [4Fe-4S](2+/1+) clusters. Enamidase, a bifunctional Fe-Zn enzyme belonging to the amidohydrolase family, mediates hydrolysis of 1,4,5,6-tetrahydro-6-oxonicotinate to ammonia and (S)-2-formylglutarate. NADH-dependent reduction of the latter to (S)-2-(hydroxymethyl)glutarate is catalyzed by a member of the 3-hydroxyisobutyrate/phosphogluconate dehydrogenase family. A [4Fe-4S]-containing serine dehydratase-like enzyme is predicted to form 2-methyleneglutarate. After the action of the coenzyme B(12)-dependent 2-methyleneglutarate mutase and 3-methylitaconate isomerase, an aconitase and isocitrate lyase family pair of enzymes, (2R,3S)-dimethylmalate dehydratase and lyase, completes the pathway. Genes corresponding to the first three enzymes of the E. barkeri nicotinate catabolism were identified in nine Proteobacteria.

Searching for Intermediates in the Carbon Skeleton Rearrangement of 2-Methyleneglutarate to (R)-3-Methylitaconate Catalyzed by Coenzyme B12-Dependent 2-Methyleneglutarate Mutase from Eubacterium barkeri

Coenzyme B(12)-dependent 2-methyleneglutarate mutase from the strict anaerobe Eubacterium barkeri catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Proteins with mutations in the highly conserved coenzyme binding-motif DXH(X)(2)G(X)(41)GG (D483N and H485Q) exhibited decreased substrate turnover by 2000-fold and >4000-fold, respectively. These findings are consistent with the notion of H485 hydrogen-bonded to D483 being the lower axial ligand of adenosylcobalamin in 2-methyleneglutarate mutase. (E)- and (Z)-2-methylpent-2-enedioate and all four stereoisomers of 1-methylcyclopropane-1,2-dicarboxylate were synthesized and tested, along with acrylate, with respect to their inhibitory potential. Acrylate and the 2-methylpent-2-enedioates were noninhibitory. Among the 1-methylcyclopropane-1,2-dicarboxylates only the (1R,2R)-isomer displayed weak inhibition (noncompetitive, K(i) = 13 mM). Short incubation (5 min) of 2-methyleneglutarate mutase with 2-methyleneglutarate under anaerobic conditions generated an electron paramagnetic resonance (EPR) signal (g(xy) approximately 2.1; g(z) approximately 2.0), which by analogy with the findings on glutamate mutase from Clostridium cochlearium [Biochemistry, 1998, 37, 4105-4113] was assigned to cob(II)alamin coupled to a carbon-centered radical. At longer incubation times (>1 h), inactivation of the mutase occurred concomitant with the formation of oxygen-insensitive cob(II)alamin (g(xy) approximately 2.25; g(z) approximately 2.0). In order to identify the carbon-centered radical, various (13)C- and one (2)H-labeled substrate/product molecules were synthesized. Broadening (0.5 mT) of the EPR signal around g = 2.1 was observed only when C2 and/or C4 of 2-methyleneglutarate was labeled. No effect on the EPR signals was seen when [5'-(13)C]adenosylcobalamin was used as coenzyme. The inhibition and EPR data are discussed in the context of the addition-elimination and fragmentation-recombination mechanisms proposed for 2-methyleneglutarate mutase.

Rotation of the exo-methylene group of (R)-3-methylitaconate catalyzed by coenzyme B(12)-dependent 2-methyleneglutarate mutase from Eubacterium barkeri.

