B12-dependent enzymes

Figure 15.8 (a) Structure and (b) alternative conformations of cobalamine found in B12-dependent enzymes. The functional group R is deoxyadenosine in AdoCbl, methyl in MeCbl and -CN in vitamin B12. (From Bannerjee and Ragsdale, 2003. Reprinted with permission from Annual Reviews.)... 

As pointed out earlier a third class of B12-dependent enzymes, present in anaerobic microbes, carry out reductive dehalogenation reactions, which play an important role in the detoxification of chlorinated aliphatic and aromatic compounds, among which are many important man-made pollutants. The role of B12 in this class of enzymes is not clear— possibly by formation of an organocobalt adduct, as in the case of methyltransferases or alternatively by the corrinoid serving as an electron donor. 

If our postulates are correct the most interesting feature of P-450 is the manner in which the protein has adjusted the coordination geometry of the iron and then provided near-neighbour reactive groups to take advantage of the activation generated by the curious coordination. Vallee and Williams (68) have observed this situation in zinc, copper and iron enzymes and referred to it as an entatic state of the protein. It is also apparent that some such adjustment of the coordination of cobalt occurs in the vitamin B12 dependent enzymes. As a final example we have looked at the absorption spectra of chlorophyll for its spectrum is in many respects very like that of a metal-porphyrin. This last note is intended to stress the features of chlorophyll chemistry which parallel those of P-450. 

In 1980 Poston (PI) proposed that vitamin B12 was required for the conversion of the branched-chain amino acid p-leucine to leucine. He found circulating P-leucine levels elevated in patients with vitamin B12 deficiency. The concentration of leucine on the other hand was found to be much lower. He suggested that 2,3-aminomutase, which catalyzes the interconversion of P-leucine and leucine, is a vitamin B12-dependent enzyme which is consequently reduced in patients with pernicious anemia. The enzyme has been found in the liver of several animals and in human leucocytes, and in vitro experiments have shown it to be adenosylcobalamin dependent (P2). 

In mammals, there are only three vitamin B12 -dependent enzymes methionine synthetase, methylmalonyl CoA mutase, and leucine aminomutase. The enzymes use different coenzymes methionine synthetase uses methylcobal-amin, and cobalt undergoes oxidation during the reaction methylmalonyl CoA mutase and leucine aminomutase use adenosylcobalamin and catalyze the formation of a 5 -deoxyadenosyl radical as the catalytic intermediate. 

Ludwig ML and Matthews RG (1997) Structure-based perspectives on B12-dependent enzymes. Annual Reviews of Biochemistry 66,269-313. 

Babior, B. M., Moss, T. H., Orme-Johnson, W. H., and Beinert, H., 1974, The mechanism of action of edianolamine ammonia-lyase, a B12- dependent enzyme. The participation of paramagnetic species in die catalytic deamination of 2-aminopropanol. J. Biol. Chem. 249 4537n4544. 

The known coenzyme Bi2-dependent enzymes all perform chemical transformations in enzymatic radical reactions that are difficult to achieve by typical organic reactions. Homolytic cleavage of the Co bond of the protein-bound coenzyme B12 (3) to a 5 -deoxy-5 -adenosyl radical (9) and cob(n)alamin (5) is the entry to reversible H-abstraction reactions involving the 5 -position of the radical (9). Indeed, homolysis of the Co bond is the thermally most easily achieved transformation of coenzyme B12 (3) in neutral aqueous solution (with a homolytic (Co-C)-BDE of about 30 kcal mol ). However, to be relevant for the observed rates of catalysis by the coenzyme B12-dependent enzymes, the homolysis of the Co-C bond of the protein-bound coenzyme (3) needs to be accelerated by a factor of about 10 , in the presence of a substrate. Coenzyme B12 might then be considered, first of aU, to be a structurally sophisticated, reversible source for an alkyl radical, whose Co bond is labihzed in the protein-bound state (Figure 8), and the first major task of the... 

