CoA mutase

Methylmalonyl-CoA mutase is of special importance because it is the only known AdoCbl-dependent enzyme that is found in animals and man, as well as in bacteria. It catalyses the reversible interconversion of (R)-methylmalonyl-CoA and succinyl-CoA (Fig. 21). 

The steric course of the methylmalonyl-CoA mutase reaction, as it affects the C-2 of methylmalonyl-CoA, can be elucidated by determining the absolute configuration of the substrate and of a suitably labelled product. The former problem was solved by two groups [30,31]. Briefly, the (25) configuration of the epimeric product obtained from enzymic carboxylation of propionyl-CoA was established and the (2R) configuration of the mutase-active methylmalonyl-CoA followed per ex-clusionem. 

Sprinson and coworkers [30] conducted the methylmalonyl-CoA mutase reaction in deuterium oxide using a crude mitochondrial preparation. The presence of methylmalonyl-CoA epimerase insured that (1) all substrate molecules incorporated one atom of deuterium into position 2, and (2) in the course of the reaction the (2R)-epimer of methylmalonyl-CoA was continuously supplied by epimerization of the (25)-epimer, which was in turn generated by the enzymic carboxylation of propionyl-CoA. Alkaline hydrolysis of the product and subsequent purification furnished succinic acid which was mainly monodeuterated (70% 2H,-, 15% 2H2-labelled and 13% unlabelled species). A positive ORD curve revealed its (5) configuration indicating stereochemical retention for the AdoCbl-dependent rearrangement (Fig. 22). No plausible explanation could be offered for the formation of doubly deuterated and unlabelled species. Essentially the same results were later obtained with a highly purified mutase preparation from Propionibacterium sher-manii (J. Retey, unpublished). 

The chiral monodeuterated ethanols 28 and 33 were obtained by Simon s method [33] and their tosylates, 29 and 34, reacted with malonic ester anion to afford 30 and 35. The expected inversion of configuration in the malonic ester synthesis was confirmed by decarboxylating the derived acids 31 and 36 to (35)- and (3R)-[3-2H,]butanoic acids, respectively, the chiroptical properties of which were already known [34]. The chirally deuterated CoA esters 32 and 37, prepared from 31 and 36, were rearranged on methylmalonyl-CoA mutase from P. shermanii and, after hydrolysis, the methylsuccinate products were isolated. In a parallel experiment the 

In the 1 H-NMR spectrum (270 MHz) the methine and the two methylene protons of methylsuccinic add gave rise to a well-resolved ABC system. In the two enantiomers 38 and 39 the H atoms HR ,R/HSlS and H5(R/HWe.v are pair-wise reflection equivalent, i.e., identical in the NMR spectrum, whereas the diastereotopic geminal protons can be distinguished by their different chemical shifts. For the assignment of the signals to the diastereotopic protons, reference compounds of known configuration were synthesized by treating mesaconic and citraconic acids (40 and 42) with deuterated diimide. The known syn-addition of deuterium [35,36] afforded the racemic but stereospecifically dideuterated methylsuccinic acids (41 and 43) (Fig. 25). 

The second B 12-dependent enzyme to be discussed is methylmalonyl-CoA mutase which catalyzes the transformation of (/ )-methylmalonyl-CoA to succinyl-CoA [69]  

This step is the culmination of a reaction sequence in which propionyl-CoA, a toxic metabolite derived from the degradation of fats, is removed from circulation. Carboxylation of propionyl-CoA gives (5)-methylmalonyl-CoA, which is epimerized to (/ )-methylmalonyl-CoA. Conversion of the (7 )-isomer to succinyl-CoA allows further metabolism via the Krebs cycle [74]. Methylmalonyl-CoA mutase is also the only adenosylcobalamin-dependent enzyme known to participate in human metabolism, and as such has received significant study [30, 37, 38]. 

Acceptance of the bound free-radical hypothesis in this instance results in the radical rearrangement shown in reaction 7 [47, 75]  

As with the 2-methyleneglutarate mutase system, the detailed computational investigation of the methylmalonyl-CoA mutase system is somewhat complex. We therefore continue to use the model system approach, and replace the SCoA and carboxylate groups by hydrogen atoms. This simplification results in the degenerate rearrangement of the 3-propanal radical (8) [69, 76]  

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Mg-" Alcohol dehydrogenase Hexokinase 5 -Deoxyadenosylcobalamin (vitamin Big) H atoms and alkyl groups Me thy Im alony 1-CoA mutase... 

