Enzyme methylmalonyl-CoA mutase

In mammals and in the majority of bacteria, cobalamin regulates DNA synthesis indirectly through its effect on a step in folate metabolism, catalyzing the synthesis of methionine from homocysteine and 5-methyltetrahydrofolate via two methyl transfer reactions. This cytoplasmic reaction is catalyzed by methionine synthase (5-methyltetrahydrofolate-homocysteine methyl-transferase), which requires methyl cobalamin (MeCbl) (253), one of the two known coenzyme forms of the complex, as its cofactor. 5 -Deoxyadenosyl cobalamin (AdoCbl) (254), the other coenzyme form of cobalamin, occurs within mitochondria. This compound is a cofactor for the enzyme methylmalonyl-CoA mutase, which is responsible for the conversion of T-methylmalonyl CoA to succinyl CoA. This reaction is involved in the metabolism of odd chain fatty acids via propionic acid, as well as amino acids isoleucine, methionine, threonine, and valine. 

Odd-chain fatty acids are an exception. While they are relatively rare in the diet, odd-chain-length fatty acids end up at propionyl-CoA (C3). Propionyl-CoA is carboxylated by propionyl-CoA carboxylase to give methylmalonyl-CoA. Methylmalonyl-CoA is rearranged to succinyl-CoA by the enzyme methylmalonyl-CoA mutase, a vitamin-B12-requiring enzyme. 

The other reaction that requires vitamin B12 is isomerization of methylmalonyl-CoA to succinyl-CoA by the enzyme methylmalonyl-CoA mutase (Figure 33-2B). In vitamin B12 deficiency, this conversion cannot take place and the... 

Isomerases that are dependent on coenzyme B12 constitute the largest subfamily of Bi2 enzymes and are components of a number of fermentative pathways in microbes [10, 11]. A single member of this group of enzymes, methylmalonyl-CoA mutase, is found in both bacteria and in mammals where it is a mitochondrial enzyme involved in the catabolism of odd-chain fatty acids, branched chain amino... 

Heptamethylcob3rrinate cobester (13) and peripherically functionalized derivatives of 13 were used as catalysts to mimic the rearrangement catalyzed by the coenzyme Bi2-dependent enzyme methylmalonyl-CoA mutase (see Fig. 13) (45). In these studies, the reductive transformation of bromomethyl-malonates to succinates, catalyzed by 13, and similar rearrangement reactions were observed (45). 

In the fermentation the sequence occurs as shown in Fig. 2. The carboxyl is transferred from methylmalonyl CoA to pyruvate yielding oxalacetate and propionyl CoA. The oxalacetate is reduced to sucdnate. CoA then is transferred from the propionyl CoA to sucdnate and the methylmalonyl CoA is regenerated from the resulting sucdnyl CoA. The net result of this cycle is the conversion of pyruvate to propionate. In studying the cycle we investigated the conversion of sucdnyl CoA to methylmalonyl CoA by the corrinoid enzyme, methylmalonyl CoA mutase, but space limitation prevents discussion of this interesting enzyme. 

In a meantime, primiparous cows were fed the same basal diet than in the experiment described previously and supplemented daily with folic acid and rumen-protected methionine. The effects of weekly intramuscular injections of saline (0.95 NaCl) or 10 mg of vitamin Bj2 on milk production were monitored from 4 to 18 weeks of lactation. Supplementary vitamin Bj2 tended to increase milk yield from 28.5 to 31.1 kg/d and increased energy-corrected milk as well as milk yields of solids, fat and lactose but had no effect on dry matter intake or milk composition. Furthermore, packed cell volume and blood haemoglobin increased and serum methylmalonic acid decreased in cows that received vitamin Bj2 injections. The first observation suggests that low vitamin B 2 supply interfered with folate metabolism because folic acid deficiency, through its role in DNA synthesis, affects hematopoiesis (Bills et al., 1992). The second one indicates that low vitamin Bj2 supply interfered with the other vitamin B j 2-dependent enzyme, methylmalonyl-CoA mutase. These findings supported the hypothesis that vitamin Bj2 supply is suboptimal in early lactation (Girard and Matte, 2005) and may have limited the potential role of folic acid in these first studies. 

D-Methylmalonyl-CoA, the product of this reaction, is converted to the L-isomer by methylmalonyl-CoA epunerase (Figure 24.19). (This enzyme has often and incorrectly been called methylmalonyl-CoA racemase. It is not a racemase because the CoA moiety contains five other asymmetric centers.) The epimerase reaction also appears to involve a carbanion at the a-position (Figure 24.20). The reaction is readily reversible and involves a reversible dissociation of the acidic a-proton. The L-isomer is the substrate for methylmalonyl-CoA mutase. Methylmalonyl-CoA epimerase is an impressive catalyst. The for the proton that must dissociate to initiate this reaction is approximately 21 If binding of a proton to the a-anion is diffusion-limited, with = 10 M sec then the initial proton dissociation must be rate-limiting, and the rate constant must be... 

