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The subunit structure of methylmalonyl-CoA mutase from Propionibacterium shermanii.

ABSTRACT: 5'-Deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase was purified to homogeneity from Propionibacterium shermanii by a simplified procedure. The native enzyme has an apparent Mr of 165,000, similar to the enzyme from other sources but larger than previously reported. It consists of two non-identical subunits, of Mr 79,000 and 67,000 respectively. The smaller subunit is apparently not a proteolytic fragment of the larger one. The final preparation usually contained some inactive mutase, bearing a tenaciously bound cobalamin species. This protein proved to be readily separable from apoenzyme by fast protein liquid chromatography on anion-exchange columns.

SUBMITTER: Francalanci F

PROVIDER: S-EPMC1146866 | biostudies-other | 1986 Jun

REPOSITORIES: biostudies-other

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

Project description: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.

| S-EPMC1138676 | biostudies-other

Adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii. Active holoenzyme produced from Escherichia coli.

Project description:The linked structural genes coding for both subunits of adenosylcobalamin-dependent methylmalonyl-CoA mutase from the Gram-positive bacterium Propionibacterium shermanii have been altered by site-directed mutagenesis and placed under the control of an inducible phage-T7-specific plasmid promoter in Escherichia coli. Conditions have been found under which both alpha- and beta-subunits are produced in soluble form, in near 1:1 ratio, and assemble to form apo-mutase totalling about 5% of the total cellular protein. Methylmalonyl-CoA mutase purified from these cells could be readily converted into the holoenzyme by addition of adenosylcobalamin. The active holoenzyme apparently crystallizes in the same space group as an inactive corrinoid-containing form of the enzyme obtained previously.

| S-EPMC1131574 | biostudies-other

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

Project description: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.

| S-EPMC1138674 | biostudies-other

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

Project description: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.

| S-EPMC1132309 | biostudies-other

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

Project description: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.

| S-EPMC1138675 | biostudies-other

Purification and characterization of methylmalonyl-CoA epimerase from Propionibacterium shermanii.

Project description:Methylmalonyl-CoA epimerase, which specifically interconverts the (2R)- and (2S)- epimers of methylmalonyl-CoA, was purified 95-fold from Propionibacterium shermanii by a new method that affords apparently homogeneous enzyme, in 80-100mg quantities, in yields representing about 40% of the activity in cell-free extracts. The specific activity of the purified enzyme, 10.1 mukat/mg, is much greater than previously reported. Native methylmalonyl-CoA epimerase has Mr about 33000, and apparently consists of two identical subunits. The purified enzyme is stable indefinitely when stored at -20 degrees C and pH 8.5, but contrary to previous reports it is not unusually acid-stable. The activity of methylmalonyl-CoA epimerase is increased by Co2+, and to a smaller extent by Ni2+, Mn2+ and Zn2+.

| S-EPMC1163141 | biostudies-other

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. The reaction of (2R)-methylmalonyl-CoA in tritiated water.

Project description:The reaction catalysed by methylmalonyl-CoA epimerase from Propionibacterium shermanii was studied in tritiated water, in the direction with (2R)-methylmalonyl-CoA as substrate, under 'irreversible' conditions. After partial reaction, even when most of the substrate had been converted into product (isolated as propionyl-CoA) essentially no solvent tritium appeared in residual (2R)-methylmalonyl-CoA. The product, however, did contain tritium, and the specific radioactivity of the (2S)-epimer was deduced to be 0.33 times that of the solvent. These results provide further support for the mechanism proposed for the epimerase-catalysed reaction in the accompanying paper [Leadlay & Fuller (1983) Biochem. J. 213, 635-642], in which two enzyme bases act respectively as proton donor and acceptor. The observed low discrimination against solvent tritium entering the product can be accounted for by a mechanism in which the release of product is slow, and the re-protonation step on the enzyme is reversible, without leading to isotopic exchange with the solvent.

| S-EPMC1152179 | biostudies-other

Proton transfer in methylmalonyl-CoA epimerase from Propionibacterium shermanii. Studies with specifically tritiated (2R)-methylmalonyl-CoA as substrate.

Project description:(2R)-Methyl[2-3H]malonyl-CoA was used as the substrate for methylmalonyl-CoA epimerase from Propionibacterium shermanii, under conditions where the (2S)-methylmalonyl-CoA product was removed enzymically as fast as it was formed, and the fate of the label was monitored at different extents of reaction. Very little, if any, tritium is found attached to the C-2 position in the (2S)-epimer product (isolated as propionyl-CoA). Evidently, the hydrogen atom of the new C-H bond in the product is essentially solvent-derived. The rate of tritium release into the solvent is lower than the rate of product formation, and shows a primary kinetic tritium-isotope effect on kcat./Km of 2.3 +/- 0.1. The specific radioactivity of the remaining substrate rises slowly during the epimerase-catalysed reaction, and this provides an independent estimate of the primary kinetic tritium-isotope effect on kcat./Km of 1.6 +/- 0.5. These results, taken together, indicate that the mechanistic pathway of the epimerase-catalysed reaction resembles that established for proline racemase [Cardinale & Abeles, (1968) Biochemistry 7, 3970-3978], in which two enzyme bases are involved in catalysis. One base removes the proton from the substrate, the second provides the new proton, and there is no fast isotopic exchange between enzyme-bound intermediates and solvent protons.

| S-EPMC1152178 | biostudies-other

Primary structure and activity of mouse methylmalonyl-CoA mutase.

Project description:Methylmalonyl-CoA mutase (MCM) is an adenosylcobalamin-dependent enzyme that catalyses isomerization between methylmalonyl-CoA and succinyl-CoA (3-carboxypropionyl-CoA). Genetic deficiency of this enzyme in man causes an often fatal disorder of organic acid metabolism termed mut methylmalonicacidaemia. We report cloning of a mouse MCM cDNA and the characterization of its primary structure and biological function. Mouse MCM in fibroblasts and crude liver extracts exhibits activity and reaction kinetics similar to those of the human enzyme. The predicted amino acid sequence of mouse MCM exhibits 94% identity with its human homologue and considerable identity with a prokaryotic MCM. Transfection of the mouse cDNA into cultured cells constitutes an active apoenzyme and can complement genetic deficiency of the apoenzyme in cells from patients with mut methylmalonicacidaemia. These results establish that mouse MCM is homologous to human MCM in structure and function and provides a basis for using the mouse as a model for studying this enzyme and its deficiency state.

| S-EPMC1149575 | biostudies-other

Itaconyl-CoA forms a stable biradical in methylmalonyl-CoA mutase and derails its activity and repair.

Project description:Itaconate is an immunometabolite with both anti-inflammatory and bactericidal effects. Its coenzyme A (CoA) derivative, itaconyl-CoA, inhibits B12-dependent methylmalonyl-CoA mutase (MCM) by an unknown mechanism. We demonstrate that itaconyl-CoA is a suicide inactivator of human and Mycobacterium tuberculosis MCM, which forms a markedly air-stable biradical adduct with the 5'-deoxyadenosyl moiety of the B12 coenzyme. Termination of the catalytic cycle in this way impairs communication between MCM and its auxiliary repair proteins. Crystallography and spectroscopy of the inhibited enzyme are consistent with a metal-centered cobalt radical ~6 angstroms away from the tertiary carbon-centered radical and suggest a means of controlling radical trajectories during MCM catalysis. Mycobacterial MCM thus joins enzymes in the glyoxylate shunt and the methylcitrate cycle as targets of itaconate in pathogen propionate metabolism.

| S-EPMC7070230 | biostudies-literature

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