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... 

Based on the mechanism for the methylmalonyl-CoA mutase (Eigure 24.21), write reasonable mechanisms for the reactions shown below. 

Methylmalonyl-CoA mutase 5 -deoxyadenosylco-balamin is part of dimethylbenzimidazolecobamide coenzyme, a constituent of methylmalonyl-CoA mutase. This mutase catalyses the isomerization of methylmalonyl-CoA to succinyl-CoA (anaplerotic reaction of the citric acid cycle). 

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-... 

Ratnatilleke A, JW Vrijbloed, JA Robinson (1999) Cloning and sequencing of the coenzyme B,2-binding domain of isobutyryl-CoA mutase from Streptomyces cinnamonensis. Reconstitution of mutase activity and characterization of the recombinant enzyme produced in Escherichia coli. J Biol Chem 274 31679-31685. 

Monooxygenase, Isopenicillin N synthase, Glutathione peroxidase, Methylmalonyl-CoA mutase, PLP-dependent b-lyase... 

The present chapter reviews applications in biocatalysis of the ONIOM method. The focus is on studies performed in our research group, in most cases using the two-layer ONIOM(QM MM) approach as implemented in Gaussian [23], The studied systems include methane monooxygenase (MMO), ribonucleotide reductase (RNR) [24, 25], isopenicillin N synthase (IPNS) [26], mammalian Glutathione peroxidase (GPx) [27,28], Bi2-dependent methylmalonyl-CoA mutase [29] and PLP-dependent P-lyase [30], These systems will be described in more detail in the following sections. ONIOM applications to enzymatic systems performed by other research groups will be only briefly described. 

However, in some cases the reaction coordinate actually extends from the initial active-site selection into the protein, and the same-configuration solution is not adequate. One example appears in the study of Methylmalonyl-CoA mutase described below. Another drawback of a static optimization scheme is that it... 

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. 

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. 

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. 

Reeves, A.R., Britain, I.A., Cemota, W.H. et al. (2006) Effects of methylmalonyl-CoA mutase gene knockouts on erythromycin production in carbohydrate-based and oil-based fermentations of Saccharopolyspora ery-thraea. Journal of Industrial Microbiology and Biotechnology, 33, 600-609. 

Makes propionyl-CoA, which is metabolized by propionyl-CoA carboxylase (biotin) and methylmalonyl-CoA mutase (B12) to give succinyl-CoA. 

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. 

Cobalamin-c disease remethylation of homocysteine to methionine also requires an activated form of vitamin B12. In the absence of normal B12 activation, homocystinuria results from a failure of normal vitamin B12 metabolism. Complementation analysis classifies defects in vitamin B12 metabolism into three groups cblC (most common), cblD and cblF. Most individuals become ill in the first few months or weeks of life with hypotonia, lethargy and growth failure. Optic atrophy and retinal changes can occur. Methylmalonate excretion is excessive, but less than in methylmalonyl-CoA mutase deficiency, and without ketoaciduria or metabolic acidosis. 

The fibroblasts do not convert cyanocobalamin or hydroxocobalamin to methylcobalamin or adenosyl-cobalamin, resulting in diminished activity of both N5-methyltetrahydrofolate homocysteine methyltransferase and methylmalonyl-CoA mutase. Supplementation with hydroxocobalamin rectifies the aberrant biochemistry. The precise nature of the underlying defect remains obscure. Diagnosis should be suspected in a child with homocystinuria, methylmalonic aciduria, megaloblastic anemia, hypomethioninemia and normal blood levels of folate and vitamin B12. A definitive diagnosis requires demonstration of these abnormalities in fibroblasts. Prenatal diagnosis is possible. 

Historically, the intervention of tunneling has usually been invoked when the observed KIE exceeded limits set by semi-classical theory. A recent example is the hydrogen atom transfer step in methylmalonyl-CoA mutase (MCM) catalyzed... 

Cya n ocobalamin (Bir) Homocysteine methyltransferase Methylmalonyi CoA mutase Methionine, SAM Odd-carbon fatty acids, Val, Met, He, Thr MCC pernicious anemia. Also in aging, especially with poor nutrition, bacterial overgrowth of terminal ileum, resection of the terminal ileum secondary to Crohn disease, chronic pancreatitis, and, rarely, vegans, or infection with D. latum Megaloblastic (macrocytic) anemia Progressive peripheral neuropathy. ... 

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