Characterization of methylmalonyl-CoA mutase involved in the propionate photoassimilation of Euglena gracilis Z.

Significant accumulation of the methylmalonyl-CoA mutase apoenzyme was observed in the photosynthetic flagellate Euglena gracilis Z at the end of the logarithmic growth phase. The apoenzyme was converted to a holoenzyme by incubation for 4 h at 4 degrees C with 10 microM 5'-deoxyadenosylcobalamin, and then, the holoenzyme was purified to homogeneity and characterized. The apparent molecular mass of the enzyme was calculated to be 149.0 kDa +/- 5.0 kDa using Superdex 200 gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme yielded a single protein band with an apparent molecular mass of 75.0 kDa +/- 3.0 kDa, indicating that the Euglena enzyme is composed of two identical subunits. The purified enzyme contained one mole of prosthetic 5'-deoxyadenosylcobalamin per mole of the enzyme subunit. Moreover, we cloned the full-length cDNA of the Euglena enzyme. The cDNA clone contained an open reading frame encoding a protein of 717 amino acids with a calculated molecular mass of 78.3 kDa, preceded by a putative mitochondrial targeting signal consisting of nine amino acid residues. Furthermore, we studied some properties and physiological function of the Euglena enzyme.

The putative coenzyme B12-dependent methylmalonyl-CoA mutase from potatoes is a phosphatase.

The reported presence of a coenzyme B12-dependent methylmalonyl-CoA mutase in potatoes has been reexamined. The enzyme converting methylmalonyl-CoA was purified to electrophoretic homogeneity. Examination of the reaction product by 1H, 31P NMR and mass spectrometry revealed that it was methylmalonyl-3'-dephospho-CoA. The phosphatase enzyme needs neither coenzyme B12 nor S-adenosylmethionine as a cofactor.

Occurrence of 5'-deoxyadenosylcobalamin and its physiological function as the coenzyme of methylmalonyl-CoA mutase in a marine eukaryotic microorganism, Schizochytrium limacinum SR21.

A marine eukaryotic microorganism, Schizochytrium limacinum SR21, had the ability to absorb and accumulate exogenous cobalamin, which was converted to the cobalamin coenzymes 5'-deoxyadenosylcobalamin (20.1%) and methylcobalamin (29.6%). A considerably high activity (about 38 mU/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) involved in amino acid and odd-chain fatty acid metabolism was found in the cell homogenate of S. limacinum SR21. The enzyme was purified to homogeneity and characterized.

Purification and characterization of methylmalonyl-CoA mutase from a photosynthetic coccolithophorid alga, Pleurochrysis carterae.

Low activity (about 4 mU/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM; EC 5.4.99.2) was found in a cell homogenate of a photosynthetic coccolithophorid alga, Pleurochrysis carterae. Most of the enzyme occurred as the apo-enzyme, which was labile during purification. The holo-enzyme, which was converted from the apo-enzyme by incubation with 10 microM 5'-deoxyadenosylcobalamin at 4 degrees C in the dark, was purified to homogeneity and partially characterized. An apparent molecular mass for the enzyme of 150+/-5 kDa was calculated by Superdex 200 pg gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave a single protein band with an apparent molecular mass of 80+/-5 kDa, indicating that the P. carterae enzyme occurs as a homodimer. Some properties of methylmalonyl-CoA mutase from P. carterae were studied.

Purification and characterization of homodimeric methylmalonyl-CoA mutase from Sinorhizobium meliloti.

High activity (>60 munit/mg protein) of 5'-deoxyadenosylcobalamin-dependent methylmalonyl-CoA mutase (EC 5.4.99.2) was constantly found during growth of a strain of the root-nodule-forming bacterium Sinorhizobium meliloti harboring an extra plasmid-encoded copy of the methylmalonyl-CoA-mutase-encoding bhbA gene. The enzyme was purified to homogeneity and characterized. The purified enzyme was found to be a colorless apo-form. The apparent molecular weight of the enzyme was calculated to be 165,000+/-5,000 by Superdex 200 HR gel filtration. SDS-PAGE of the purified enzyme resolved one protein band with an apparent molecular mass of 80.0+/-2.0 kDa, indicating that the S. meliloti enzyme is composed of two identical subunits. The NH(2)-terminal sequence was identical to that predicted from the bhbA nucleotide sequence. Monovalent cations were required for enzyme activity.