2-Methyleneglutarate mutase from the anaerobe Eubacterium (Clostridium) barkeri is an adenosylcobalamin (coenzyme B(12))-dependent enzyme that catalyzes the equilibration of 2-methyleneglutarate with (R)-3-methylitaconate. Two possibilities for the mechanism of the carbon skeleton rearrangement of the substrate-derived radical to the product-related radical are considered. In both mechanisms an acrylate group migrates from C-3 of 2-methyleneglutarate to C-4. In the "addition-elimination" mechanism this 1,2-shift occurs via an intermediate, a 1-methylenecyclopropane-1,2-dicarboxylate radical, in which the migrating acrylate is simultaneously attached to both C-3 and C-4. In the "fragmentation-recombination" mechanism the migrating group, a 2-acrylyl radical, becomes detached from C-3 before it starts bonding to C-4. In an attempt to distinguish between these two possibilities we have investigated the action of 2-methyleneglutarate mutase on the stereospecifically deuterated substrates (Z)-3-methyl[2'-(2)H(1)]itaconate and (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate. The enzyme catalyzes the equilibration of both compounds with their corresponding E-isomers and with a 1:1 mixture of the corresponding (E)- and (Z)-2-methylene[2'-(2)H(1)]glutarates, as shown by monitoring of the reactions with (1)H and (2)H NMR. In the initial phase of the enzyme-catalyzed equilibration a significant excess (8-11%) of (E)-3-methyl[2'-(2)H(1)]itaconate over its equilibrium value was observed ("E-overshoot"). The E-overshoot was only 3-4% with (Z)-3-[2'-(2)H(1),methyl-(2)H(3)]methylitaconate because the presence of the deuterated methyl group raises the energy barrier from 3-methylitaconate to the corresponding radical. The overshoot is explained by postulating that the migrating acrylate group has to overcome an additional energy barrier from the state leading back to the substrate-derived radical to the state leading forward to the product-related radical. It is concluded that the fragmentation-recombination mechanism can provide an explanation for the results in terms of an additional energy barrier, despite the higher calculated activation energy for this pathway.

A novel reaction between adenosylcobalamin and 2-methyleneglutarate catalyzed by glutamate mutase.

We describe a novel reaction of adenosylcobalamin that occurs when adenosylcobalamin-dependent glutamate mutase is reacted with the substrate analogue 2-methyleneglutarate. Although 2-methyleneglutarate is a substrate for the closely related adenosylcobalamin-dependent enzyme 2-methyleneglutarate mutase, it reacts with glutamate mutase to cause time-dependent inhibition of the enzyme. Binding of 2-methyleneglutarate to glutamate mutase initiates homolysis of adenosylcobalamin. However, instead of the adenosyl radical proceeding to abstract a hydrogen from the substrate, which is the next step in all adenosylcobalamin-dependent enzymes, the adenosyl radical undergoes addition to the exo-methylene group to generate a tertiary radical at C-2 of methyleneglutarate. This radical has been characterized by EPR spectroscopy with regiospecifically (13)C-labeled methyleneglutarates. Irreversible inhibition of the enzyme appears to be a complicated process, and the detailed chemical and kinetic mechanism remains to be elucidated. The kinetics of this process suggest that cob(II)alamin may reduce the enzyme-bound organic radical so that stable adducts between the adenosyl moiety of the coenzyme and 2-methyleneglutarate are formed.

Elucidation of the coenzyme binding mode of further B 12-dependent enzymes using a base-off analogue of coenzyme B 12

(Coβ-5′-Deoxyadenosin-5′-yl)-( p-cresyl)cobamide (Ado-PCC), a base-off analogue of coenzyme B 12 (Ado-Cbl), was used to elucidate the coenzyme B 12 binding mode of glutamate mutase, 2-methyleneglutarate mutase and ethanolamine ammonia–lyase (EAL). Ado-PCC functions as excellent coenzyme for the carbon skeleton rearrangements with apparent K m values of 200 and 10 nM for glutamate and 2-methyleneglutarate mutases, respectively. The corresponding values for Ado-Cbl are 60 and 54 nM, respectively. In contrast, Ado-PCC showed no coenzyme activity with EAL but was a competitive inhibitor with respect to Ado-Cbl. The K i value for Ado-PCC was 26 nM, the apparent K m value for Ado-Cbl was 30 nM. These results are in agreement with the notion that in glutamate and 2-methyleneglutarate mutases, a conserved histidine residue of the protein is coordinated to the cobalt atom of coenzyme B 12, whereas in EAL the dimethylbenzimidazole residue of the coenzyme itself serves as ligand.

