Insertional Inactivation of Methylmalonyl Coenzyme A (CoA) Mutase and Isobutyryl-CoA Mutase Genes in Streptomyces cinnamonensis: Influence on Polyketide Antibiotic Biosynthesis

The coenzyme B(12)-dependent isobutyryl coenzyme A (CoA) mutase (ICM) and methylmalonyl-CoA mutase (MCM) catalyze the isomerization of n-butyryl-CoA to isobutyryl-CoA and of methylmalonyl-CoA to succinyl-CoA, respectively. The influence that both mutases have on the conversion of n- and isobutyryl-CoA to methylmalonyl-CoA and the use of the latter in polyketide biosynthesis have been investigated with the polyether antibiotic (monensin) producer Streptomyces cinnamonensis. Mutants prepared by inserting a hygromycin resistance gene (hygB) into either icmA or mutB, encoding the large subunits of ICM and MCM, respectively, have been characterized. The icmA::hygB mutant was unable to grow on valine or isobutyrate as the sole carbon source but grew normally on butyrate, indicating a key role for ICM in valine and isobutyrate metabolism in minimal medium. The mutB::hygB mutant was unable to grow on propionate and grew only weakly on butyrate and isobutyrate as sole carbon sources. (13)C-labeling experiments show that in both mutants butyrate and acetoacetate may be incorporated into the propionate units in monensin A without cleavage to acetate units. Hence, n-butyryl-CoA may be converted into methylmalonyl-CoA through a carbon skeleton rearrangement for which neither ICM nor MCM alone is essential.     

IcmF Is a Fusion between the Radical B12 Enzyme Isobutyryl-CoA Mutase and Its G-protein Chaperone

Coenzyme B₁₂ is used by two highly similar radical enzymes, which catalyze carbon skeleton rearrangements, methylmalonyl-CoA mutase and isobutyryl-CoA mutase (ICM). ICM catalyzes the reversible interconversion of isobutyryl-CoA and n-butyryl-CoA and exists as a heterotetramer. In this study, we have identified >70 bacterial proteins, which represent fusions between the subunits of ICM and a P-loop GTPase and are currently misannotated as methylmalonyl-CoA mutases. We designate this fusion protein as IcmF (isobutyryl-CoA mutase fused). All IcmFs are composed of the following three domains: the N-terminal 5′-deoxyadenosylcobalamin binding region that is homologous to the small subunit of ICM (IcmB), a middle P-loop GTPase domain, and a C-terminal part that is homologous to the large subunit of ICM (IcmA). The P-loop GTPase domain has very high sequence similarity to the Methylobacterium extorquens MeaB, which is a chaperone for methylmalonyl-CoA mutase. We have demonstrated that IcmF is an active ICM by cloning, expressing, and purifying the IcmFs from Geobacillus kaustophilus, Nocardia farcinica, and Burkholderia xenovorans. This finding expands the known distribution of ICM activity well beyond the genus Streptomyces, where it is involved in polyketides biosynthesis, and suggests a role for this enzyme in novel bacterial pathways for amino acid degradation, myxalamid biosynthesis, and acetyl-CoA assimilation.     