Purification and characterization of methylmalonyl-CoA mutase from a methanol-utilizing bacterium, Methylobacterium extorquens NR-1.

High activity (about 50 mU x mg protein(-1)) of methylmalonyl-CoA mutase (82-95% apo-enzyme) was constantly found during the cell growth of a methanol-utilizing bacterium, Methylobacterium extorquens NR-1. The apo-enzyme was purified to homogeneity and characterized. The purified enzyme was colorless. An apparent Mr of M. extorquens NR-1 enzyme was calculated to be 150,000 +/- 5,000 by Superdex 200 HR gel filtration. SDS-polyacrylamide gel electrophoresis of the purified enzyme gave two protein bands with an apparent Mr of 85.000 +/- 2,000 and 70,000 +/- 2,000, indicating that the M. extorquens NR-1 enzyme is composed of two nonidentical subunits. NH2-terminal amino acid sequences of the small and large subunits of M. extorquens NR-1 enzyme showed no significant homology to those of the enzyme from other species. Some enzymological properties of the M. extorquens NR-1 enzyme were studied.

Stabilization of radical intermediates by an active-site tyrosine residue in methylmalonyl-CoA mutase.

The adenosylcobalamin-dependent methylmalonyl-CoA mutase catalyzes the reversible rearrangement of methylmalonyl-CoA into succinyl-CoA by a free-radical mechanism. The recently solved X-ray crystal structure of methylmalonyl-CoA mutase from Propionibacterium shermanii has shown that tyrosine 89 is an active-site residue involved in substrate binding. The role of tyrosine 89, a conserved residue among methylmalonyl-CoA mutases, has been investigated by using site-directed mutagenesis to replace this residue with phenylalanine. The crystal structure of the Tyr89Phe mutant was determined to 2.2 A resolution and was found to be essentially superimposable on that of wild-type. Mutant and wild-type enzyme have very similar KM values, but kcat for the Tyr89Phe mutant is 580-fold lower than for wild-type. The rate of release of tritium from 5'-[3H]adenosylcobalamin during the enzymatic reaction and its rate of appearance in substrate and product were measured. The tritium released was found to partition unequally between methylmalonyl-CoA and succinyl-CoA, in a ratio of 40:60 when the reaction was initiated by addition of methylmalonyl-CoA and in a ratio of 10:90 when the reaction was initiated by addition of succinyl-CoA. The overall release of tritium was four times faster when succinyl-CoA was used as substrate. The tritium isotope effect on the enzyme catalyzed hydrogen transfer, measured with methylmalonyl-CoA as a substrate, was kH/kT = 30, which is within the expected range for a full primary kinetic tritium isotope effect. The different partitioning of tritium, dependent upon which substrate was used, and the normal value for the kinetic tritium isotope effect contrast markedly with the behavior of wild-type mutase. It appears that the loss of a single interaction involving the hydroxyl group of tyrosine 89 both affects the stability of radical intermediates and decreases the rate of interconversion of the substrate- and product-derived radicals.

Adenosylcobalamin-dependent methylmalonyl-CoA mutase isozymes in the photosynthetic protozoon Euglena gracilis Z.

The photosynthetic protozoon Euglena gracilis Z contains adenosylcobalamin-dependent methylmalonyl-CoA mutase (MCM) involved in propionate metabolism. The specific activity of the Euglena mutase was about 6.5-fold greater in propionate-adapted Euglena cells than in photoautotrophic cells (control). Although the control cells contained only one mutase (apparent M(r) 72,000), the propionate-adapted cells contained two mutases with M(r) values of 72,000 and 17,000; both enzymes were located in the mitochondria. These results provide evidence that propionate-adapted Euglena contains two MCM isozymes. The induced mutase (M(r) 17,000) permits photoassimilation of propionate.

Evidence from electron paramagnetic resonance spectroscopy of the participation of radical intermediates in the reaction catalyzed by methylmalonyl-coenzyme A mutase.