EXAFS Data Indicate a “Normal” Axial Cobalt−Nitrogen Bond of the Organo-B12 Cofactor in the Two Coenzyme B12-Dependent Enzymes Glutamate Mutase and 2-Methyleneglutarate Mutase

A key step in the catalytic cycle of coenzyme B12-dependent enzymes is the homolysis of the cofactor's organometallic bond, leading to the formation of a 5‘-deoxyadenosyl radical. For the adenosylcobalamin-dependent enzyme methylmalonyl CoA mutase (MCM), it has been suggested that this step is mediated by a protein-induced lengthening of the cofactor's axial cobalt−nitrogen bond, in trans position to the scissile organometallic bond. In fact, such a lengthening was first observed in the crystal structure of MCM (Mancia et al. Structure 1996, 4, 339−350) and was later confirmed by an analysis of EXAFS data on the same protein in frozen solution (Scheuring et al. J. Am. Chem. Soc. 1997, 119, 12192−12200). Here, we report the results of an EXAFS study on the related coenzyme B12-dependent enzymes glutamate mutase from Clostridium cochlearium and 2-methyleneglutarate mutase from Clostridium barkeri. Both apoenzymes were overproduced from E. coli and reconstituted with methylcobalamin (MeCbl) to yield inactive...

On the Mechanism of Action of Vitamin B12: Theoretical Studies of the 2-Methyleneglutarate Mutase Catalyzed Rearrangement

Ab initio molecular orbital theory is used to investigate the coenzyme-B12-dependent rearrangement of 2-methyleneglutarate to (R)-3-methylitaconate catalyzed by 2-methyleneglutarate mutase. We use ...

Rotation of the exo-Methylene Group of 2-Methyleneglutarate Catalyzed by Coenzyme B12-Dependent 2-Methyleneglutarate Mutase from Clostridium barkeri

ADVERTISEMENT RETURN TO ISSUEPREVCommunicationNEXTRotation of the exo-Methylene Group of 2-Methyleneglutarate Catalyzed by Coenzyme B12-Dependent 2-Methyleneglutarate Mutase from Clostridium barkeriChristopher H. Edwards, Bernard T. Golding, Friedrich Kroll, Birgitta Beatrix, Gerd Bröker, and Wolfgang BuckelView Author Information Department of Chemistry, Bedson Building The University of Newcastle upon Tyne, Newcastle upon Tyne NE1 7RU, U.K. Laboratorium für Mikrobiologie, Fachbereich Biologie Philipps-Universität, D-35032 Marburg, Germany Cite this: J. Am. Chem. Soc. 1996, 118, 17, 4192–4193Publication Date (Web):May 1, 1996Publication History Received8 November 1995Published online1 May 1996Published inissue 1 January 1996https://doi.org/10.1021/ja9537641Copyright © 1996 American Chemical SocietyRIGHTS & PERMISSIONSArticle Views87Altmetric-Citations11LEARN ABOUT THESE METRICSArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. These metrics are regularly updated to reflect usage leading up to the last few days.Citations are the number of other articles citing this article, calculated by Crossref and updated daily. Find more information about Crossref citation counts.The Altmetric Attention Score is a quantitative measure of the attention that a research article has received online. Clicking on the donut icon will load a page at altmetric.com with additional details about the score and the social media presence for the given article. Find more information on the Altmetric Attention Score and how the score is calculated. Share Add toView InAdd Full Text with ReferenceAdd Description ExportRISCitationCitation and abstractCitation and referencesMore Options Share onFacebookTwitterWechatLinked InReddit Read OnlinePDF (149 KB) Get e-AlertsSupporting Info (2)»Supporting Information Supporting Information SUBJECTS:Carbene compounds,Hydrogen isotopes,Organic compounds,Peptides and proteins,Resonance structures Get e-Alerts

Glutamate and 2-methyleneglutarate mutase: from microbial curiosities to paradigms for coenzyme B12-dependent enzymes