When a spectator turns killer: suicidal electron transfer from cobalamin in methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase belongs to the class of adenosylcobalamin (AdoCbl)-dependent carbon skeleton isomerases and catalyzes the rearrangement of methylmalonyl-CoA to succinyl-CoA. In this study, we have evaluated the contribution of the active site residue, R207, in the methylmalonyl-CoA mutase-catalyzed reaction. The R207Q mutation results in a 10(4)-fold decrease in k(cat) and >30-fold increase in the K(M) for the substrate, methylmalonyl-CoA. R207 and the active site residue, Y89, are within hydrogen bonding distance to the carboxylate of the substrate. In the closely related isomerase, isobutyryl-CoA mutase the homologous residues are F80 and Q198, respectively. We therefore characterized the ability of the double mutant (Y89F/R207Q) of methylmalonyl-CoA mutase as well as of the single mutants (Y89F and R207Q) to catalyze the rearrangement of n-butyryl-CoA to isobutyryl-CoA. While none of the mutant enzymes is capable of isomerizing these substrates, the R207Q (single and double) mutants exhibited irreversible inactivation upon incubation with either n-butyryl-CoA or isobutyryl-CoA. The two products observed during inactivation under both aerobic and strictly anaerobic conditions were 5'-deoxyadenosine and hydroxocobalamin, which suggested internal electron transfer from cob(II)alamin to the substrate or the 5'-deoxyadenosyl radical. Deuterium transfer from substrate to deoxyadenosine demonstrated that the substrate radical is formed and is presumably the acceptor in the electron-transfer reaction from cob(II)alamin. These studies provide evidence for the critical role of active site residues in controlling radical reactivity and thereby suppressing inactivating side reactions.     

Ethylmalonyl-CoA mutase from Rhodobacter sphaeroides defines a new subclade of coenzyme B12-dependent acyl-CoA mutases

Coenzyme B(12)-dependent mutases are radical enzymes that catalyze reversible carbon skeleton rearrangement reactions. Here we describe Rhodobacter sphaeroides ethylmalonyl-CoA mutase (Ecm), a novel member of the family of coenzyme B(12)-dependent acyl-CoA mutases, that operates in the recently discovered ethylmalonyl-CoA pathway for acetate assimilation. Ecm is involved in the central reaction sequence of this novel pathway and catalyzes the transformation of ethylmalonyl-CoA to methylsuccinyl-CoA in combination with a second enzyme that was further identified as promiscuous ethylmalonyl-CoA/methylmalonyl-CoA epimerase. In contrast to the epimerase, Ecm is highly specific for its substrate, ethylmalonyl-CoA, and accepts methylmalonyl-CoA only at 0.2% relative activity. Sequence analysis revealed that Ecm is distinct from (2R)-methylmalonyl-CoA mutase as well as isobutyryl-CoA mutase and defines a new subfamily of coenzyme B(12)-dependent acyl-CoA mutases. In combination with molecular modeling, two signature sequences were identified that presumably contribute to the substrate specificity of these enzymes.     

MeaA, a putative coenzyme B12-dependent mutase, provides methylmalonyl coenzyme A for monensin biosynthesis in Streptomyces cinnamonensis

The ratio of the major monensin analogs produced by Streptomyces cinnamonensis is dependent upon the relative levels of the biosynthetic precursors methylmalonyl-coenzyme A (CoA) (monensin A and monensin B) and ethylmalonyl-CoA (monensin A). The meaA gene of this organism was cloned and sequenced and was shown to encode a putative 74-kDa protein with significant amino acid sequence identity to methylmalonyl-CoA mutase (MCM) (40%) and isobutyryl-CoA mutase (ICM) large subunit (36%) and small subunit (52%) from the same organism. The predicted C terminus of MeaA contains structural features highly conserved in all coenzyme B12-dependent mutases. Plasmid-based expression of meaA from the ermE* promoter in the S. cinnamonensis C730.1 strain resulted in a decreased ratio of monensin A to monensin B, from 1:1 to 1:3. Conversely, this ratio increased to 4:1 in a meaA mutant, S. cinnamonensis WM2 (generated from the C730.1 strain by insertional inactivation of meaA by using the erythromycin resistance gene). In both of these experiments, the overall monensin titers were not significantly affected. Monensin titers, however, did decrease over 90% in an S. cinnamonensis WD2 strain (an icm meaA mutant). Monensin titers in the WD2 strain were restored to at least wild-type levels by plasmid-based expression of the meaA gene or the Amycolatopsis mediterranei mutAB genes (encoding MCM). In contrast, growth of the WD2 strain in the presence of 0.8 M valine led only to a partial restoration (<25%) of monensin titers. These results demonstrate that the meaA gene product is significantly involved in methylmalonyl-CoA production in S. cinnamonensis and that under the tested conditions the presence of both MeaA and ICM is crucial for monensin production in the WD2 strain. These results also indicate that valine degradation, implicated in providing methylmalonyl-CoA precursors for many polyketide biosynthetic processes, does not do so to a significant degree for monensin biosynthesis in the WD2 mutant.     