Recombinant methylmalonyl-coenzyme A (CoA) mutase from Propionibacterium shermanii has been purified 20-fold to near homogeneity in a highly active form. Neither the apoenzyme (the form in which the enzyme is isolated) nor the holoenzyme (reconstituted with the cofactor, adenosylcobalamin) has an electron paramagnetic resonance (EPR) spectrum associated with it. However, the addition of either the substrate, methylmalonyl-CoA, or the product, succinyl-CoA, results in the appearance of a transient EPR signal. The signal has hyperfine features that indicate coupling of the unpaired electron to the cobalt nucleus. In the presence of [CD3]methylmalonyl-CoA, an EPR signal is also seen and is similar to that obtained in the presence of protiated substrate. Power saturation studies reveal the presence of two components, a slow relaxing species (with an apparent g value of 2.11) and a fast relaxing species (with an apparent g value of 2.14) that can be partially resolved at low temperature and high power. The EPR-active intermediate is observed under catalytic conditions and is approximately midway in its resonance position between a free radical and cob(II)alamin. It is postulated to represent an exchange-coupled cob(II)alamin ... free radical pair. The signal bears close resemblance to those observed with partially dehydrated polycrystalline adenosylcobalamin following laser photolysis (Ghanekar, V.D., Lin, R.J., Coffman, R.E., and Blakley, R.L. (1981) Biochem. Biophys. Res. Commun. 101, 215-221) and with the adenosylcobalamin-dependent ribonucleotide reductase under freeze-quench conditions (Orme-Johnson, W.H., Beinert, H., and Blakley, R.L. (1974) J. Biol. Chem. 249, 2338-2343). When cob(II)alamin is generated under noncatalytic conditions (i.e. in the presence of propionyl-CoA or by electrochemical reduction of enzyme-bound hydroxocob-(III)alamin), a different EPR signal is observed with g = 2.26 and g = 2.00, typical of base-on cob(II)alamin.

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

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.

Assay of methylmalonyl CoA mutase with high-performance liquid chromatography.

An assay for methylmalonyl CoA mutase activity is described. Succinyl CoA produced in this method is separated from the substrate, methylmalonyl CoA, by reverse-phase high-performance liquid chromatography and is quantified. This method is useful to differentiate mutase apoenzyme deficiency (mut0, mut-) and the defect in deoxyadenosylcobalamin synthesis using fibroblasts cultured in high concentration of supplementary hydroxocobalamin. In methylmalonic acidemia, measurement of lymphocytes mutase activity offers therapeutical and prognostic informations.

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.

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

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.

Isolation and characterization of DL-methylmalonyl-coenzyme A racemase from rat liver.

Certain amino acids and other compounds are metabolized via propionyl-CoA----D-methylmalonyl-CoA----L-methylmalonyl- CoA----succinyl-CoA----tricarboxylic acid cycle. D-Methylmalonyl-CoA can also be converted to methylmalonic acid and coenzyme A by a specific hydrolase that does not act on L-methylmalonyl-CoA [R.J. Kovachy, S.D. Copley, and R.H. Allen (1983) J. Biol. Chem. 258, 11415-11421]. Because little is known about mammalian DL-methylmalonyl-CoA racemase and because it is involved in the flow of D-methylmalonyl-CoA to L-methylmalonyl-CoA----tricarboxylic acid cycle (versus to methylmalonic acid), we developed a new assay and purified rat liver racemase 23,000-fold to homogeneity. The molecular weight of the racemase is 32,000 and it contains two subunits of Mr 16,000 that are not connected by disulfide bonds. The rat liver and the rat and human white blood cell racemase are immunologically related. They are completely inactivated by EDTA and can be activated by the addition of Co+2, with 50% activation occurring at a concentration of 0.2 microM. Lower levels for maximal activation were obtained with higher concentrations of Co+3, Fe+2, and Mn+2. Other metals such as Zn+2, Cu+2, Cu+1, and Cd+2 completely inhibited racemase even in the presence of equal concentrations of Co+2. The purified racemase appears to bind 1 mol Co/mol subunit.

Biogenesis of the mitochondrial enzyme methylmalonyl-CoA mutase. Synthesis and processing of a precursor in a cell-free system and in cultured cells.