Glutamate and 2-methyleneglutarate mutase: from microbial curiosities to paradigms for coenzyme B12-dependent enzymes

On the Role of two Different Cobalt(II) Species in Coenzyme B12-Dependent 2-Methyleneglutarate Mutase fromClostridium barkeri

Purified 2-methyleneglutarate mutase from Clostridium barkeri contains adenosylcobalamin (coenzyme B12) and varying amounts of oxygen-stable cob(II)alamin. The content of the latter was estimated by EPR spectroscopy at 6-11% of the total cobalamin (2-4 mol/mol enzyme). Tryptic digestion of the enzyme liberated the prosthetic groups, cob(II)alamin being oxidized by air to aquocobalamin. HPLC analysis of the released cobamides from several preparations revealed > 90% adenosylcobalamin and < 10% aquocobalamin. Treatment of active 2-methyleneglutarate mutase with 8M urea followed by gelfiltration yielded an inactive enzyme from which 50% of the adenosylcobalamin and up to 70% of the cob(II)alamin was removed. Addition of adenosylcobalamin to the urea-treated enzyme resulted in complete reactivation, but the content of cob(II)alamin was not increased. These data suggest that the oxygen-stable cob(II)alamin is not involved in catalysis. In the presence of the competitive inhibitor itaconate (methylenesuccinate, Ki = 0.7mM), an alteration of the UV/visible spectrum at 470 nm as well as a new line in the EPR spectrum of the enzyme (around g = 2.1) was observed. The results indicate the formation of an unusual, oxygen sensitive Co(II) species during catalysis. The EPR signal of the oxygen-stable cob(II)alamin (gx,y = 2.24) remained unchanged under those conditions.

Adenosylcobalamin and cob(II)alamin as prosthetic groups of 2-methyleneglutarate mutase from Clostridium barkeri.

The ultraviolet/visible spectrum of the pure pink-orange 2-methyleneglutarate mutase from Clostridium barkeri between 300-600 nm showed the presence of cobalamins; notably the peaks at 470 and 528 nm were indicative of oxygen-stable cob(II)alamin and adenosylcobalamin (coenzyme B12), respectively. Using the absorption coefficients of the isosbestic points at 340, 393 and 489 nm, the total cobalamin content was estimated as 3.7 +/- 0.3 mol/mol tetrameric enzyme (m = 300 kDa). Denaturation with 8 M urea in the presence of 2 mM dithiothreitol followed by gel chromatography and renaturation afforded an inactive enzyme which contained 40-50% of the initially bound cobalamin. This preparation could be reactivated to 95-100% by addition of adenosylcobalamin. The cobalamins were removed to 85% from the mutase by denaturation with 8 M urea in the presence of 1 M cyanide (pH 12) with irreversible loss of activity. 2-Methyleneglutarate mutase was inactivated by incubation with aquo-, cyano- or methylcobalamin; up to 50% of the activity was recovered by addition of adenosylcobalamin. Upon incubation of the mutase with [5'-3H]adenosylcobalamin about 30% of the total cobalamin was exchanged by the tritium-labelled cofactor without loss of activity. During aerobic catalysis the enzyme became sensitive towards oxygen which was accompanied by loss of activity and formation of aquocobalamin from adenosylcobalamin. EPR spectroscopy demonstrated the presence of 0.8 mol base-on cob(II)alamin/mol enzyme. Upon addition of 2-methyleneglutarate a second EPR signal of about equal intensity at g = 2.13 arose. The question of whether the oxygen-stable cob(II)alamin participates in catalysis or its complex with the enzyme represents an inactive form is currently under investigation.

Purification of the coenzyme B 12-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 Δ -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 Δ-isomerase after incubation with the mild detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulphonate (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 (> 95% purity) which had a pink-orange colour (λ max 280, 375, 470 and 532 nm). The enzyme was active in the absence of coenzyme B 12(adenosylcobalamin) and contained 2.1 mol of this coenzyme per homotetramer (molecular mass, m = 300 kilodalton).

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.