Assembly and protection of the radical enzyme, methylmalonyl-CoA mutase, by its chaperone

MeaB is a recently described P-loop GTPase that plays an auxiliary role in the reaction catalyzed by the radical B12 enzyme, methylmalonyl-CoA mutase. Defects in the human homologue of MeaB result in methylmalonic aciduria, but the role of this protein in coenzyme B12 assimilation and/or utilization is not known. Methylmalonyl-CoA mutase catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA that uses reactive radical intermediates that are susceptible to oxidative inactivation. In this study, we have examined the influence of MeaB on the kinetics of the reaction catalyzed by methylmalonyl-CoA mutase and on the thermodynamics of cofactor binding. MeaB alone has a modest effect on the affinity of the mutase for the 5'-deoxyadenosylcobalamin (AdoCbl) cofactor, increasing it 2-fold from 404 +/- 71 to 210 +/- 22 nM. However, in the presence of GDP, the affinity for the cofactor decreases 5-fold to 1.89 +/- 0.33 microM, while in the presence of guanosine 5'(beta-gamma imino)triphosphate, a nonhydrolyzable analogue of GTP, the binding of AdoCbl to the mutase is not detected. Protection against oxidative inactivation of the mutase by MeaB is dependent upon the presence of nucleotides with the MeaB/GDP and MeaB/GTP complexes decelerating the rate of formation of oxidized cofactor by 3- and 15-fold, respectively. This study suggests that MeaB functions in the GTP-dependent assembly of holomethylmalonyl-CoA mutase and subsequent protection of radical intermediates during catalysis.     

Uptake and physiological function of vitamin B12 in a photosynthetic unicellular coccolithophorid alga, Pleurochrysis carterae

The photosynthetic coccolithophoid alga, Pleurochrysis (Hymenomonas) carterae, could take up and accumulate exogenous vitamin B12, most of which was converted into the coenzyme forms of vitamin B12. Two vitamin B12-dependent enzyme activities (methylmalonyl-CoA mutase, 2.6+/-0.4 nmol/min/mg protein and methionine synthase, 85.1+/-38.9 pmol/min/mg protein) could be found in a cell homogenate of the vitamin B12-supplemented alga. Most of the methylmalonyl-CoA mutase activity and 19.2% of the vitamin B12 accumulated by the algal cells were recovered in the macromolecular fractions with Mr of 150 kDa, although the remaining vitamin B12 was found only in free vitamin B12 fractions.     

Novel B(12)-Dependent Acyl-CoA Mutases and Their Biotechnological Potential

The recent spate of discoveries of novel acyl-CoA mutases has engendered a growing appreciation for the diversity of 5'-deoxyadenosylcobalamin-dependent rearrangement reactions. The prototype of the reaction catalyzed by these enzymes is the 1,2 interchange of a hydrogen atom with a thioester group leading to a change in the degree of carbon skeleton branching. These enzymes are predicted to share common architectural elements: a Rossman fold and a triose phosphate isomerase (TIM)-barrel domain for binding cofactor and substrate, respectively. Within this family, methylmalonyl-CoA mutase (MCM) is the best studied and is the only member found in organisms ranging from bacteria to man. MCM interconverts (2R)-methylmalonyl-CoA and succinyl-CoA. The more recently discovered family members include isobutyryl-CoA mutase (ICM), which interconverts isobutyryl-CoA and n-butyryl-CoA; ethylmalonyl-CoA mutase, which interconverts (2R)-ethylmalonyl-CoA and (2S)-methylsuccinyl-CoA; and 2-hydroxyisobutyryl-CoA mutase, which interconverts 2-hydroxyisobutyryl-CoA and (3S)-hydroxybutyryl-CoA. A variant in which the two subunits of ICM are fused to a G-protein chaperone, IcmF, has been described recently. In addition to its ICM activity, IcmF also catalyzes the interconversion of isovaleryl-CoA and pivalyl-CoA. This review focuses on the involvement of acyl-CoA mutases in central carbon and secondary bacterial metabolism and on their biotechnological potential for applications ranging from bioremediation to stereospecific synthesis of C2-C5 carboxylic acids and alcohols, and for production of potential commodity and specialty chemicals.     