Methylmalonyl CoA mutase, leucine aminomutase, and methionine synthase (Figure 45-14) are vitamin Bj2-dependent enzymes. Methylmalonyl CoA is formed as an intermediate in the catabolism of valine and by the carboxylation of propionyl CoA arising in the catabolism of isoleucine, cholesterol, and, rarely, fatty acids with an odd number of carbon atoms—or directly from propionate, a major product of microbial fer-... 

Methylmalonyl-CoA mutase (MCM) catalyzes a radical-based transformation of methylmalonyl-CoA (MCA) to succinyl-CoA. The cofactor adenosylcobalamin (AdoCbl) serves as a radical reservoir that generates the S -deoxyadenosine radical (dAdo ) via homolysis of the Co—C5 bond [67], The mechanisms by which the enzyme stabilizes the homolysis products and achieve an observed 1012-fold rate acceleration are yet not fully understood. Co—C bond homolysis is directly kineti-cally coupled to the proceeding hydrogen atom transfer step and the products of the bond homolysis step have therefore not been experimentally characterized. 

Carboxylation of propionyl-CoA is accomplished by propionyl-CoA carboxylase (biotin, which is the carboxyl group carrier, serves as a coenzyme for this enzyme) the presence of ATP is also required. The methylmalonyl-CoA formed is converted by methylmalonyl-CoA mutase (whose coenzyme, deoxyadenosylcobalamin, is a derivative of vitamin B]2) to succinyl-CoA the latter enters the Krebs cycle. 

The vitamin cobalamin (vitamin Bjj) is reduced and activated in the body to two forms, adeno-sylcobalamin, used by methylmalonyl CoA mutase, and methylcobalamin, formed from methyl-THF in the N-methyl THF-homocysteine methyltransferase reaction. These are the only two enzymes that use vitamin (other than the enzymes that reduce and add an adenosyl group to it). 

Vitamin B12 consists of a porphyrin-like ring structure, with an atom of Co chelated at its centre, linked to a nucleotide base, ribose and phosphoric acid (6.34). A number of different groups can be attached to the free ligand site on the cobalt. Cyanocobalamin has -CN at this position and is the commercial and therapeutic form of the vitamin, although the principal dietary forms of B12 are 5 -deoxyadenosylcobalamin (with 5 -deoxyadeno-sine at the R position), methylcobalamin (-CH3) and hydroxocobalamin (-OH). Vitamin B12 acts as a co-factor for methionine synthetase and methylmalonyl CoA mutase. The former enzyme catalyses the transfer of the methyl group of 5-methyl-H4 folate to cobalamin and thence to homocysteine, forming methionine. Methylmalonyl CoA mutase catalyses the conversion of methylmalonyl CoA to succinyl CoA in the mitochondrion. 

Oxidation of unsaturated fatty acids requires two additional enzymes enoyl-CoA isomerase and 2,4-dienoyl-CoA reductase. Odd-number fatty acids are oxidized by the /3-oxidation pathway to yield acetyl-CoA and a molecule of propionyl-CoA This is carboxylated to methylmalonyl-CoA, which is isomerized to succinyl-CoA in a reaction catalyzed by methylmalonyl-CoA mutase, an enzyme requiring coenzyme B12. 

The structure of the E. coli enzyme (Fig. 16-24) shows methylcobalamin bound in a base-off conformation, with histidine 759 of the protein replacing dimethylbenzimidazole in the distal coordination position on the cobalt. This histidine is part of a sequence Asp-X-His-X-X-Gly that is found not only in methionine synthase but also in methylmalonyl-CoA mutase, glutamate mutase, and 2-methyleneglutarate mutase. However, diol dehydratase lacks this sequence and binds adenosylcobalamin with the dimethylbenz-imidazole-cobalt bond intact.417... 

The product of acetyl-CoA carboxylase reaction, malonyl-CoA, is reduced via malonate semialdehyde to 3-hydroxypropionate, which is further reductively converted to propionyl-CoA. Propionyl-CoA is carboxylated to (S)-methylmalonyl-CoA by the same carboxylase. (S)-Methylmalonyl-CoA is isomerized to (R)-methylmal-onyl-CoA, followed by carbon rearrangement to succinyl-CoA by coenzyme B 12-dependent methylmalonyl-CoA mutase. Succinyl-CoA is further reduced to succinate semialdehyde and then to 4-hydroxybutyrate. The latter compound is converted into two acetyl-CoA molecules via 4-hydroxybutyryl-CoA dehydratase, a key enzyme of the pathway. 4-Hydroxybutyryl-CoA dehydratase is a [4Fe-4S] cluster and FAD-containing enzyme that catalyzes the elimination of water from 4-hydroxybutyryl-CoA by a ketyl radical mechanism to yield crotonyl-CoA [34]. Conversion of the latter into two molecules of acetyl-CoA proceeds via normal P-oxidation steps. Hence, the 3-hydroxypropionate/4-hydroxybutyrate cycle (as illustrated in Figure 3.5) can be divided into two parts. In the first part, acetyl-CoA and two bicarbonate molecules are transformed to succinyl-CoA, while in the second part succinyl-CoA is converted to two acetyl-CoA molecules. 