Methylmalonyl-CoA mutase (EC 5.4.99.2; mutase), a cytoplasmically synthesized mitochondrial matrix enzyme, is translated in a cell-free system programmed with rat liver RNA as a larger precursor polypeptide, designated pre-mutase, which appears to be 3-4 kDa larger than the subunit of purified mutase (77.5 kDa). When pre-mutase is incubated with intact rat liver mitochondria, it is taken up by them and proteolytically processed to the size of the mature subunit. The overall reaction is inhibited by compounds such as dinitrophenol which disrupt mitochondrial energy metabolism. The final, proteolytic step can be carried out by the mitochondrial matrix in the presence of added Zn2+ and is inhibited by metal ion chelators and by certain protease inhibitors (e.g. leupeptin and p-aminobenzamidine). Newly synthesized mutase was also detected in intact, cultured Buffalo rat liver cells labeled with [3H] leucine in the presence of dinitrophenol. When dinitrophenol is removed in a pulse-chase protocol, the accumulated pre-mutase is rapidly (t1/2 = 6-9 min) converted to mutase. On the other hand, when the chase is performed in the presence of the inhibitor, the labeled pre-mutase persists for greater than 5 h. This long term stability of pre-mutase contrasts sharply with the instability previously reported for unprocessed precursors of two other mitochondrial enzymes.

Source of methylmalonyl-coenzyme A for erythromycin synthesis: methylmalonyl-coenzyme A mutase from Streptomyces erythreus.

Streptomyces erythreus produces erythromycin, presumably from methylmalonyl-coenzyme A (CoA), which could be generated by the isomerization of succinyl-CoA. In S. erythreus cultures, [1,4-14C,2,3-3H]succinate was incorporated into erythromycin with a doubling of the 3H/14C ratio. This result is consistent with the hypothesis that succinyl-CoA is isomerized to methylmalonyl-CoA before incorporation into the macrocyclic lactone of erythromycin. The presence of methylmalonyl-CoA mutase, which catalyzes this isomerization, was demonstrated in cell-free extracts prepared from this organism. Consistent with the suggested role for this enzyme, methylmalonyl-CoA mutase activity increased over 12-fold at the time of the most rapid antibiotic production, and the activity level drastically declined when the antibiotic production ceased. The mutase was partially purified from this organism with DEAE-cellulose, ammonium sulfate precipitation, and affinity chromatography on a B12-coenzyme Sepharose column. The enzyme was stimulated 2.5-fold by the addition of B12-coenzyme. The enzyme showed a typical Michaelis-Menten type substrate saturation patterns, with KmS of 0.31 mM and 0.09 microM for methylmalonyl-CoA and B12-coenzyme, respectively, and a V of 0.5 mumol/min per mg. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed a major band with a molecular weight of 63,000. The properties of this enzyme appear to be fairly similar to those of the mutase previously obtained from other sources.

Purification and characterization of methylmalonyl-CoA mutase from Ascaris lumbricoides.

Methylmalonyl CoA mutase from Ascaris lumbricoides has been purified to homogeneity. The mutase is homogeneous as judged by equilibrium sedimentation and polyacrylamide gel electrophoresis. The worm mutase is a glycoprotein with a mol. wt of 147,000 +/- 3500 composed of two identical or very similar subunits. One molecule of adenosylcobalamin is tightly bound to each subunit. The mutase from Ascaris is not affected by exposure to light, cyanide ion or intrinsic factor and is not inhibited by iodoacetate and rho-hydroxymercuribenzoate. The kinetic constants of this mutase for (R,S)methylmalonyl CoA are Km = 4.2 X 10(-5) M and Vmax = 4.73 mumol/mg/min.

Isolation and characterization of methylmalonyl-CoA mutase from human placenta.

Methylmalonyl-CoA mutase, one of two known cobalamin-dependent enzymes present in mammalian tissues, has been isolated from 2.5 kg of human placenta utilizing affinity chromatography on 5'-deoxyadenosylcobalamin-Sepharose as the major purification step. The enzyme gives a single band on polyacrylamide disc gel electrophoresis. The Mr of the enzyme is 145,000 and it has two subunits of Mr = 72,000. Amino acid analysis reveals major differences from other human cobalamin-binding proteins. Based on x-ray fluorescence, the enzyme has 2 mol of cobalamin bound/mol of enzyme. In contrast to purified cobalamin transport proteins, most of the cobalamin bound to the enzyme is not released by boiling at low pH in the presence of KCN, or dialysis against 7.5 M guanidine containing 0.2 M dithiothreitol, or both, suggesting the possibility that cobalamin may be covalently attached to the purified enzyme. Both precipitating antibodies and antibodies that inhibit enzyme activity have been raised in a chicken.