Thermodynamic and kinetic characterization of Co-C bond homolysis catalyzed by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Methylmalonyl-CoA mutase is a member of the family of coenzyme B(12)-dependent isomerases and catalyzes the 1,2-rearrangement of methylmalonyl-CoA to succinyl-CoA. A common first step in the reactions catalyzed by coenzyme B(12)-dependent enzymes is cleavage of the cobalt-carbon bond of the cofactor, leading to radical-based rearrangement reactions. Comparison of the homolysis rate for the free and enzyme-bound cofactors reveals an enormous rate enhancement which is on the order of a trillion-fold. To address how this large rate acceleration is achieved, we have examined the kinetic and thermodynamic parameters associated with the homolysis reaction catalyzed by methylmalonyl-CoA mutase. Both the rate and the amount of cob(II)alamin formation have been analyzed as a function of temperature with the protiated substrate. These studies yield the following activation parameters for the homolytic reaction at 37 degrees C: DeltaH(f)() = 18.8 +/- 0.8 kcal/mol, DeltaS(f)() = 18.2 +/- 0.8 cal/(mol.K), and DeltaG(f)() = 13.1 +/- 0.6 kcal/mol. Our results reveal that the enzyme lowers the transition state barrier by 17 kcal/mol, corresponding to a rate acceleration of 0.9 x 10(12)-fold. Both entropic and enthalpic factors contribute to the observed rate acceleration, with the latter predominating. The substrate binding step is exothermic, with a DeltaG of -5.2 kcal/mol at 37 degrees C, and is favored by both entropic and enthalpic factors. We have employed the available kinetic and spectroscopic data to construct a qualitative free energy profile for the methylmalonyl-CoA mutase-catalyzed reaction.     

Energetics of interaction between the G-protein chaperone, MeaB, and B12-dependent methylmalonyl-CoA mutase

MeaB is an auxiliary protein that supports the function of the radical B(12)-dependent enzyme, methylmalonyl-CoA mutase, although its precise role is not understood. Mutations in the human homolog of MeaB, MMAA, lead to methylmalonic aciduria, an inborn error of metabolism that can be fatal. To obtain insights into the function of this recently discovered protein, we have characterized the entropic and enthalpic contributions to DeltaGdegree (assoc) for complexation of MeaB (in the presence and absence of nucleotides) with methylmalonyl-CoA mutase (in the presence and absence of cofactor). The dissociation constant for binding of methylmalonyl-CoA mutase and MeaB ranges from 34 +/- 4 to 524 +/- 66 nm, depending on the combination of nucleotide and mutase form. Holomutase binds MeaB 15-fold more tightly when the nonhydrolyzable GTP analog, GMPPNP, is bound versus GDP. In contrast, the apomutase binds MeaB with similar affinity in the presence of either nucleotide. Our studies reveal that a large structural rearrangement accompanies interaction between these proteins and buries between approximately 4000 and 8600A(2) of surface area, depending on the combination of ligands in the active sites of the two proteins. Furthermore, we demonstrate that MeaB binds GTP and GDP with similar affinity (K(d) of 7.3 +/- 1.9 and 6.2 +/- 0.7 microm, respectively at 20 degrees C) and has low intrinsic GTPase activity (approximately 0.04 min(-1) at 37 degrees C), which is stimulated approximately 100-fold by methylmalonyl-CoA mutase. These studies provide insights into the energetics of interaction between the radical enzyme methylmalonyl-CoA mutase and MeaB, which are discussed.     