Chemistry of B12-Dependent Enzyme Reactions Metallo-Enzymes and Metallo-Proteins, Chemistry of Nitric Oxide, Biological Targets of NMR Overview of Applications in Chemical Biology NMR for Proteins... 

The second class of B12-dependent enzymes is represented by the aminomutases. All reactions in this class involve the 1,2-shift of an amino group. More often than not, the substrates involved in these transformations are the all-important a-amino acids. A typical such example is provided by L-leucine-2,3-amino mutase (reaction d in Scheme 2). 

Fatty acids that contain double bonds or odd numbers of carbon atoms require ancillary steps to be degraded. An isomerase and a reductase are required for the oxidation of unsaturated fatty acids, whereas propionyl CoA derived from chains with odd numbers of carbon atoms requires a vitamin B12-dependent enzyme to be converted into succinyl CoA ... 

Reiizer, R., Gruber, K., Jogl, G., Wagner, U. G., Bothe, H., Buckel, W., Kratky, C. (1999) Glutamate mutase from Clostridium cochlea-rium the structure of a coenzyme B12-dependent enzyme provides new mechanistic insights. Structure Fold. Des. 7, 891-902. 

Gruber, K., Reitzer, R., Kratky, C. (2001) Radical shuttling in a protein Ribose pseudorotation controls alkyl-radical transfer in the coenzyme B12 dependent enzyme glutamate mutase, Angew. Chem. Int. Ed. Engl. 40, 3377-3380. 

The x-ray analysis of MMCM was the first crystal structure of a coenzyme B12-dependent enzyme [18,163,194,195]. The study concerned the 150 kDa heterodimeric MMCM from P. shermanii and showed the B -cofactor to be bound base-off/His-on. The a-side of the corrin-bound cobalt center was coordinated to the histidine of the regulatory triad His-Asp-Lys. As in MetH the nucleotide tail of the boimd corrinoid was tightly inserted into the protein and the corrinoid was boimd at an interface between two domains. A rather flat corrin ligand with a ligand-folding comparable to that in imidazolyl-cobamides was revealed [31,68]. 

Poston JM. Coenzyme B12 dependent enzymes in potatoes Leucine 2,3 aminoinutase and methylmalonyl-CoA mutase. Phytochem 1978 17 40I -402. 

Methyltransferases. The catalysis of the transfer of a methyl group is an important role of enzyme-bound vitamin B12 derivatives in human, animal, and bacterial metabolism (2,3,10,49). B12-dependent, enzyme-controlled methyl group transfer reactions are key steps in the cobamide-dependent methylations of homocysteine to methionine (10,49), in the metabolic formation of methane from other Ci compounds in methanogenic bacteria (56,57), and in the fixation of carbon dioxide via the acetyl coenzyme A pathway (58). 

The rearrangement described above by Barker using the coenzyme B12-dependent enzyme glutamate mutase is a most remarkable reaction. Until very recently, no analogous chemical reaction was known. In fact, elucidation of the structure of the coenzyme form of vitamin B12 did not clarify its mechanism. Beside this transformation, nine distinct enzymatic reactions requiring coenzyme B12 as cofactor are known. Most of which are without precedent in terms of organic reactions. They are listed in Fig. 6.11. In choosing vitamin B12 derivatives as coenzymes, enzymes appear to have reached a peak of chemical sophistication which would be difficult to mimic by the chemist. 

Y., Yasuoka, N., and Toraya, T. (2002) Substrate-induced conformational change of a coenzyme B12-dependent enzyme crystal structure of the substrate-free form of diol dehydratase. Biochemistry, 41 (42), 12607-12617. 

Just as a set of reactions may require a given cocatalyst, sometimes a set of enzymes require a given coenzyme. For example, coenzyme B, a Co-containing cofactor, is required for activity in a number of B12 dependent enzymes. 

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