Crystallization Studies of 5'-deoxyadenosyl Radical Enzymes

ion from a cysteine thiol, which then catalyzes the reduction of ribonucleotides (Figure 1.3). While the overall mechanism of this reaction varies greatly from that of the isomerases, in every case, the reaction involves abstraction of a hydrogen atom by Ado*. 1.2.3. Adenosylcobalamin transport and activation For AdoCbl-dependent isomerases in the absence of substrate, the cobalamin homolysis products are not observable in solution; however, upon substrate binding, the homolytic cleavage rate is increased a trillionfold (4). An important question in the study of AdoCbl enzymes is how the enzyme avoids generating potentially harmful radicals in the absence of substrate, and how the substrate binding increases the cleavage rate so dramatically. In addition, the organism must avoid generating radicals from AdoCbl before it is loaded on the enzyme. This is important both in protecting the cell from radical damage and protecting the cofactor from destruction, since cobalamin biosynthesis requires a lot of energy. The cobalamin transport system in humans has been studied extensively, and it appears as though cobalamin is essentially always protein-bound in the body (5). While many different AdoCbl isomerases are found in bacteria, humans use AdoCbl in only one enzyme, MCM, which performs a carbon skeleton rearrangement to convert methylmalonyl-CoA into succinyl-CoA. If this enzyme is inactive, methylmalonic aciduria, a potentially fatal condition, can result. It has been suggested that human adenosyltransferase (hATR) both catalyzes the reaction that forms AdoCbl from cobalamin and adenosine 5'-triphosphate (ATP) and acts as a chaperone, handing off AdoCbl to MCM (6). This delivery service would prevent side reactions that could occur if the AdoCbl was floating around in the cell. Once the AdoCbl is in place on the enzyme, it is also necessary to control reactivity to prevent radical damage to the enzyme. In the case of MCM, this is achieved in part through the coupling of substrate binding to C-Co bond homolysis (7), and in part through protection of the enzyme by MeaB, a chaperone that binds to MCM and prevents inactivation (8). 1.3. Adenosylmethionine radical enzymes AdoMet has long been known as a methylating agent used in many pathways in the cell (reviewed in 9). More recently, enzymes that use AdoMet as a free radical initiator have been characterized (reviewed in 1). AdoMet radical enzymes participate in biosynthetic and catabolic pathways and are present in all three kingdoms of life. All AdoMet radical enzymes contain a CxxxCxxC motif, with the three cysteines coordinating three of the irons in the [4Fe-4S] cluster (10). The fourth iron of the [4Fe-4S] cluster is coordinated by AdoMet itselt by its amino group and carboxylate oxygen (11). 1.3.1. General mechanism The cluster must be reduced from its resting state of [4Fe-4S] 2+ to [4Fe-4S]'+ for activity; in E. coli, this reduction is catalyzed by flavodoxin (12). The reduced cluster transfers an electron to AdoMet, reductively cleaving the carbon-sulfur bond to produce methionine and Ado*, while simultaneously regenerating the [4Fe-4S]2+ cluster (Figure 1.4). The carbon-sulfur bond cannot be cleaved homolytically as in AdoCbl-dependent enzymes because the bond is too strong (greater than 60 kcal/mol) (13). After cleavage, the 5'deoxyadenoxyl radical abstracts a hydrogen atom from the substrate. After the reaction occurs, in some cases (lysine 2,3-aminomutase (2,3-LAM), spore photoproduct lyase), the AdoMet is re-formed (1); in other cases (lipoate synthase (LipA), class III RNR, Biotin Synthase (BioB)), methionine and 5 '-deoxyadenosine are products of the reaction (1). 1.3.2. Reactions catalyzed by AdoMet radical enzymes AdoMet radical enzymes catalyze reactions on a huge variety of substrates, ranging from as small as the single amino acid lysine to large proteins (Figure 1.5). These reactions include amino group migration, carbon-carbon bond cleavage, carbon-sulfur bond formation, alcohol oxidation, and glycyl radical formation. While the substrates vary greatly in size and the reactions vary greatly in outcome, all are initiated by the abstraction of a hydrogen atom. In the class III RNR activating enzyme and pyruvate formate lyase activating enzyme, as well as other enzymes, the AdoMet radical enzyme abstracts a hydrogen atom from a glycine residue in another protein; this glycyl radical then goes on to catalyze another reaction. In the case of class III RNR, this glycyl radical abstracts a hydrogen atom from a cysteine, forming the thiyl radical necessary for ribonucleotide reduction with a similar mechanism to the other classes of RNR. 1.4. Adenosyl radical chemistry and enzymology AdoMet radical enzymes use a [4Fe-4S] cluster and AdoMet to create Ado*, while adenosylcobalamin (AdoCbl)-dependent isomerases also generate Ado* using coenzyme B12. Both AdoMet radical enzymes and AdoCbl isomerases can have TIM barrel folds or partial TIM barrel folds where the radical chemistry occurs (14). TIM barrels were first discovered in the enzyme triose phosphate isomerase and are ubiquitous in nature. They are most often involved in energy metabolism, but are present in at least 28 different enzyme classes. A full TIM barrel consists of an (oW)8 motif. The #-sheets line the interior of the barrel, which can protect reactive intermediates from solvent. While in the solved structures of AdoCbl enzymes, the TIM barrel is the full (4o)8 barrel (15, 16, 17, 18, 19, 20), three out of the four structures of AdoMet radical enzymes have a threequarters (01)6 barrel (21, 22, 23, 24). Superposition of the TIM barrel of AdoCbldependent diol dehydratase with that of the AdoMet radical enzyme BioB shows that the ring of diol dehydratase's AdoCbl occupies the same position as the [4Fe-4S] cluster in