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].     

Homology modeling of human methylmalonyl-CoA mutase: a structural basis for point mutations causing methylmalonic aciduria.

Point mutations in the human gene encoding coenzyme B12 (adenosylcobalamin)-dependent methylmalonyl-CoA mutase give rise to an inherited disorder of propionic acid metabolism termed mut methylmalonic aciduria. Almost all such mutations alter amino acids in the homodimeric human enzyme that are identical to residues in the catalytic alpha-subunit of the heterodimeric methylmalonyl-CoA mutase from the bacterium Propionibacterium shermanii, to which the mature human enzyme shows an overall 65% sequence identity. To explore how specific mutations might cause the observed clinical phenotype, 12 known mutations were mapped onto a three-dimensional homology model of the subunit of the human enzyme, generated using the program MODELLER on the basis of the recently published 2.0 A X-ray crystal structure of the P. shermanii methylmalonyl-CoA mutase. Eight mutations are found in the C-terminal B12-binding domain, of which 4 (G623R, G626C, G630E, G703R) are in direct contact with the corrin and are clustered around the histidine ligand (H627) provided by the protein to coordinate the cobalt atom of the B12 cofactor. Introduction of a side chain, particularly one that is charged, at any of these positions is expected to disrupt the flavodoxin-like fold and severely impair its binding of B12. Mutation at either of two other highly conserved glycine residues in this domain (G648D, G717V) also disrupts critical elements in the fold as would the introduction of an additional positive charge in the mutation H678R. Mutation of an arginine in a solvent-exposed loop to a hydrophobic residue (R694W) is also pathogenic. The remaining mutations have been mapped to the N-terminal region of the mutase, two of which introduce a buried, uncompensated charge, either near the subunit interface (A377E), or near the narrow channel through which acyl-CoA esters gain access to the active site (W105R). The extreme N-terminus of methylmalonyl-CoA mutase is predicted to make extensive contacts with the other subunit, and a mutant in this region (R93H) may prevent the correct assembly of the dimer.     

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.     

Structure and dynamics of the B12-binding subunit of glutamate mutase from Clostridium cochlearium.

Glutamate mutase (Glm) is an adenosylcobamide-dependent enzyme that catalyzes the reversible rearrangement of (2S)-glutamate to (2S, 3S)-3-methylaspartate. The active enzyme from Clostridium cochlearium consists of two subunits (of 53.6 and 14.8 kDa) as an alpha2beta2 tetramer, whose assembly is mediated by coenzyme B12. The smaller of the protein components, GlmS, has been suggested to be the B12-binding subunit. Here we report the solution structure of GlmS, determined from a heteronuclear NMR-study, and the analysis of important dynamical aspects of this apoenzyme subunit. The global fold and dynamic behavior of GlmS in solution are similar to those of the corresponding subunit MutS from C. tetanomorphum, which has previously been investigated using NMR-spectroscopy. Both solution structures of the two Glm B12-binding subunits share striking similarities with that determined by crystallography for the B12-binding domain of methylmalonyl CoA mutase (Mcm) from Propionibacterium shermanii, which is B12 bound. In the crystal structure a conserved histidine residue was found to be coordinated to cobalt, displacing the endogenous axial ligand of the cobamide. However, in GlmS and MutS the sequence motif, Asp-x-His-x-x-Gly, which includes the cobalt-coordinating histidine residue, and a predicted alpha-helical region following the motif, are present as an unstructured and highly mobile loop. In the absence of coenzyme, the B12-binding site apparently is only partially formed. By comparing the crystal structure of Mcm with the solution structures of B12-free GlmS and MutS, a consistent picture on the mechanism of B12 binding has been obtained. Important elements of the binding site only become structured upon binding B12; these include the cobalt-coordinating histidine residue, and an alpha helix that forms one side of the cleft accommodating the nucleotide 'tail' of the coenzyme.     