Vitamin B12 is a biologically active corrinoid, a group of cobalt-containing compounds with macrocyclic pyrrol rings. Vitamin B12 functions as a cofactor for two enzymes, methionine synthase and L-methylmalonyl coenzyme A (CoA) mutase. Methionine synthase requires methylcobalamin for the methyl transfer from methyltetrahydrofolate to homocysteine to form methionine tetrahy-drofolate. L-methylmalonyl-CoA mutase requires adenosylcobalamin to convert L-methylmalonyl-CoA to succinyl-CoA in an isomerization reaction. An inadequate supply of vitamin B12 results in neuropathy, megaloblastic anemia, and gastrointestinal symptoms (Baik and Russell, 1999). 

Although the fatty acid oxidation scheme works neatly for even-numbered chain lengths, it can t work completely for fatty acids that contain an odd number of carbons. P-oxidation of these compounds leads to propionyl-CoA and acetyl-CoA, rather than to two acetyl-CoA at the final step. The propionyl-CoA is not a substrate for the TCA cycle or other simple pathways. Propionyl-CoA undergoes a carboxylation reaction to form methylmalonyl-CoA. This reaction requires biotin as a cofactor, and is similar to an essential step in fatty acid biosynthesis. Methylmalonyl-CoA is then isomerized by an epimerase and then by methylmalonyl-CoA mutase—an enzyme that uses Vitamin Bi2 as a cofactor—to form succinyl-CoA, which is a TCA-cycle intermediate. 

Methylmalonyl-CoA mutase is a cobalamin-linked enzyme of mitochondria that catalyzes the isomerization of methylmalonyl-CoA to succinyl-CoA. A reduction of this enzyme due to vitamin B12 deficiency will result in a metabolic block with the urinary excretion of methylmalonic acid, and the measurement of this metabolite has been used to confirm a deficiency of vitamin B12. The test has also been useful in investigating rare abnormalities of this enzyme that result in the excretion of methylmalonic acid in the presence of adequate vitamin B12. Given an oral loading dose of valine or isoleucine will increase the urinary excretion of methylmalonic acid in patients with a vitamin B12 deficiency (G4). However, Chanarin and his colleagues (CIO) found that one-quarter of their patients with pernicious anemia excreted a normal concentration of methylmalonic acid even after a loading dose of valine. Normal subjects excrete up to 15 mg of methylmalonic acid in their urine over a 24-hour period (Cll). 

S-Methylmalonyl-CoA mutase (EC 5.4.99.2) is a deoxyadenoxyladen-osylcobalamin-dependent enzyme of mitochondria required to catalyze the conversion of methylmalonyl-CoA to succinyl-CoA. A decrease in the activity of methylmalonyl-CoA mutase leads to the urinary excretion of large amounts of methylmalonic acid (C22). The biochemical lesion may be at the mutase level due to an abnormality of apoenzyme protein or an inability to elaborate the required coenzyme form of vitamin B12> i.e., adenosyl-cobalamin. In rare cases the abnormality may be due to an inability to convert the d form of methylmalonyl-CoA mutase to the l form as a result of a defective racemase (EC 5.1.99.1) (Kll). In patients, the nature of the abnormality can be determined by tissue culture studies (D13) and by clinical trial, since patients with a defect in adenosylcobalamin production will show clinical improvement when treated with very large doses of vitamin B12 (Mil). 

Propionate formation in cestodes probably proceeds essentially via reversal of the reactions required for the conversion of propionate to succinate in animal tissues (Fig. 5.10). Two of the enzymes involved, propionyl-CoA carboxylase and methylmalonyl-CoA mutase have been demonstrated in the mitochondria of S. mansonoides (643, 884). An acyl-CoA carboxylase, which can catalyse the carboxylation of propionyl-CoA, has also been isolated from this worm (533). The proposed pathway of propionate formation is associated with net ATP synthesis... 

Answer One of the enzymes necessary for the conversion of propionate to oxaloacetate is methylmalonyl-CoA mutase (see Fig. 17-11). This enzyme requires as an essential cofactor the cobalt-containing coenzyme B12, which is synthesized from vitamin B12. A cobalt deficiency in animals would result in coenzyme B12 deficiency. 

Answer The catabolism of the carbon skeletons of valine, isoleucine, and methionine is impaired because of the absence of a functional methylmalonyl-CoA mutase. This enzyme requires coenzyme B12 as a cofactor, and a deficiency of this vitamin leads to elevated methylmalonic acid levels (methylmalonic acidemia). The symptoms and effects of this deficiency are severe (see Table 18-2 and Box 18-2). 

---