Generating a 5-Deoxyadenosyl Radical

There are no direct chemical models for cleaving a sulfiir-carbon bond using iron-sulfur clusters however, several suggestions have been advanced to explain how a S -deoxyadenosyl S -radical might be generated from SAM. One of the earliest proposals implicated an 

An attractive feature of this mechanism with respect to LAM is that it can be easily tested experimentally. On formation of a p3-S-adenosyl bond and termination of the reaction by acid denaturation of the protein, 5 -deoxy 5 -thioadenosine would be expected to be among the products formed. LAM was incubated with 5-[8- C]adenosyl-methionine under turnover conditions for various lengths of time, and then the reaction was terminated by the addition of trichloroacetic acid. Subsequent isolation and analysis by HPLC of the cleavage products of SAM showed that no radioactivity migrated with an authentic sample of 5 -deoxy 5 -thioadenosine.  

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Spore Photoproduct Lyase. The DNA in spores is in A-form because of dehydration. As a consequence, when spores are exposed to UV, the stereochemistry of the bases is not conducive to the formation of cyclobutane pyrimidine dimers or (6-4) photoproducts. Instead, UV induces the formation of 5-thyminyl-5,6-dihydrothymine or spore photoproduct (SP). This lesion is repaired by a 40-kDa enzyme called SP lyase. The enzyme is an iron-sulfur [4Fe-4S] protein that employs S-adenosyl-methionine (AdoMet) as a catalytic cofactor and carries out repair by a radical mechanism (6) (Fig. 2). In this mechanism, the reduced [Fe-S] center cleaves AdoMet to generate a 5 -deoxyadenosyl radical intermediate and methionine. The radical then abstracts an H-atom from C-6 of the SP. The resulting substrate radical undergoes bond cleavage to generate a product radical. The latter abstracts an H-atom from the 5 deoxyadenosine to form canonical thymines and a 5 deoxyadenosyl radical. Finally, the catalytic cycle is closed by electron transfer back to the [Fe-S] cluster concomitant with the formation of AdoMet (6). 

Figure 22.14. Formation of a 5"-Deoxyadenosyl Radical. The methylmalonyl Co A mutase reaction begins with the homolytic cleavage of the bond joining Co3+ to a carbon of the ribose of the adenosine moiety. The cleavage generates a 5 -deoxyadenosyl radical and leads to the reduction of 0 + to 0 +.

The cleavage generates a 5 -deoxyadenosyl radical and leads to the reduction of... 

A widely accepted mechanism of coenzyme Bir dependent rearrangements encompasses, as the initial step, the homolytic cleavage of the carbon-cobalt bond to generate the 5 -deoxyadenosyl radical. The thermodynamic and kinetic aspects of this and related processes involving the homolysis of transition metal-carbon bonds are discussed. 

Figure 11. Mechanisms for generating a 5 -deoxyadenosyl 5 -radical. (A) Model invoking an iron-carbon bond (B) model invoking a n,3-S-carbon bond (C) model invoking electron transfer from the iron-sulfur cluster to SAM.

Figure 15 Cleavage of S-adenosyl-L-methionine by enzymes within the radicai SAM superfamiiy. The reductive cleavage reaction generates a 5 -deoxyadenosyl 5 -radical and L-methionine, a spectator in the reaction. The physiological electron donor is the flavodoxin/flavodoxin reductase reducing system, with electrons deriving ultimately from NADPH. Artificial electron donors such as sodium dithionite and 5-deazaflavin plus light can also effect reduction in in vitro activity determinations.

Figure 16 Proposed mechanism of the LipA reaction. Cataiysis occurs on an octanoyi substrate attached in an amide linkage to a lipoyl carrier protein (LCP). In the first half-reaction, reductive cleavage of SAM to generate a 5 -deoxyadenosyl 5 -radical allows for hydrogen atom abstraction at C6 of the fatty acyl chain. The proposed source of sulfur is a second [4Fe-4S] cluster, housed in the CX4CX5C motif conserved in lipoyl synthases. In the second half-reaction, the second 5 -deoxyadenosyl 5 -radical abstracts a hydrogen atom from C8 of the fatty acyl chain, allowing for sulfur insertion at this position. The addition of two protons affords lipoic acid in its reduced form.