Controlling the reactivity of radical intermediates by coenzyme B(12)-dependent methylmalonyl-CoA mutase

Adenosylcobalamin or coenzyme B(12)-dependent enzymes are members of the still relatively small group of radical enzymes and catalyse 1,2-rearrangement reactions. A member of this family is methylmalonyl-CoA mutase, which catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA and, unlike the others, is present in both bacteria and animals. Enzymes that catalyse some of the most chemically challenging reactions are the ones that tend to deploy radical chemistry. The use of radical intermediates in an active site lined with amino acid side chains that threaten to extinguish the reaction by presenting alternative groups for abstraction poses the conundrum of how the enzymes control their reactivity. In this review, insights into this issue that have emerged from kinetic, mutagenesis and structural studies are described for methylmalonyl-CoA mutase.     

Crystal structure of substrate complexes of methylmalonyl-CoA mutase.

X-ray crystal structures of methylmalonyl-CoA mutase in complexes with substrate methylmalonyl-CoA and inhibitors 2-carboxypropyl-CoA and 3-carboxypropyl-CoA (substrate and product analogues) show that the enzyme-substrate interactions change little during the course of the rearrangement reaction, in contrast to the large conformational change on substrate binding. The substrate complex shows a 5'-deoxyadenine molecule in the active site, bound weakly and not attached to the cobalt atom of coenzyme B12, rotated and shifted from its position in the substrate-free adenosylcobalamin complex. The position of Tyralpha89 close to the substrate explains the stereochemical selectivity of the enzyme for (2R)-methylmalonyl-CoA.     

The error in the cryptic stereospecificity of methylmalonyl-CoA mutase. The use of carba-(dethia)-coenzyme A substrate analogues gives new insight into the enzyme mechanism

A preparation containing 80.0 +/- 0.5% (2RS)-methylmalonyl-carba-(dethia)-CoA and 20.0 +/- 0.5% propionyl-carba-(dethia)-CoA was reacted in buffered deuterium oxide with catalytic amounts of coenzyme B12, methylmalonyl-CoA mutase and methylmalonyl-CoA epimerase. The rearrangement of the methylmalonyl-carba-(dethia)-CoA to succinyl-carba-(dethia)-CoA was monitored by recording 500-MHz 1H-NMR spectra in short time intervals. After reaching equilibrium (approximately equal to 28 min) the products showed chemical stability for about 17 h, i.e. succinyl species did not undergo the spontaneous hydrolysis encountered with normal succinyl-CoA. In the pre-equilibrium stage only about 66% of the produced succinyl-CH2CoA was the expected monodeuterated species. The remainder was 15.5% unlabelled and 18.3% 3,3-dideuterated. After reaching equilibrium a continuous deuterium incorporation (washing-in) from the solvent to the products was observed and quantified. The time course of the appearance of unlabelled, mono-, di- and trideuterated succinyl-CH2CoA species was determined by assigning and integrating the isotope-shifted 1H signals from the various species. Furthermore, mutase catalyses slow deuterium incorporation into first the methylene and then the methyl group of propionyl-CH2CoA. On the basis of these data it was concluded that methylmalonyl-CoA mutase and epimerase are responsible for continuous deuterium incorporation and multiple incorporation occurs when the backward reaction (succinyl-CH2CoA----methylmalonyl-CH2CoA) becomes important. To account for all of the results obtained with dethia and natural substrates we propose a new mutase mechanism whereby the enzyme can retain full stereospecificity at C-3 of succinyl while an internal 1,2-H shift to give a C-2 succinyl radical is responsible for partial scrambling of diastereotopic protons at C-3. This mechanism successfully predicts the observed deuterium disproportionation in succinyl species and the order of appearance of di- and trideuterated products via the washing-in process.     