An immunological study of corrinoid proteins from bacteria revealed homologous antigenic determinants of a soluble corrinoid-dependent methyltransferase and corrinoid-containing membrane proteins from Methanobacterium species

The hypothesis of common epitopes in corrinoid-dependent enzymes was tested by a monospecific polyclonal antiserum against the 33 kDa corrinoid-containing membrane protein from Methanobacterium thermoautotrophicum Marburg. Cross-reaction was detected with the 33 kDa and the 31 kDa subunits of the corrinoid-containing enriched 5-methyl-H4MPT: 5-hydroxybenzimidazolyl cobamide methyltransferase from the cytoplasmic fraction and a 33 kDa protein from the membrane fraction of Methanobacterium thermoauto-trophicum ΔH. This indicates that both proteins have similar antigenic determinants and that they may have similar function as methyltransfer proteins. Also a soluble 20 kDa protein of yet unknown function from Clostridium barkeri cross-reacted with the antiserum. No cross-reactions were observed with the purified corrinoid-containing 2-methyleneglutarate mutase from C. barkeri, the corrinoid/iron-sulfur protein from C. thermoaceticum, the carbon monoxide dehydrogenases from C. thermoaceticum and Methanothrix soehngenii, and the corrinoid-binding protein intrinsic factor from porcine gastric mucosa. Also cell extracts from the corrinoid-rich bacteria Sporomusa ovata, Methanolobus tindarius, Chloroflexus aurantiacus, Propionibacterium shermanii, the membrane fraction and the cytoplasmic fraction of Methanococcus voltae or extracts from human liver, contained no antibody combining sites others than with the preimmunological serum. These findings indicate, that many corrinoid-containing proteins from bacteria have no common antigenic determinants.

Assay and purification of the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri.

A continuous spectrophotometric assay was developed for the adenosylcobalamin-dependent 2-methyleneglutarate mutase from Clostridium barkeri. Thereby the product (R)-3-methylitaconate is converted by the delta-isomerase from the same organism to 2,3-dimethylmaleate which absorbs at 240 nm, much higher than both parent compounds (delta epsilon = 3.7 mM-1.cm-1). In addition a discontinuous assay using the facile formation of 2,3-dimethylmaleic anhydride in aqueous solution at pH 0-1 (delta epsilon = 4.0 mM-1.cm-1 at 256 nm) was established. The mutase and the isomerase were purified together by chromatography on quaternary-amine-Sepharose (Q-Sepharose) and on cyanocobalamin-agarose. The enzymes were separated and obtained in homogenous forms by preparative PAGE in non-denaturing buffer. Both enzymes appear to be homotetramers with subunits of 70 kDa (mutase) and 50 kDa (isomerase). The equilibrium constants for both reactions were determined at I = 0.1 M and 25 degrees C: K1, app = [(R)-3-methylitaconate].[2-methyleneglutarate]-1 = 0.26 +/- 0.04, K2,app = [2,3-dimethylmaleate].[(R)-3-methylitaconate]-1 = 7.40 +/- 0.21.

On the steric course of the adenosylcobalamin-dependent 2-methyleneglutarate mutase reaction in Clostridium barkeri.

The enzymatically active enantiomer of 3-methylitaconate in Clostridium barkeri has (R)-configuration. This was checked by fermentation of the racemate and reisolation of the (S)-enantiomer. In addition (R)-3-methylitaconate was synthesized by enzymatic isomerisation of 2,3-dimethylmaleate which was protonated at the Si-face. 2-Methylene[2-2H1]glutarate was synthesized via (R)-3-methyl[3-2H1]itaconate by brief incubation of 2,3-dimethylmaleate with a cell-free extract of Clostridium barkeri in 2H2O. The predominantly monodeuterated compound was oxidized to (S)-[2-2H1]succinate as analysed by circular dichroism. The results demonstrate that 2-methyleneglutarate mutase catalyses the reversible migration of an acryloyl residue from the alpha-carbon to the beta-carbon of propionate with inversion of configuration at the alpha-carbon.