Figure 12. Working hypothesis for the generation of a 5 -deoxyadenosyl 5 -radical.

Removal of hydrogen from the substrate by the 5 -deoxyadenosyl radical, generating a substrate radical. It is not clear whether the dehydrogenation of the substrate occurs simultaneously with the cleavage of the Co-C bond or whether the 5 -deoxyadenosyl radical catalyzes this step. 

If ES involves a radical pair, the recombination rate of ES fe) is possible to be influenced by an external magnetic field. On the other hand, ki and k should be independent of the field. Harkins and Grisssom [4] studied MFEs on the conversion of unlabeled and deuterated ethanolamine to acetaldehyde and ammonia in bacteria by ethanolamine ammonia lyase. In this reaction, AdoCbP acts as a coenzyme and a radical pair is easily generated through the enzyme-induced homolysis of the C-Co bond. The escape 5 -deoxyadenosyl radical from the pair initiates the conversion reaction. They measured MFEs on the Vmax and Vmax/Km valucs at 25°C and obtained the results as shown in Fig. 15-4. The Vmax value was independent of B up to 0.25 T. This is reasonable because kj should be independent of B. On the other hand, the Vmax/ m values of the unlabeled and deuterated systems exhibited decreases of 25 % (at 0.1 T) and 60 % (at 0.15 T), respectively. These magnetically induced deceases can be explained by the HFCM, where k2 should be increased by such low fields as 0.1-0.15 T. At higer fields, the values were found to increase from their minimum... 

Recently, a glycerol dehydratase was discovered in the anaerobic bacterium Clostridium butyricum, whose active site contains a glycyl radical formed by the action of the 5 -deoxyadenosyl radical on a specific glycine residue of the protein (20). The 5 -deoxyadenosyl radical is generated not from coenzyme Bi2, but by one-electron reduction of the stmcturally much simpler molecule 5-adenosylmethionine (SAM), named poor man s B12 by Barker. Hence, this glycerol dehydratase performs the same reaction, probably with a similar pathway, to the coenzyme B 12-dependent glycerol dehydratase. 

Figure 30 Mechanism for L-lyslne/L-/3-lyslne Isomerization catalyzed by LAM. The 5 -deoxyadenosyl radical (Ado - CH2 ) Is generated from the homolytic cleavage of SAM at the [4Fe S] cluster site. Reproduced with permission from P. A. Frey M. D. Ballinger G. H. Reed, Biochem. Soc. Trans. 1998, 26 (3), 304-310.

As the field of SAM-dependent radical mechanisms developed, the radical SAM superfamily became recognized.The radical SAM enzymes were found to employ a different chemical strategy for generating the 5 -deoxyadenosyl, reductive cleavage of SAM by the unique [4Fe-4S] clusters in members of this superfamily. To date, more than 2800 members of this superfamily have been found, and more than 40 distinct biological reactions have been attributed to them. ... 

The critical functionality of radical SAM enzymes arises through the generation of the 5 -deoxyadenosyl radical intermediate. Nature has exploited this chemical reactivity in one other system, that of the radical Bi2-dependent enzymes, which utilize 5 -deoxyadenosylcobalamin (AdoCbl), also known as coenzyme B12, to catalyze isomerization reactions (Figure 3)." Homolytic cleavage of the cobalt(III)-deoxyadenosine bond results in a cobalt(II) center and the 5 -deoxyadenosyl radical, which generates a substrate-derived radical via the abstraction of a hydrogen atom from the substrate molecule, just as it does in the [4Fe S] reductive... 

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