Coenzyme B12-dependent enzymes in potatoes: Leucine 2,3-aminomutase and methylmalonyl-coa mutase

Extracts of potato tubers contain endogenous activities of two coenzyme B₁₂-dependent enzymes, leucine 2,3-aminomutase and methylmalonyl-CoA mutase. These activities are stimulated by the addition of coenzyme B₁₂ and are inhibited by intrinsic factor. The inhibition is overcome by coenzyme B₁₂.     

On the mechanism of action of methylmalonyl-CoA mutase. Change of the steric course on isotope substitution

When (methyl-2H3)methylmalonyl-CoA was reacted with partially purified methylmalonyl-CoA mutase, 1H-NMR revealed that about 24% of the migrating deuterium was lost after 88% conversion. When [methyl-3H]methylmalonyl-CoA was incubated with highly purified methylmalonyl-CoA mutase, tritium exchange with the medium depended on added methylmalonyl-CoA epimerase. With highly purified preparations of methylmalonyl-CoA mutase, effective tritium exchange from [5'-3H]adenosylcobalamin to water required the addition of methylmalonyl-CoA epimerase and of substrate (e.g. succinyl-CoA). By addition of [14C]succinyl-CoA to a partially purified preparation of methylmalonyl-CoA mutase, it was shown that the mutase binds one substrate molecule very tightly. Coupling the mutase reaction with the transcarboxylase reaction and using variously labelled succinyl-CoA as substrate, revealed that only (2R)- and not (2S)-methylmalonyl-CoA will be formed by the mutase with a kinetic isotope effect of 3.5 using (2H4)succinyl-CoA. When (1-13C) propionyl-CoA was reacted with a mixture of highly purified methylmalonyl-CoA carboxylase, epimerase and mutase, 13C-NMR signals were obtained for the thioester carbonyl of succinyl-CoA (relative intensity 100%) and of methylmalonyl-CoA (5%) as well as for the carboxyl of free succinic acid (27%) and of succinyl-CoA (less than 4.5%). Thus very little, if any, migration of the CoA from one carboxyl to the other appears to take place. (1,4-13C2)Succinic acid and (1,4-13C2)succinyl-CoA were synthesised and their 13C-NMR chemical shifts were exactly determined. Evidence is provided for a strict stereospecificity of the mutase toward the (2R)-epimer of methylmalonyl-CoA and for an incomplete stereospecificity toward the two diastereotopic 3-H atoms of succinyl-CoA. The latter, combined with a high intramolecular isotope discrimination, causes rapid washing-out of the migrating 2H and 3H to water and slow washing-in from the medium. Whenever migration of protium from the sterically less preferred 3-pro(S)- position of succinyl-CoA occurs and simultaneously a heavy isotope is maneuvered from the migratable 3-pro(R)- position into the labile alpha-position of methylmalonyl-CoA, the substitution by the COSCoA group takes place with inversion of configuration. When the sterically preferred 3-pro(R)-hydrogen atom migrates, the previously reported stereochemical retention occurs. A mechanistic and stereochemical scheme is discussed that fully accounts for all observations.     

Crystallization and preliminary diffraction data for adenosylcobalamin-dependent methylmalonyl-CoA mutase from Propionibacterium shermanii

Pink crystals of methylmalonyl-CoA mutase from Propionibacterium shermanii, a coenzyme B12 (5'-deoxyadenosylcobalamin)-dependent enzyme, have been obtained by the hanging-drop method in two different forms. One form lies in the space group P21, with unit cell dimensions a = 122 A, b = 160 A and c = 90 A, with beta = 104 degrees (1 A = 0.1 nm). There are two alpha beta dimers in the asymmetric unit. The crystals diffract to 3.2 A resolution and are suitable for high resolution X-ray diffraction studies.